The entire first verse corresponds to the white policeman's perspective. As you will listen (and read), Eminem's words are explicit in reporting the cop's viewpoint. The truth cannot be sugarcoated: "we (the cops) ain't gonna lie to you" and don't like the sight of you".

Setting the scene: the song was released as a single from the album Revival (2017). Just as The Blacker The Berry, the song is deeply rooted in contemporary America and ferociously addresses systemic racism, police brutality and white supremacy — from the perspective of someone who is a white man. The song was chosen as it perfectly encapsulates and maximizes all elements noted in previous songs.

Since the song was released only two years after The Blacker The Berry, the historical context is (almost) the same, characterized by the Black Lives Matter movement (see note on The Blacker The Berry), except for a significant change in the presidency. Indeed, Barack Obama's presidency ended in January 2017, when Republican Donald Trump was inaugurated as President.

So, why is he a hypocrite? "he refers to himself as a hypocrite for the contrast between the code of righteousness to which he ideologically subscribes and the sinfulness of his […] violence." https://open.library.ubc.ca/media/stream/pdf/24/1.0300649/4 As he clarified in a Rolling Stone interview, "he started thinking about his own time in the streets and all the wrong he's done. So he started writing a new verse, in which he turned the microscope on himself. How can he criticize America for killing young black men […] when young black men are often just so good at it?" https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_

In 2012, Travyon Martin, a 17-year-old black teenager, was killed on his way back home by a white neighborhood watch volunteer, George Zimmerman. The man acted against the police's instructions, followed Trayvon and shot him to then declare that he did it as a form of self-defense. Zimmerman was charged with second-degree murder: however, he was declared "not guilty". The case inspired a civil rights movement around issues such as race, justice and police brutality in America that would have later become worldwide known as "Black Lives Matter". In the words of former U.S. President Obama, "that was the start of America looking inward […], coming to terms with what has always been our original sin." https://youtu.be/lJynpEzXCY8?si=hTUlaeMllztuIxBf https://youtu.be/pG8FC1Nv18g?si=KvEljPV-Csd3OuPq

It was said tragedy that inspired this song: "Lamar was flipping through the channels on his tour bus when he saw on the news a report that a 16-year-old named Travyon martin had been shot to death in a Florida subdivision." The murder, the singer explained, "put a whole new anger inside me […] It made me remember how I felt. Being harassed, my partners being killed." https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_

Said acronym has different meanings; however, given the context, I inferred it stays for "Black Entertainment Television", which provides entertainment programs with a focus on Black culture. https://www.bet.com/

Marcus Garvey (1887-1940) was a Jamaican activist, mostly known for having founded the Universal Negro Improvement Association (UNIA). He encouraged Black nationalism through the celebration of African culture and history and advocated for a "back to Africa" movement. https://www.archives.gov/research/african-americans/individuals/marcus-garvey

Here the reference is to the Black Panther Party: do you remember which singer (or which song) among the previous ones mentioned it?

Starting from the 1970s, other African-American gangs emerged to defend themselves from the Crips: the Brims and the Pirus were two of them. The name comes from Piru Street in Compton, where the gang originated. They eventually united in a larger gang under the name of Bloods, which is now one of the major L.A. based gang. https://www.ojp.gov/ncjrs/virtual-library/abstracts/crips-and-bloods https://www.dictionary.com/culture/slang/piru

Lamar himself "grew up surrounded by gangs. Some of his close friends were West Side Pirus […] and his mom says her brothers were Compton Crips." https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_

If you are interested in discovering the gang's structure and delve more into their origins, here is a detailed (and very interesting If I may add) Intelligence report: https://info.publicintelligence.net/BloodsStreetGangIntelligenceReport.pdf.

Kendrick Lamar was born and raised in Compton, California, situated south of Los Angeles. The Crips he mentions are a modern criminal organization that became active in 1969 throughout Los Angeles high schools. The Compton Crips are a faction of the gang currently active in Compton. Violent fights with other gangs (mainly the Bloods) for drug dealing, smuggling and prostitution control has been going on for decades. https://web.stanford.edu/class/e297c/poverty_prejudice/gangcolor/lacrips.htm https://study.com/academy/lesson/crips-gang-history-locations.html

If you are interested in the topic, here is a link to a YouTube documentary that delves into Crip neighborhoods in Compton: https://youtu.be/iwfUCR8MbBQ?si=LWfyT8x7n5HbXVi9

The Desert Eagle is a semi-automatic gun.

The wordplay here is based on the meaning of the word black: it can mean African-American but it is also the symbolical color of evilness.

It means I am going to in a process of word evolution which may be the following: I am going to be > I'm gonna > Imma > I'ma. http://ima.urbanup.com/2146

According to you and to what we have been examined in the explanatory part, what does this line refer to? Clue: focus on the word "penitentiary".

These lines pose a critique towards societal impositions: Lamar feels irrelevant and deprived of any importance in American society. However, this feeling entirely depends on what white people have been telling him. Once again, double consciousness dominates the self.

Lamar refuses to accept that Blacks are "doomed from the start": making use of a sport metaphor, he speaks in terms of a "race", in which everyone begins from the same starting point.

The. https://jamaicanpatwah.com/term/di/963

The American English version would be: "How don't you see the whip, left scar on our back". https://jamaicanpatwah.com/term/nuh/1055 https://jamaicanpatwah.com/term/di/963 https://jamaicanpatwah.com/term/pon/1257

Cah means because. I hypothesize that it may be the outcome of an evolution process like the following: because > 'cause > cah. https://jamaicanpatwah.com/term/cah/4365

"This Black proverb appears as early as 1929 in the title of Wallace Thurman’s novel, The Blacker the Berry. Most agree that the phrase is meant as an affirmation of the richness and beauty of Black people and of darker skin Blacks. In many ways it is a counter response to the pervasive celebration of white or lighter skin Black Americans. The phrase appears in Tupac Shakur’s 1993 song “Keep Your Head Up,” and continues to flow through Black culture as a form of praise and affirmation." https://thedig.howard.edu/all-stories/save-culture-slang-exploration-black-language-expressions

See the annotation I made on the same term in 2Pac's song "Changes".

As you may have gathered by now, Lamar's song is unfiltered: although he acknowledges the hierarchy that forces his community to remain "at the bottom of mankind", he does not feel inferior. On the contrary, he is proud of his identity and his African ancestry, so much as he does not hesitate in judging the oppressors.

After having described some features stereotypically connected with being African-American, Lamar asks white people a rhetorical question ("you hate me, don't you?") since he already knows the answer. In such a perspective, there seems to be no glimpse of hope for Black people: they are hated and their culture is at risk of being "terminated". Nevertheless, Lamar does not renounce to his voice and gives space to the rage that feels since he was a teenager. https://open.library.ubc.ca/media/stream/pdf/24/1.0300649/4

Lamar's viewpoint is crystal clear: not only is there a social hierarchy in America, but also he identifies black as the ones "at the bottom". There is no possible equality in this scenario.

Once again, the you corresponds to white people listening the song.

This sentence functions as an explanation of the previous one: Lamar claims that he may be experiencing life in a schizophrenic way but blames whites (the ideal interlocutors in this scenario) for it.

Schizophrenia is a mental illness, in "characterized by disruptions in thought processes, perceptions, emotional responsiveness, and social interactions". https://www.nimh.nih.gov/health/statistics/schizophrenia However, in this case the term is more likely to refer to W.E.B. Du Bois' double consciousness. This notion hints at the idea that black people possess a double identity: the first one is tied to being African-American, whereas the other corresponds to the perspective of the White oppressors. As a consequence, their sense of self is fragmented. Hence, the reference to the fragmented self of schizophrenic people. https://study.com/learn/lesson/web-du-bois-double-consciousness-overview-background-examples.html#:~:text=Double%20consciousness%20is%20the%20feeling%20of%20having%20two%20social%20identities,%2C%20and%20treatment%20by%2C%20Caucasians. https://open.library.ubc.ca/media/stream/pdf/24/1.0300649/4

Morn' is the abbreviation of morning. https://dictionary.cambridge.org/dictionary/english/morn

Springsteen is historically accurate here: during the second half of the 19th century, most of U.S. immigrants came from Ireland, Poland and Germany (including many Jews). Moreover, the 1900 census corroborates the same thesis, showing that European immigrants mostly migrated from Italy and Germany, as well as Russia, Austria-Hungary and Britain. https://www.gilderlehrman.org/history-resources/spotlight-primary-source/map-foreign-born-population-united-states-1900#:~:text=According%20to%20the%201900%20census,Austria%2DHungary%2C%20and%20Russia. https://www.loc.gov/classroom-materials/united-states-history-primary-source-timeline/rise-of-industrial-america-1876-1900/immigration-to-united-states-1851-1900/ https://immigrationhistory.org/lesson-plan/european-migration/

Curiosity: Bruce Springsteen is actually a third generation immigrant and Zerilli was his mother's last name. His grandparents came to the United States from Vico Equestre (NA) in the beginning of the 20th century. https://napoli.corriere.it/notizie/cultura-e-tempo-libero/24_febbraio_02/torneremo-a-danzare-abbracciati-l-addio-di-bruce-springsteen-a-mamma-adele-era-originaria-di-vico-equense-8106e8ac-f4bf-415c-b8f1-162269453xlk.shtml

Curiosity: Springsteen's great-great-grandfather was named Richard McNicholas and emigrated from Ireland to the United States. https://www.independent.ie/entertainment/music/land-of-hope-and-dreams-untangling-the-puzzle-of-bruce-springsteens-irish-ancestry/a1466026818.html

Spires are "the upper tapering part of something". https://www.merriam-webster.com/dictionary/spires

This verse hints at other American myths: self-reliance, individualism and the self-made man. In short, these three combined point at the idea that working hard, putting effort and invest in one's self will eventually lead to (economic) success, independently from your initial condition.

Your turn: do you think that working hard is enough to achieve success? In other words, does success exclusively depend on effort or there may be other uncontrollable factors involved? Can poverty be overcome in this way?

Merriam-Webster defines faucet as "a fixture for drawing or regulating the flow of liquid especially from a pipe."

Gutter is defined as "a trough or groove to catch and direct something" or, more specifically, "a trough along the eaves to catch and carry off rainwater". In Italian it would be something like canali di scolo. https://www.merriam-webster.com/dictionary/gutters

The's is not an actual English word. In this case, it is probable that the singer means there are.

The song opens with the story of a man who migrates to the United States to seek fortune: he is speaking directly to his girlfriend, to whom he promises to send money as soon as possible. https://voices.pitt.edu/TeachersGuide/Unit10/American%20Land.htm

Starting from "the late 1800s, people in many parts of the world decided to leave their homes and immigrate to the United States. Fleeing crop failure, land and job shortages, rising taxes, and famine, many came to the U. S. because it was perceived as the land of economic opportunity". https://www.loc.gov/classroom-materials/united-states-history-primary-source-timeline/rise-of-industrial-america-1876-1900/immigration-to-united-states-1851-1900/

From a strictly grammatical point of view, this expression is wrong: two negative forms (didn't and no) are not acceptable in a sentence. The correct form should be either "My mama did not raise any fools" or "My mama raised no fool". However, double negative (also called negative concord) is used in some varieties of English (in this case, African-American Vernacular English) to intensify the meaning of the sentence. In other words, the construction is a sub-standard form used mainly to give more emphasis and expressive force to what is being said. It should be avoided in written and formal writing, but it is normal to use in oral speech, especially in African-American communities.

Curiosity: labeling an expression or an usage as "wrong" or "correct" is rarely a matter of language and more frequently a matter of (social) prestige: normally, it is considered "right" the variant of the language that is used by educated, white people; in contrast, "wrong" expressions are often the ones employed by minorities, uneducated or stigmatized groups. Long story short: people think they are judging an incorrect linguistic form, when, in fact, they are stigmatizing the community that uses it.

https://www.merriam-webster.com/dictionary/double%20negative https://web.archive.org/web/20100810125721/https://www.american.edu/cas/tesol/resources/upload/Kirby_Philippa.pdf

Informal alternative of you written to mimic the pronunciation in oral speech. https://www.merriam-webster.com/dictionary/ya

In this case, the expression is a synonym of hold back that is prevent someone to do something, "to hinder the progress or achievement". In Italian, it would translate to ostacolare, tenere indietro. https://www.merriam-webster.com/dictionary/hold%20back

(To) Jack up has multiple meanings; however, given the content of the song, the most probable meaning in this context is "to treat or confront (someone) in a harassing, rough, or overly aggressive and typically unwarranted manner" or "to beat up or hurt (someone)". https://www.merriam-webster.com/dictionary/jack%20up

Given what has been already noted on the previous song, do you remember what is the war on drugs? Which President of the United States promoted it?

The 1990s were a decade of unrest and, sadly, or great military violence. The main conflict that occurred in the Middle East is the Persian Gulf War (1990-1991), an international war fought between Iraq, Kuwait and the Unites States, which intervened when Kuwait was invaded by Iraqi forces. During the 1990s, other Middle East conflicts include: 1. the Iraqi Kurdish Civil War (1994-1997); 2. the Yemeni Civil War (1994); 3. the Operation Desert Fox (1998), which consisted in the U.S. bombing of Iraq.

https://www.britannica.com/event/Persian-Gulf-War#:~:text=The%20Persian%20Gulf%20War%2C%20also,Kuwait%20on%20August%202%2C%201990 https://www.bbc.co.uk/bitesize/guides/zhsssk7/revision/5

At the time in which 2Pac sang, there was a sharp rivalry among hip-hop singers of the East Coast and those of the West Coast, in which Shakur himself was implicated with and died for. Moreover, streets feuds among gangs were ferocious, mostly due to the control of drug markets. If you are interested in looking more into the topic, here is a 1990 short news special on L.A. youth gangs: https://youtu.be/-W_jhoknV1Y?si=8YSEmzi6PiZFwn5l.

Gotta is the informal abbreviation of got to, which is, in turn, a more informal alternative to have to. https://www.merriam-webster.com/dictionary/gotta

Again, similar to the "We gotta start makin' changes" verse, there seems to be glimpse of hope through intentional actions to improve the world.

(To) Make a G is the abbreviation of making a grand, where grand stands for a thousand dollars. https://dictionary.cambridge.org/dictionary/english/grand

This verse and the following ones delve into a moral dilemma: the only way to survive in this ruthless America for Black people is by "operating the easy way", which means going behind the law. Already introduced at the beginning of the song, the idea of committing crimes as a result of poverty and lack of resources is here confirmed. The "G" is made illegally, by selling drugs to a child. When confronted with the immorality of his action, the man answers that he "gotta get paid", meaning that morals and ethics come second when the priority is making ends meet. https://americansongwriter.com/the-painful-meaning-behind-the-song-changes-by-tupac/

It means "What does a mother have to do?" This verse alludes to what has been examined in the "Setting the scene" section: the single-parent families.

What topic, already mentioned in the discussion, does this sentence point to?

What 2Pac considered impossible actually occurred in 2009, when Barack Obama was elected President of the United States and then again in 2012, when he was reelected. Nevertheless, his presidency was filled with conspiracy theories (especially revolving around his being born in the Unites States) and controversies that tried to undermine the figure of the President. Ultimately, this may suggest that the United States was not ready for a black President.

Here, the condemnation towards the lack of equality and straight-up racism in America is explicit.

As you can note, 2Pac seems picking up where Hornsby left off: he shares the same disillusionment towards the possibility of equality in the United States. The entire song, as a matter of fact, denounces the hardships of being Black in an America that refuses you. https://americansongwriter.com/the-painful-meaning-behind-the-song-changes-by-tupac/

In this disheartening scenario, 2Pac seeks refuge in the pleasant memories of an idealized childhood, which is perceived as the only moment in space and time where he may find solace.

There is a missing word here: what is it? Clue: the following verb is in the present tense, so…

Do you notice anything strange in this line? For an answer, see the comment on the line "My mama didn't raise no fool".

This is directly connected to what has been said about the pervasiveness of drugs (of crack, in particular) among poor families.

Just as the previous term, this one is charged with a derogatory connotation, so much as it is preferred to refer to it with the euphemistic expression "N-word", which emcompasses both this word and its -er variant. The phenomenon that allowed the erosion of the -er ending is called r-dropping: the -r is replaced by a schwa (ə), an indistinct vowel pronounced "uh". These terms have always been linked with white supremacy, racism and white power. Even though the two terms may seem synonyms, in fact there is a difference: the -er ending word is strictly connected with the all-encompassing hatred and contempt towards black people, whereas the second one is perceived as a term of endearment when uttered by someone belonging to the Black community. Indeed, starting from the 1980s, the word has underwent a process of reclaiming (also called semantic inversion or looping) which corresponds to "taking a word meant as a slur and reappropriating it as a term of endearment" (https://www.washingtonpost.com/sf/national/2014/11/09/the-n-word-an-entrenched-racial-slur-now-more-prevalent-than-ever/?utm_term=.1590a4928864). This strategy allows the originally oppressive term to be re-semantized (that is, to acquire a new meaning) and used to celebrate the community's unique identity and humanity as "an act of redemption by black folk. The word survives on the conditions that black folks have inscribed for it and nobody else can take that. And it becomes violent when other people try to take it and use it." Indeed, white people "have created the word in the first place, but […] they have lost the power to use it with impunity, they have lost the power to reclaim it." […] "If you understand the history of the word and how it's been used, it's not for white people to use […] So if you're not black you can't do that. You actually can't use the word in the way that we use it. It's not possible, because you're not in that space. So any other usage of it is completely wrong." (https://www.bbc.com/news/stories-53749800). https://www.merriam-webster.com/dictionary/N-word https://share.google/2p6rElVA4Vin0v2cC https://www.dailydot.com/irl/how-not-to-use-the-n-word/

The word is notoriously a derogatory term to indicate African-Americans. However, "both Negro and Negress are sometimes used by Black people in self-reference, but use of either term by others is offensive". https://www.merriam-webster.com/dictionary/Negro

In the society in which the narrative voice lives, both poverty and blackness have a negative connotation. In other words, being Black adds up to being poor.

The verb blast can have multiple meanings; in this case, the most probable is shoot (sparare in Italian) https://www.merriam-webster.com/dictionary/blast#dictionary-entry-2

Disclaimer: sensitive content.

In a world dominated by racism and in which nothing changes, the singer wonders whether the struggles he has to cope with on a daily basis make living worth it. He even contemplates the possibility to end his life in the same verse. If you have ever felt the same way the singer does in this verse, please seek help at https://azzurro.it/ or at least talk with an adult or a teacher.

Note how don't is paired with a singular noun, law. "Don't is occasionally used in American English speech and in historical writing as a contraction of does not (as in, "He don't know where he is going."), but this use is now considered improper and should be avoided. Remember that in modern speech and writing, don't cannot be used in the third person singular." https://www.britannica.com/dictionary/eb/qa/don-t-and-doesn-t

Hornsby's posture seems clear: in these verses, he states that legal measures can only "go so far", that is, they can only operate up to a certain extent. He hints at the fact that another change has to occur: a cultural one. According to the author, indeed, equality can be achieved only in a co-constructive process that implicates the law on one side, and the culture on the other. In other words, what Hornsby is trying to state is that acts can be signed into law and assure people their rights, but it is equally important that people change their mindsets.

The "law" in this verse could allude to: * the Civil Rights Act passed in 1964 by President Lyndon Johnson. It "prohibited discrimination in public places, provided for the integration of schools and other public facilities, and made employment discrimination illegal" (https://www.archives.gov/milestone-documents/civil-rights-act#:~:text=This%20act%2C%20signed%20into%20law%20by%20President,most%20sweeping%20civil%20rights%20legislation%20since%20Reconstruction.) In other words, it made segregation in all its forms illegal. * the Economic Opportunity Act which was introduced in the same year. It "aimed at facilitating education, health, employment, and general welfare for impoverished Americans" (https://www.britannica.com/event/American-civil-rights-movement). It was part of the so-called "War on Poverty", a program endorsed by President Johnson and which intended to reduce poverty in the United States, along with making the country more equitable. https://www.britannica.com/topic/War-on-Poverty

It is probable that the song hints at both Acts, rather than pointing at just one.

In this sad scene, a new character is introduced: a man in a silk suit. He represents the rich and wealthy people who do not have to wait in line for economic help and, for this reason, hurries by (that is, he moves rapidly). His perspective is biased: he blames those in line for being poor and encourages them to "get a job". Therefore, Hornsby's intention is that of criticizing social polarization and inequality, apart from biased convictions that link poverty to laziness.

Your turn: is the man in a silk suit right? Are poor people always lazy or are there any other reasons for poverty?

The reason that is provided as for why they are waiting and need financial support is their poverty. The expression "buy a job" may refer to the fact that they cannot afford college education, in a modern scenario in which education assures better job opportunities; in a darker perspective, it might also mean that they have no connections to secure themselves a job through nepotism or bribery.

As an adjective, welfare means "of, relating to, or concerned with welfare and especially with improvement of the welfare of disadvantaged social groups" (https://www.merriam-webster.com/dictionary/welfare). Dime, instead, is a synonym of coin (https://www.merriam-webster.com/dictionary/dime). The combination of the the two words refers to the economic assistance provided by the government to families in financial distress.

This first stanza describes a specific image: the songs opens with a symbolical "line" where people in distress are waiting to receive some help from the government.

Here is the chorus of the song: to all the questions posed, the songwriter is not able to answer. In fact, the answer is as elusive as the wind and impossible to grasp. https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind

Another interesting element to point out is the relationship that Dylan seems to have with the audience: as previously remarked, this song converts Dylan into a spokesman of the common people, a "primus inter pares" if you wish, who makes use of his art to denounce inequality and social struggles. He does not think of himself as a custodian of a hidden truth no one else has discovered: just as everybody else, he does not have a clue about why discrimination and wars occur.

Your turn: if there are no answers, why asking questions? Do you think there is value in asking questions that cannot be answered?

"The mountain […] is a symbol of those human institutions that keep war and oppression in place. The stony mountain is all that resists change: the shape of government and history, certainly, but also the rocky terrain inside people's hearts. The slow, persistent erosive power of the ocean, on the other hand, symbolizes the action of internal and external change." https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind

Here, the reference to the Vietnam war is explicit through the usage of the key word cannonballs.

The dove is a white bird that corresponds to colomba in Italian. It usually symbolizes peace. https://dictionary.cambridge.org/dictionary/english/dove <br /> "There's also a reference here to a specific dove: the biblical dove of Noah's Ark, which flew out from the Ark to seek dry land and returned bearing a hopeful olive branch. The symbolism of the dove here suggests that the change the speaker hopes for may not be easy to come by" (https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind).

Notice how the structure "before […] how many" is repeated throughout all the song. https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind

The repetition of "yes" at the beginning of many verses indicates the usage of a specific rhetorical device — the anaphora. https://www.merriam-webster.com/dictionary/anaphora

This is the opening of the song, which starts and procedes by asking multiple rhetorical questions to the listeners. In this case, Bob Dylan is calling into question the essence of humanity by describing an activity as simple as walking. The "roads" may hint at the long history of difficulties and hardships that Black people have experienced. The call to equality is unmistakable. https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind

According to you, what makes us human?

Setting the scene: the song was released in 1963 and included in the album The Freewhelin' Bob Dylan. All of Dylan's "most famous political songs were written [...] between January 1962 and October 1963" and "those [...] fixed him in the popular imagination" as a protest songwriter (Lynskey, 2010, 67). The historical context in which the song was released is essential to understand its meaning: during the 1950s the civil rights movement against racial segregation started to gain momentum, reaching its highest point precisely in 1963. Indeed, in that year: * Desegregation protests spread throughout the Southern states over more than 100 cities. The most famous was the March on Washington: it was organized by the "Big Six" of the civil rights movement (Martin Luther King Jr., James Farmer, J. Lewis, P. Randolph, R. Wilkins, W. Young), attended by more than 250.000 protesters (including Bob Dylan). It was in this occasion that the renowned "I have a dream" speech took place. * On June 12, President Kennedy announced he would present a civil rights bill to Congress, which was eventually passed the following year under the name of the Civil Rights Act. https://www.britannica.com/event/American-civil-rights-movement https://www.britannica.com/event/American-civil-rights-movement

It is important to bear in mind that protests were not only motivated by the insufferable racial segregation, but they were also anti-war oriented: the Vietnam war (1955-1975), which had been raging for ten years by the mid-1960s, was felt as an unnecessary conflict, especially by students and young people, who were the ones recruited in the U.S. army. Indeed, "the average age of an American soldier in Vietnam was 19" (https://www.bbc.co.uk/bitesize/guides/z6dk8hv/revision/4 ).

The entire first verse corresponds to the white policeman's perspective. As you will listen (and read), Eminem's words are explicit in reporting the cop's viewpoint. The truth cannot be sugarcoated: "we (the cops) ain't gonna lie to you" and don't like the sight of you".

In the opening of the song, Eminem simulates a scene in which a white policeman stops a car driven by a black young man.

Setting the scene: the song was released as a single from the album Revival (2017). Just as The Blacker The Berry, the song is deeply rooted in contemporary America and ferociously addresses systemic racism, police brutality and white supremacy — from the perspective of someone who is a white man. The song was chosen as it perfectly encapsulates and maximizes all elements noted in previous songs.

Since the song was released only two years after The Blacker The Berry, the historical context is (almost) the same, characterized by the Black Lives Matter movement (see note on The Blacker The Berry), except for a significant change in the presidency. Indeed, Barack Obama's presidency ended in January 2017, when Republican Donald Trump was inaugurated as President.

So, why is he a hypocrite? "he refers to himself as a hypocrite for the contrast between the code of righteousness to which he ideologically subscribes and the sinfulness of his […] violence." https://open.library.ubc.ca/media/stream/pdf/24/1.0300649/4 As he clarified in a Rolling Stone interview, "he started thinking about his own time in the streets and all the wrong he's done. So he started writing a new verse, in which he turned the microscope on himself. How can he criticize America for killing young black men […] when young black men are often just so good at it?" https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_

In 2012, Travyon Martin, a 17-year-old black teenager, was killed on his way back home by a white neighborhood watch volunteer, George Zimmerman. The man acted against the police's instructions, followed Trayvon and shot him to then declare that he did it as a form of self-defense. Zimmerman was charged with second-degree murder: however, he was declared "not guilty". The case inspired a civil rights movement around issues such as race, justice and police brutality in America that would have later become worldwide known as "Black Lives Matter". In the words of former U.S. President Obama, "that was the start of America looking inward […], coming to terms with what has always been our original sin." https://youtu.be/lJynpEzXCY8?si=hTUlaeMllztuIxBf https://youtu.be/pG8FC1Nv18g?si=KvEljPV-Csd3OuPq

It was said tragedy that inspired this song: "Lamar was flipping through the channels on his tour bus when he saw on the news a report that a 16-year-old named Travyon martin had been shot to death in a Florida subdivision." The murder, the singer explained, "put a whole new anger inside me […] It made me remember how I felt. Being harassed, my partners being killed." https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_

Said acronym has different meanings; however, given the context, I inferred it stays for "Black Entertainment Television", which provides entertainment programs with a focus on Black culture. https://www.bet.com/

February is the Black History Month: introduced in 1926 by C. G. Woodson (who helped established African American Studies), the birthmonth of former U.S. President Abraham Lincoln and reformer F. Douglass has been dedicated to celebration of African-American history and culture. Every year, the Association for the Study of African American Life and History (ASALH) chooses a theme to focus on. Last year, for example, the theme revolved around labour. https://parade.com/living/black-history-month-themes https://www.weforum.org/stories/2024/02/black-history-month-what-is-it-and-why-do-we-need-it/

Marcus Garvey (1887-1940) was a Jamaican activist, mostly known for having founded the Universal Negro Improvement Association (UNIA). He encouraged Black nationalism through the celebration of African culture and history and advocated for a "back to Africa" movement. https://www.archives.gov/research/african-americans/individuals/marcus-garvey

This expression precedes proof of Kendrick Lamar's effort and interest in promoting African-American culture: * he supports and remembers the Black Panther Party; * he lectures Georgia State college students about pivotal figures in the African-American community, such as Marcus Garvey; * he celebrates Black History month as it were his birthday.

However, the expression "don't matter how much" (just as the haunting repetition of being "the biggest hypocrite of 2015") signals that somehow his committment to the cause is not enough: why? The singer forces the audience to wait until the last lines of the song to clarify the reasons of his hypocrisy.

Starting from the 1970s, other African-American gangs emerged to defend themselves from the Crips: the Brims and the Pirus were two of them. The name comes from Piru Street in Compton, where the gang originated. They eventually united in a larger gang under the name of Bloods, which is now one of the major L.A. based gang. https://www.ojp.gov/ncjrs/virtual-library/abstracts/crips-and-bloods https://www.dictionary.com/culture/slang/piru

Lamar himself "grew up surrounded by gangs. Some of his close friends were West Side Pirus […] and his mom says her brothers were Compton Crips." https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_

If you are interested in discovering the gang's structure and delve more into their origins, here is a detailed (and very interesting If I may add) Intelligence report: https://info.publicintelligence.net/BloodsStreetGangIntelligenceReport.pdf.

Kendrick Lamar was born and raised in Compton, California, situated south of Los Angeles. The Crips he mentions are a modern criminal organization that became active in 1969 throughout Los Angeles high schools. The Compton Crips are a faction of the gang currently active in Compton. Violent fights with other gangs (mainly the Bloods) for drug dealing, smuggling and prostitution control has been going on for decades. https://web.stanford.edu/class/e297c/poverty_prejudice/gangcolor/lacrips.htm https://study.com/academy/lesson/crips-gang-history-locations.html

If you are interested in the topic, here is a link to a YouTube documentary that delves into Crip neighborhoods in Compton: https://youtu.be/iwfUCR8MbBQ?si=LWfyT8x7n5HbXVi9

Southern Black Africans presents four major ethnic divisions; one of them is the Nguni, which, in its turn, can be divided into four groups: Zulu and Xhosa are two of them. https://sahistory.org.za/article/xhosa https://sahistory.org.za/article/zulu Although they share a common history, they were implicated in a civil war from 1990 to 1994. The reason behind this conflict is, actually, linguistic and tied to colonialism: the two languages were "created" by colonizers and African interpreters. Before African colonialism, indeed, there weren't any written languages and people did not distinguish themselves on a linguistic basis, but rather on social belonging. This does not mean that "Zulu and Xhosa identities didn’t exist before the languages were well defined, rather that the identities were transformed when these languages came into existence." https://theconversation.com/zulu-vs-xhosa-how-colonialism-used-language-to-divide-south-africas-two-biggest-ethnic-groups-204969

The Desert Eagle is a semi-automatic gun.

Tyrone is originally Irish name, but recently is increasingly more common for boys in African-American communities. Since its strong association to blacks, it is also at the center of discrimination: compared to Tyrone, "white" names are twice as likely to receive responses. Also Darius is a typical African-American name. https://www.irishexaminer.com/news/arid-10108995.html https://www.behindthename.com/name/darius

In this scenario dominated by manslaughter and perverse traditions, the pervasive double consciousness that haunts the singer seems to falter: the American heritage, which also entails this bitter history or racism, is too heavy for the singer to carry. Ultimately, he's African.

In this last verse, there is no possible misunderstanding: Lamar directly attacks centuries of racism, whose thousands of deaths he (rightly) labels as genocide. Another striking expression is that of "generational hatred": the singer evokes an atmosphere in which racism is like a twisted tradition which, instead of passing down from father to son, passes down from generation to generation, destroying people in the meantime.

It means I am going to in a process of word evolution which may be the following: I am going to be > I'm gonna > Imma > I'ma. http://ima.urbanup.com/2146

The American English version would be: "How don't you see the whip, left scar on our back". https://jamaicanpatwah.com/term/nuh/1055 https://jamaicanpatwah.com/term/di/963 https://jamaicanpatwah.com/term/pon/1257

Cah means because. I hypothesize that it may be the outcome of an evolution process like the following: because > 'cause > cah. https://jamaicanpatwah.com/term/cah/4365

This entire verse is written in Jamaican patois, that is, "an English-lexified creole language spoken by the majority of Jamaicans". As you will see, some words may be intuitive, but others are definitely not. While I was looking for the lyrics of the song, I found other anglicized versions which were certainly more comprehensible, but, I am afraid, less faithful to the singer's intention. Consequently, I opted for the original, more complex version. Why has the singer recurred to Jamaican patois? My hypothesis is that he features it as a way to give importance and centrality to a marginal community through its language.

Dem is they. https://jamaicanpatwah.com/term/dem/961

Lamar mangles (storpia, in Italian) the words of the proverb: this modified version seems to represent a counterpart to the the original one. If the first one celebrates and honors Black culture, this one seems to connect to the brutality of reality, in which violence dominates and is, apparently, the only possible response.

"This Black proverb appears as early as 1929 in the title of Wallace Thurman’s novel, The Blacker the Berry. Most agree that the phrase is meant as an affirmation of the richness and beauty of Black people and of darker skin Blacks. In many ways it is a counter response to the pervasive celebration of white or lighter skin Black Americans. The phrase appears in Tupac Shakur’s 1993 song “Keep Your Head Up,” and continues to flow through Black culture as a form of praise and affirmation." https://thedig.howard.edu/all-stories/save-culture-slang-exploration-black-language-expressions

See the annotation I made on the same term in 2Pac's song "Changes".

This word is a highly offensive, derogatory term used to insult Black people by comparing them to animals. The likening of a person to an ape, a monkey or a gorilla is a discriminatory practice that takes the name of simianization. Simianization dates back to the Middle Ages and has progressively taken a racist turn and started only to indicate black-skinned individuals. Reasons for this association may include the prevalence of apes in Africa and the aesthetic difference between whites and blacks. In any case and whatever the reason may be, it is a form of degradation and dehumanization. In this case, it is probable that Lamar is aiming for a reclaiming (that is, semantic inversion) of the term. In other words, the same reasoning applied for "nigga" in 2Pac's "Changes" is applicable here: the singer uses this term to affirm his identity as an African-American. https://www.dictionary.com/browse/simianization https://theconversation.com/comparing-black-people-to-monkeys-has-a-long-dark-simian-history-55102

After having described some features stereotypically connected with being African-American, Lamar asks white people a rhetorical question ("you hate me, don't you?") since he already knows the answer. In such a perspective, there seems to be no glimpse of hope for Black people: they are hated and their culture is at risk of being "terminated". Nevertheless, Lamar does not renounce to his voice and gives space to the rage that feels since he was a teenager. https://open.library.ubc.ca/media/stream/pdf/24/1.0300649/4

Once again, the you corresponds to white people listening the song.

Here Lamar denounces a decade-long rage for anti-Black racism and police brutality he himself was a victim of. In a Rolling Stone interview, Lamar declared that as a teenager "the majority of my interactions with the police were not good […] there were a few good ones who were actually protecting the community. But then you have the ones from the Valley. They never met me in my life, but since I'm a kid […] they wanna slam me on the hood of the car. Sixteen years old […] Even if he's not a good kid, that don't give you the right to slam a minor on the ground or pull a pistol on him. " […] Indeed, "police pull guns on him on two occasions. The first when he was 17." https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_

Schizophrenia is a mental illness, in "characterized by disruptions in thought processes, perceptions, emotional responsiveness, and social interactions". https://www.nimh.nih.gov/health/statistics/schizophrenia However, in this case the term is more likely to refer to W.E.B. Du Bois' double consciousness. This notion hints at the idea that black people possess a double identity: the first one is tied to being African-American, whereas the other corresponds to the perspective of the White oppressors. As a consequence, their sense of self is fragmented. Hence, the reference to the fragmented self of schizophrenic people. https://study.com/learn/lesson/web-du-bois-double-consciousness-overview-background-examples.html#:~:text=Double%20consciousness%20is%20the%20feeling%20of%20having%20two%20social%20identities,%2C%20and%20treatment%20by%2C%20Caucasians. https://open.library.ubc.ca/media/stream/pdf/24/1.0300649/4

Morn' is the abbreviation of morning. https://dictionary.cambridge.org/dictionary/english/morn

Setting the scene: the song was released as a second single from the 2015 album To Pimp A Butterfly. The album (and the song as well) "is firmly in the present. It's [Lamar's] take on what it means to be young and black in America today". https://issuu.com/lawrenceambrocio5018/docs/rolling_stone_march_26_2015_usa_1_ Indeed, the album is deeply connected to contemporary issues in America (police brutality, systemic racism and inequality) and, in particular, to Black Lives Matter. Black Lives Matter is a "Black-centered political-movement-building project" that surged in 2013 as a reaction to the Travyon Martin murder. The core beliefs of the movement include: * the affirmation of the importance of Black Lives against police brutality and any other racist manifestation; * transforming the present world in which "Black lives are systemically targeted for deliberate and indirect demise"; * affirmation of Black humanity and contributions; * abolition of mass incarceration to the detriment of Black people; * collective safety.

https://blacklivesmatter.com/our-history/

Here the central critique to the lack of equality and the debunking of the immigration myths appears evident: in particular, Springsteen makes uses of the anaphora "they died" to underscore the timeless sacrifice of immigrants, who are identified with the makers of the United States ("the hands that build the country"). This last part of the song completely overturns what Springsteen has sung so far: there are no streets paved with gold, no diamonds in the sidewalks or "treasure for the taking". What remains is work "to bones and skin". Once again, Springsteen has not departed from the historical truth: despite their hopes, European immigrants who landed in the United States did not improve their status. In other words, if they were poor, they stayed as such; “past European immigrants often struggled when they first arrived, and most of them did not succeed in reaching the American Dream within their lifetimes.” https://ui.charlotte.edu/story/streets-gold-debunking-american-immigration-myths/ Moreover, research shows that from 1880 to 1920 immigrants "were the mainstay of the American industrial workforce". https://pmc.ncbi.nlm.nih.gov/articles/PMC2760060/#abstract1 Making home in the American Land was, therefore, impossible for them because America itself rejected and discriminated immigrants after exploiting them for its own industrial and economic growth. Indeed, "often stereotyped and discriminated against, many immigrants suffered verbal and physical abuse because they were "different." […] The newcomers helped transform American society and culture, demonstrating that diversity, as well as unity, is a source of national strength." https://www.loc.gov/classroom-materials/united-states-history-primary-source-timeline/rise-of-industrial-america-1876-1900/immigration-to-united-states-1851-1900/

Springsteen's radical claim is that such discrimination and sacrifice are not limited to a distant past, but have continued up until now ("they're still dyin' now"). When Springsteen released the song, the country was actually undergoing a period of decline in immigration due to measures taken after the terrorist attacks of September 11, 2001 such as the Patriot Act. https://library.law.howard.edu/civilrightshistory/immigration/history. However, it is true that immigration continues to be a divisive topic in the United States, especially in the current presidency: what do you know about Trump's immigration policies?

Springsteen is historically accurate here: during the second half of the 19th century, most of U.S. immigrants came from Ireland, Poland and Germany (including many Jews). Moreover, the 1900 census corroborates the same thesis, showing that European immigrants mostly migrated from Italy and Germany, as well as Russia, Austria-Hungary and Britain. https://www.gilderlehrman.org/history-resources/spotlight-primary-source/map-foreign-born-population-united-states-1900#:~:text=According%20to%20the%201900%20census,Austria%2DHungary%2C%20and%20Russia. https://www.loc.gov/classroom-materials/united-states-history-primary-source-timeline/rise-of-industrial-america-1876-1900/immigration-to-united-states-1851-1900/ https://immigrationhistory.org/lesson-plan/european-migration/

Curiosity: Bruce Springsteen is actually a third generation immigrant and Zerilli was his mother's last name. His grandparents came to the United States from Vico Equestre (NA) in the beginning of the 20th century. https://napoli.corriere.it/notizie/cultura-e-tempo-libero/24_febbraio_02/torneremo-a-danzare-abbracciati-l-addio-di-bruce-springsteen-a-mamma-adele-era-originaria-di-vico-equense-8106e8ac-f4bf-415c-b8f1-162269453xlk.shtml

Curiosity: Springsteen's great-great-grandfather was named Richard McNicholas and emigrated from Ireland to the United States. https://www.independent.ie/entertainment/music/land-of-hope-and-dreams-untangling-the-puzzle-of-bruce-springsteens-irish-ancestry/a1466026818.html

This verse hints at other American myths: self-reliance, individualism and the self-made man. In short, these three combined point at the idea that working hard, putting effort and invest in one's self will eventually lead to (economic) success, independently from your initial condition.

Do you think that working hard is enough to achieve success? In other words, does success exclusively depend on effort or there may be other uncontrollable factors involved? Can poverty be overcome in this way?

Spires are "the upper tapering part of something". https://www.merriam-webster.com/dictionary/spires

"Immigrants entered the United States through several ports. Those from Europe generally came through East Coast facilities, while those from Asia generally entered through West Coast centers. More than 70 percent of all immigrants, however, entered through New York City, which came to be known as the "Golden Door." Throughout the late 1800s, most immigrants arriving in New York entered at the Castle Garden depot near the tip of Manhattan. In 1892, the federal government opened a new immigration processing center on Ellis Island in New York harbor." https://www.loc.gov/classroom-materials/united-states-history-primary-source-timeline/rise-of-industrial-america-1876-1900/immigration-to-united-states-1851-1900/

Ellis Island was chosen as the first federal facility in which immigrants were processed because of its strategic position: it was isolated, far from the mainland and, therefore, considered fitting to carefully inspect immigrants and prevent them from entering the country without being registered. Inspection process was not detached from class distinctions: only indigent, (that is, poor), third-class passengers (also referred to as "steerage") were required to undergo the inspection process as Ellis Island. What was the criterion, then? Those who boarded the ship on first or second class were presumed to be wealthy people, less likely to "become a public charge in America due to medical or legal reasons". After a long trip, which entailed staying for days in unsanitary conditions and overly crowded spaces, poor people were submitted to a minimum of 3\5 hours of inspection in the Great Hall: their health condition was examined and their origins as well as destinations were investigated. https://klagenfurtmigrationstudies.home.blog/understanding-barriers-to-immigration-by-listening-to-ellis-island-oral-histories/ https://www.statueofliberty.org/ellis-island/overview-history/. Ellis Island is now seat of a National Museum of Immigration which can be visited (https://www.statueofliberty.org/visit/); otherwise, the official website offers many online resources if you are interested in digging in the topic.

Merriam-Webster defines faucet as "a fixture for drawing or regulating the flow of liquid especially from a pipe."

Gutter is defined as "a trough or groove to catch and direct something" or, more specifically, "a trough along the eaves to catch and carry off rainwater". In Italian it would be something like canali di scolo. https://www.merriam-webster.com/dictionary/gutters

These sentences evoke popular belief among immigrants that in America there were "streets paved with gold" : this rumor (this myth if you want) created anticipation and hope for those who left their homelands in search of better opportunities. The powerful expression gave birth to a homonymous exhibit: https://www.nps.gov/elis/learn/education/streets-paved-with-gold.htm.

The song opens with the story of a man who migrates to the United States to seek fortune: he is speaking directly to his girlfriend, to whom he promises to send money as soon as possible. https://voices.pitt.edu/TeachersGuide/Unit10/American%20Land.htm

Starting from "the late 1800s, people in many parts of the world decided to leave their homes and immigrate to the United States. Fleeing crop failure, land and job shortages, rising taxes, and famine, many came to the U. S. because it was perceived as the land of economic opportunity". https://www.loc.gov/classroom-materials/united-states-history-primary-source-timeline/rise-of-industrial-america-1876-1900/immigration-to-united-states-1851-1900/

Setting the scene: the song was first released in 2006 in the We Shall Overcome: the Seeger Sessions album as a bonus live track on a special edition of the album. The recorded version was added in 2012 to the Wrecking Ball album. The song has a long genesis: it is based on Pete Seeger's "He Lies in the American Land" (1956), which, in its turn, was the translation of a text originally written by a Slovak steelworker, Andrew Kovaly, at the beginning of the 20th century. Consequently, Springsteen recovers and adapts a song that deals with a timeless topic: immigration and, ultimately, the lack of equality for those who migrate to the United States. The music itself is a mixture of typical "American" sounds (rock 'n' roll, the electric guitar...) and an Irish-like folk motive.

Since its "discovery", America has been a land of immigration; in particular, the major wave of immigrants landed in America at the end of the 19th century and the beginning of the following one. Indeed, between 1870 and 1900, nearly 12 million people arrived in the United States. Simultaneously, xenophobia and anti-immigration actions gained momentum: differentiations between "desirable" and "undesirable" immigrants based on racist assumptions around ethnicity and religion laid the foundations for the Immigration Act of 1917, which restricted immigration by imposing literacy tests and by preventing immigration from Asia and almost the entire Middle East. Context around this specific period of time is important because it is exactly in those years that Kovaly wrote "He lies in the American Land".

Compared to the previous songs, which focus mainly on African Americans and racism, this song shifts attention to inequality due to being an immigrant.

https://immigrationhistory.org/lesson-plan/european-migration/ https://voices.pitt.edu/TeachersGuide/Unit10/American%20Land.htm https://alessandroportelli.blogspot.com/2012/03/bruce-springsteen-wrecking-ball.html https://library.law.howard.edu/civilrightshistory/immigration/history

From a strictly grammatical point of view, this expression is wrong: two negative forms (didn't and no) are not acceptable in a sentence. The correct form should be either "My mama did not raise any fools" or "My mama raised no fool". However, double negative (also called negative concord) is used in some varieties of English (in this case, African-American Vernacular English) to intensify the meaning of the sentence. In other words, the construction is a sub-standard form used mainly to give more emphasis and expressive force to what is being said. It should be avoided in written and formal writing, but it is normal to use in oral speech, especially in African-American communities.

Curiosity: labeling an expression or an usage as "wrong" or "correct" is rarely a matter of language and more frequently a matter of (social) prestige: normally, it is considered "right" the variant of the language that is used by educated, white people; in contrast, "wrong" expressions are often the ones employed by minorities, uneducated or stigmatized groups. Long story short: people think they are judging an incorrect linguistic form, when, in fact, they are stigmatizing the community that uses it.

https://www.merriam-webster.com/dictionary/double%20negative https://web.archive.org/web/20100810125721/https://www.american.edu/cas/tesol/resources/upload/Kirby_Philippa.pdf

Informal alternative of you written to mimic the pronunciation in oral speech. https://www.merriam-webster.com/dictionary/ya

In this case, the expression is a synonym of hold back that is prevent someone to do something, "to hinder the progress or achievement". In Italian, it would translate to ostacolare, tenere indietro. https://www.merriam-webster.com/dictionary/hold%20back

(To) Jack up has multiple meanings; however, given the content of the song, the most probable meaning in this context is "to treat or confront (someone) in a harassing, rough, or overly aggressive and typically unwarranted manner" or "to beat up or hurt (someone)". https://www.merriam-webster.com/dictionary/jack%20up

The 1990s were a decade of unrest and, sadly, or great military violence. The main conflict that occurred in the Middle East is the Persian Gulf War (1990-1991), an international war fought between Iraq, Kuwait and the Unites States, which intervened when Kuwait was invaded by Iraqi forces. During the 1990s, other Middle East conflicts include: 1. the Iraqi Kurdish Civil War (1994-1997); 2. the Yemeni Civil War (1994); 3. the Operation Desert Fox (1998), which consisted in the U.S. bombing of Iraq.

https://www.britannica.com/event/Persian-Gulf-War#:~:text=The%20Persian%20Gulf%20War%2C%20also,Kuwait%20on%20August%202%2C%201990 https://www.bbc.co.uk/bitesize/guides/zhsssk7/revision/5

At the time in which 2Pac sang, there was a sharp rivalry among hip-hop singers of the East Coast and those of the West Coast, in which Shakur himself was implicated with and died for. Moreover, streets feuds among gangs were ferocious, mostly due to the control of drug markets. If you are interested in looking more into the topic, here is a 1990 short news special on L.A. youth gangs: https://youtu.be/-W_jhoknV1Y?si=8YSEmzi6PiZFwn5l.

Gotta is the informal abbreviation of got to, which is, in turn, a more informal alternative to have to. https://www.merriam-webster.com/dictionary/gotta

Again, similar to the "We gotta start makin' changes" verse, there seems to be glimpse of hope through intentional actions to improve the world.

(To) Make a G is the abbreviation of making a grand, where grand stands for a thousand dollars. https://dictionary.cambridge.org/dictionary/english/grand

This verse and the following ones delve into a moral dilemma: the only way to survive in this ruthless America for Black people is by "operating the easy way", which means going behind the law. Already introduced at the beginning of the song, the idea of committing crimes as a result of poverty and lack of resources is here confirmed. The "G" is made illegally, by selling drugs to a child. When confronted with the immorality of his action, the man answers that he "gotta get paid", meaning that morals and ethics come second when the priority is making ends meet. https://americansongwriter.com/the-painful-meaning-behind-the-song-changes-by-tupac/

Apart from the reference to crack, this verse has another important element to be noted: 2Pac is trying to debunk the myth that only African-Americans use drugs. In fact, this 1995 report clearly shows racial disparity in connection to arrests for drug sale and possession: https://bjs.ojp.gov/content/pub/pdf/rdusda.pdf. Although it is undeniable that the African-American community was deeply affected by the (ab)use of crack, it is also true that it was widespread among poors, including whites.

As you can note, 2Pac seems picking up where Hornsby left off: he shares the same disillusionment towards the possibility of equality in the United States. The entire song, as a matter of fact, denounces the hardships of being Black in an America that refuses you. https://americansongwriter.com/the-painful-meaning-behind-the-song-changes-by-tupac/

The "Huey" mentioned is Huey Newton was the co-founder (with Bobby Seale) of the Black Panther Party in 1966. (https://americansongwriter.com/the-painful-meaning-behind-the-song-changes-by-tupac/) The Black Panther was a 1960s revolutionary party whose original purpose was that of protecting Blacks from the attack of police officers and eventually extended its scope "to a Marxist revolutionary group that called for the arming of all African Americans, the exemption of African Americans from the draft and from all sanctions of so-called white America, the release of all African Americans from jail, and the payment of compensation to African Americans for centuries of exploitation by white Americans". (https://www.britannica.com/topic/Black-Panther-Party)

In this verse and in the previous one, the singer is making explicit reference to the police brutality in the U.S.A: excessive use of force (also culminating in murder) by police officers towards specific categories has been documented since the early 19th century. The targets have varied through the centuries, but African-Americans are historically the most targeted group because of racial implications. In this case, the singer emphasizes policemen's impunity before the law; on the contrary, they are acclaimed and welcomed as "heros". https://www.britannica.com/topic/police-brutality-in-the-United-States-2064580/Police-brutality-after-World-War-II

Just as the previous term, this one is charged with a derogatory connotation, so much as it is preferred to refer to it with the euphemistic expression "N-word", which emcompasses both this word and its -er variant. The phenomenon that allowed the erosion of the -er ending is called r-dropping: the -r is replaced by a schwa (ə), an indistinct vowel pronounced "uh". These terms have always been linked with white supremacy, racism and white power. Even though the two terms may seem synonyms, in fact there is a difference: the -er ending word is strictly connected with the all-encompassing hatred and contempt towards black people, whereas the second one is perceived as a term of endearment when uttered by someone belonging to the Black community. Indeed, starting from the 1980s, the word has underwent a process of reclaiming (also called semantic inversion or looping) which corresponds to "taking a word meant as a slur and reappropriating it as a term of endearment" (https://www.washingtonpost.com/sf/national/2014/11/09/the-n-word-an-entrenched-racial-slur-now-more-prevalent-than-ever/?utm_term=.1590a4928864). This strategy allows the originally oppressive term to be re-semantized (that is, to acquire a new meaning) and used to celebrate the community's unique identity and humanity as "an act of redemption by black folk. The word survives on the conditions that black folks have inscribed for it and nobody else can take that. And it becomes violent when other people try to take it and use it." Indeed, white people "have created the word in the first place, but […] they have lost the power to use it with impunity, they have lost the power to reclaim it." […] "If you understand the history of the word and how it's been used, it's not for white people to use […] So if you're not black you can't do that. You actually can't use the word in the way that we use it. It's not possible, because you're not in that space. So any other usage of it is completely wrong." (https://www.bbc.com/news/stories-53749800). https://www.merriam-webster.com/dictionary/N-word https://share.google/2p6rElVA4Vin0v2cC https://www.dailydot.com/irl/how-not-to-use-the-n-word/

The word is notoriously a derogatory term to indicate African-Americans. However, "both Negro and Negress are sometimes used by Black people in self-reference, but use of either term by others is offensive". https://www.merriam-webster.com/dictionary/Negro

Disclaimer: sensitive content.

In a world dominated by racism and in which nothing changes, the singer wonders whether the struggles he has to cope with on a daily basis make living worth it. He even contemplates the possibility to end his life in the same verse. If you have ever felt the same way the singer does in this verse, please seek help at https://azzurro.it/ or at least talk with an adult or a teacher.

Disclaimer: some of the punctuation marks (commas, mainly) were not present in the original lyrics of the song; I added them later. I found it as a useful, non-intrusive alteration of the text which may result in a better understanding of the content. Commas, indeed, help separating sentences and organize the content in a clearer way; in this context, I argue that they are necessary, especially for non-learner students who are approaching a song that employs a variant of English (the African-American Vernacular English) they are probably not familiar with. Apart from punctuation, the lyrics were not altered in any way.

Note how don't is paired with a singular noun, law. "Don't is occasionally used in American English speech and in historical writing as a contraction of does not (as in, "He don't know where he is going."), but this use is now considered improper and should be avoided. Remember that in modern speech and writing, don't cannot be used in the third person singular." https://www.britannica.com/dictionary/eb/qa/don-t-and-doesn-t

The "law" in this verse could allude to: * the Civil Rights Act passed in 1964 by President Lyndon Johnson. It "prohibited discrimination in public places, provided for the integration of schools and other public facilities, and made employment discrimination illegal" (https://www.archives.gov/milestone-documents/civil-rights-act#:~:text=This%20act%2C%20signed%20into%20law%20by%20President,most%20sweeping%20civil%20rights%20legislation%20since%20Reconstruction.) In other words, it made segregation in all its forms illegal. * the Economic Opportunity Act which was introduced in the same year. It "aimed at facilitating education, health, employment, and general welfare for impoverished Americans" (https://www.britannica.com/event/American-civil-rights-movement). It was part of the so-called "War on Poverty", a program endorsed by President Johnson and which intended to reduce poverty in the United States, along with making the country more equitable. https://www.britannica.com/topic/War-on-Poverty

It is probable that the song hints at both Acts, rather than pointing at just one.

Normally, will is contracted when preceded by there, here and personal pronouns (I, you, she\he\it, we, you, they) only in oral speech or informal writing. In this case, things'll https://dictionary.cambridge.org/us/grammar/british-grammar/contractions In everyday casual language, it is quite common to find will contracted even when combined with proper nouns and objects just as in this case. However, it is important to remember that it is not appropriate to use this form in formal writing. In the following video, starting at the minute 4:16, you can see (and hear) other examples similar to things'll: https://youtu.be/tXTu2tpJKaM?si=4e6I2J3wHd7On5i3&t=256

Here is the chorus of the song: at first, the singer's reaction to the widespread inquality that dominates the society seems to coincide with a resigned acceptation of the status quo. The feeling of quiet resignation amplifies as the song goes on, reaching its climax when the singer makes reference to the Civil Rights Act and, therefore, establishing a parallelism between the present and the past. However, the chorus closes with a glimpse of hope: indeed, the author invites the audience not to believe to inevitability, thus suggesting that things may change if only people started thinking and acting differently. As a matter of fact, the singer himself insists upon this last verse: "Some things will never change is a statement of resignation, but the most important line in that song is the one that comes after that: But don't you believe them. So I've always been about being strong when resignation is a possibility. Trying to pull up from that and have a positive outlook so that things can change" (http://www.musicfordemocracy.org/node/34.html).

Your turn: do you think that injustice and inequality will never be defeated?

In this sad scene, a new character is introduced: a man in a silk suit. He represents the rich and wealthy people who do not have to wait in line for economic help and, for this reason, hurries by (that is, he moves rapidly). His perspective is biased: he blames those in line for being poor and encourages them to "get a job". Therefore, Hornsby's intention is that of criticizing social polarization and inequality, apart from biased convictions that link poverty to laziness.

Your turn: is the man in a silk suit right? Are poor people always lazy or are there any other reasons for poverty?

The reason that is provided as for why they are waiting and need financial support is their poverty. The expression "buy a job" may refer to the fact that they cannot afford college education, in a modern scenario in which education assures better job opportunities; in a darker perspective, it might also mean that they have no connections to secure themselves a job through nepotism or bribery.

As an adjective, welfare means "of, relating to, or concerned with welfare and especially with improvement of the welfare of disadvantaged social groups" (https://www.merriam-webster.com/dictionary/welfare). Dime, instead, is a synonym of coin (https://www.merriam-webster.com/dictionary/dime). The combination of the the two words refers to the economic assistance provided by the government to families in financial distress.

Setting the scene: the song was released in July 1986 as a single from the band's debut album The Way It Is. It was a great success and the band won the 1987 Grammy Awards in the Best New Artist category. The success of the song has had a long-lasting effect in the music industry: it was sampled by other artists and included in songs such as 2Pac's Changes and Polo G's Wishing for a Hero. The singer has "never counted it" but he has read that his song "has now been recorded 17 times by hip-hop artists" (https://www.rollingstone.com/music/music-features/bruce-hornsby-interview-way-it-is-non-secure-connection-1036032/). In order to understand the following lyrics, it is necessary to place the song in its historical context. The 1980s were years in which several issues emerged: * The process of de-industrialization (that is, the process in which American companies moved their seats abroad, outside the country) deeply affected the job market: tens of thousands of workers lost their jobs. In particular, Blacks were the ones who suffered the most since the majority of them were employed in various industrial fields. As a consequence, poverty spread: 30% of black work force was jobless in 1982. * The conservative Reagan presidency (1981-1989) reduced federal (governmental, that is) economic support to people in need by 20%. The cut to financial measures combined with the ongoing industrial crisis was disastrous. * White supremacy movements and groups (such as the Ku Klux Klan) reignited and engaged in violent acts against African Americans, firebombing of churches and campaigns against affirmative actions programs and integration in schools. "Millions of white Americans had become convinced that “too much” had been given to blacks". * Poverty, hunger and hopelessness paved the way to the abuse of drugs; crack was especially consumed by poor Americans as it was inexpensive and easily available. As a consequence of the combination of low employment, educational poverty and drug popularity, drug dealing became the source of income for young people and violence increased significantly in Black neighborhoods.

What was the government's response? Aggravated levels of violence and crime were responded with the "War on Drugs", which entailed: 1. the elimination of parole (that is, the conditional release of a prisoner, often on the basis of good behavior in prison); 2. stricter penalties for drug sale and possession; 3. building a larger network of prisons.

Needless to say, African-Americans were most targeted. Mass incarceration as a system of control (see the "home" of the website for more on the topic) started to bloom.

https://www.amistadresource.org/the_future_in_the_present/social_and_economic_issues.html

Till means until and they are largely interchangeable; however, it is not its abbreviated form because till predates the longer word. https://www.merriam-webster.com/grammar/should-you-use-until-or-till-or-til

Here is the chorus of the song: to all the questions posed, the songwriter is not able to answer. In fact, the answer is as elusive as the wind and impossible to grasp. https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind

Another interesting element to point out is the relationship that Dylan seems to have with the audience: as previously remarked, this song converts Dylan into a spokesman of the common people, a "primus inter pares" if you wish, who makes use of his art to denounce inequality and social struggles. He does not think of himself as a custodian of a hidden truth no one else has discovered: just as everybody else, he does not have a clue about why discrimination and wars occur.

Your turn: if there are no answers, why asking questions? Do you think there is value in asking questions that cannot be answered?

"The mountain […] is a symbol of those human institutions that keep war and oppression in place. The stony mountain is all that resists change: the shape of government and history, certainly, but also the rocky terrain inside people's hearts. The slow, persistent erosive power of the ocean, on the other hand, symbolizes the action of internal and external change." https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind

This word presents a common linguistic phenomenon called g-dropping: it consists in the drop of the -g at the end of certain words. In fact, no -g is actually "dropped" because the 'g' is not even pronounced. All English speakers g-dropped, but the frequency of this phenomenon is tied to class belonging, race, sex and degree of formality. Generally, it is more common among lower social classes. http://itre.cis.upenn.edu/~myl/languagelog/archives/000878.html

What does this mean in the context of this song? Answer: By adopting g-dropping and thus language-wise, Bob Dylan positions himself in the tradition of folk music and becomes the spokesman of the people.

Here, the reference to the Vietnam war is explicit through the usage of the key word cannonballs.

The dove is a white bird that corresponds to colomba in Italian. It usually symbolizes peace. https://dictionary.cambridge.org/dictionary/english/dove <br /> "There's also a reference here to a specific dove: the biblical dove of Noah's Ark, which flew out from the Ark to seek dry land and returned bearing a hopeful olive branch. The symbolism of the dove here suggests that the change the speaker hopes for may not be easy to come by" (https://www.litcharts.com/poetry/bob-dylan/blowin-in-the-wind).

The repetition of "yes" at the beginning of many verses indicates the usage of a specific rhetorical device — the anaphora. https://www.merriam-webster.com/dictionary/anaphora

Setting the scene: the song was released in 1963 and included in the album The Freewhelin' Bob Dylan. All of Dylan's "most famous political songs were written [...] between January 1962 and October 1963" and "those [...] fixed him in the popular imagination" as a protest songwriter (Lynskey, 2010, 67). The historical context in which the song was released is essential to understand its meaning: during the 1950s the civil rights movement against racial segregation started to gain momentum, reaching its highest point precisely in 1963. Indeed, in that year: * Desegregation protests spread throughout the Southern states over more than 100 cities. The most famous was the March on Washington: it was organized by the "Big Six" of the civil rights movement (Martin Luther King Jr., James Farmer, J. Lewis, P. Randolph, R. Wilkins, W. Young), attended by more than 250.000 protesters (including Bob Dylan). It was in this occasion that the renowned "I have a dream" speech took place. * On June 12, President Kennedy announced he would present a civil rights bill to Congress, which was eventually passed the following year under the name of the Civil Rights Act. https://www.britannica.com/event/American-civil-rights-movement https://www.britannica.com/event/American-civil-rights-movement

It is important to bear in mind that protests were not only motivated by the insufferable racial segregation, but they were also anti-war oriented: the Vietnam war (1955-1975), which had been raging for ten years by the mid-1960s, was felt as an unnecessary conflict, especially by students and young people, who were the ones recruited in the U.S. army. Indeed, "the average age of an American soldier in Vietnam was 19" (https://www.bbc.co.uk/bitesize/guides/z6dk8hv/revision/4 ).

Eminem speaks directly at the heart of the white supremacist ideals, which support the idea that whites are considered "untouchable rockstars" just for the color of their skin. Regardless of the crimes they commit (and especially if they are policemen), they won't ever be charged or held accountable for them. As a matter of fact, in most of the cases in which African-Americans were victims of police brutality, white policemen were acquitted (which means assolti) of all charges.

Eminem refers to the very symbol of slavery, plantation fields.

Eminem's wording strikes again: he plays with America's ideals and bitter social reality by pairing the concluding phrase of the U.S. national anthem, the Star-Spangled Banner, ("O'er the land of the free and the home of the brave!") and the expression racist ville (that means the city of racists). https://amhistory.si.edu/starspangledbanner/the-lyrics.aspx

The oxymoronic expression reveals that America's ideals are just aspirations that never became reality.

The reference to President Donald Trump (he is a Republican) could not be more straightforward.

Notice how this part of the song echoes Lamar's lyrics "gang-banging make me kill a nigga blacker than me?": both songs highlight feuds inside the African-American community due to gang divisions. However, Eminem seems to be more reluctant to blame African-Americans: in the following lines, he underscores that said fights are rooted in problems such as single-parenting, drug abuse and struggle with addiction that lead people "with nothin(g) to lose to shoot each other for shoes".

Do you think hiring more African-American policemen would make a difference?

Eminem links high levels of crime to poor neighborhoods and socio-economic status — which singer whose song we analyzed made the same connection? Do you agree with Eminem's viewpoint?

"Once again, he tries to kill stereotypes by denouncing the “crack spot” as a hangout place for blacks. He’s also critical of naive cops who walk into black neighborhoods with no sense of understanding of their behavior or culture. Instead of walking with a fair mind and attitude, their level of fears heighten because of the unknown, and cause them to automatically be racists." https://www.billboard.com/music/rb-hip-hop/eminem-untouchable-lyrics-decoded-8062711/

The singer does not pretend to be colorblind and acknowledges the fact that he is white. Nevertheless, his ideals do not coincide with the ones supported by white suprematists; hence, the shame in being white.

bojour

Author response:

Reviewer #1:

We thank the reviewer for this important point. Beyond long reaction times, we did not originally exclude participants based on low EMA variability. We agree this is a relevant concern, particularly given the need to add small random noise to some EMA series for model convergence. In the revised manuscript, we will assess additional indicators of careless responding, including within-person EMA variability (e.g., standard deviation or proportion of modal responses) following Jaso et al., 2022 criteria. We will conduct sensitivity analyses excluding low-variability responses or participants and report whether these checks affect the robustness of the results. We will also clarify in the Discussion that minimal EMA variance may reflect either true affective stability or reduced engagement, and discuss how this ambiguity may affect interpretation.

Reviewer #2:

We thank the reviewer for raising this fundamental conceptual concern. We agree that more research is needed to fully understand the processes captured by DQRT. In the revised manuscript, we will more clearly reference and summarize prior validation work from our lab providing strong support for a cognitive characterization of DQRT as a measure of cognitive processing speed, while also explicitly acknowledging potential confounds and limitations (Teckentrup et al., 2025). We will clarify that our DQRT computation followed those validated procedures, including exclusion of extreme values above the sample-specific median + 2 SD. In addition, consistent with Reviewer #1’s comment, we will expand the Discussion of how potential careless responding and non-cognitive factors may influence DQRT. We will further tone down language implying causal inference.

References

Jaso, B. A., Kraus, N. I., & Heller, A. S. (2022). Identification of careless responding in ecological momentary assessment research: From posthoc analyses to real-time data monitoring. Psychological Methods, 27(6), 958.

Teckentrup, V., Rosická, A. M., Donegan, K. R., Gallagher, E., Hanlon, A. K., & Gillan, C. M. (2025). Digital questionnaire response time (DQRT): A ubiquitous and low-cost digital assay of cognitive processing speed. Behavior Research Methods, 57(7), 200.

eLife Assessment

This useful manuscript reports findings indicating that cell cycle progression and cytokinesis both contribute to the transition from early to late neural stem cell fates. Although orthogonal approaches would help confirm the findings, which are based on loss-of-function, the experimental evidence is convincing. Lastly, an investigation of the underlying mechanisms linking the cell cycle to temporal factor expression is still needed.

Reviewer #1 (Public review):

Summary:

Drosophila larval type II neuroblasts generate diverse types of neurons by sequentially expressing different temporal identity genes during development. Previous studies have shown that transition from early temporal identity genes (such as Chinmo and Imp) to late temporal identity genes (such as Syp and Broad) depends on the activation of the expression of EcR by Seven-up (Svp) and progression through the G1/S transition of the cell cycle. In this study, Chaya and Syed examined if the expression of Syp and EcR is regulated by cell cycle and cytokinesis by knocking down CDK1 or Pav, respectively, throughout development or at specific developmental stages. They find that knocking down CDK1 or Pav either in all type II neuroblasts throughout the development or in single type neuroblast clones after larval hatching consistently leads to failure to activate late temporal identity genes Syp and EcR. To determine whether the failure of the activation of Syp and EcR is due to impaired Svp expression, they also examined Svp expression using a Svp-lacZ reporter line. They find that Svp is expressed normally in CDK1 RNAi neuroblasts. Further, knocking down CDK1 or Pav after Svp activation still leads to loss of Syp and EcR expression. Finally, they also extended their analysis to type I neuroblasts. They find that knocking down CDK1 or Pav, either at 0 hours or at 42 hours after larval hatching, also results in loss of Syp and EcR expression in type I neuroblasts. Based on these findings, the authors conclude that cycle and cytokinesis are required for the transition from early to late late temporal identity genes in both types of neuroblasts. These findings add mechanistic details to our understanding of the temporal patterning of Drosophila larval neuroblasts.

Strengths:

The data presented in the paper are solid and largely support their conclusion. Images are of high quality. The manuscript is well-written and clear.

Weaknesses:

The authors have addressed all the weaknesses in this revision.

Reviewer #2 (Public review):

Summary:

Neural stem cells produce a wide variety of neurons during development. The regulatory mechanisms of neural diversity are based on the spatial and temporal patterning of neural stem cells. Although the molecular basis of spatial patterning is well-understood, the temporal patterning mechanism remains unclear. In this manuscript, the authors focused on the roles of cell cycle progression and cytokinesis in temporal patterning and found that both are involved in this process.

Strengths:

They conducted RNAi-mediated disruption on cell cycle progression and cytokinesis. As they expected, both disruptions affected temporal patterning in NSCs.

Weaknesses:

Although the authors showed clear results, they needed to provide additional data to support their conclusion sufficiently.

For example, they can examine the effects of cell cycle acceleration on the temporal patterning.

Reviewer #3 (Public review):

Summary:

The manuscript by Chaya and Syed focuses on understanding the link between cell cycle and temporal patterning in central brain type II neural stem cells (NSCs). To investigate this, the authors perturb the progression of the cell cycle by delaying the entry into M phase and preventing cytokinesis. Their results convincingly show that temporal factor expression requires progression of the cell cycle in both Type 1 and Type 2 NSCs in the Drosophila central brain. Overall, this study establishes an important link between the two timing mechanisms of neurogenesis.

Strengths:

The authors provide solid experimental evidence for the coupling of cell cycle and temporal factor progression in Type 2 NSCs. The quantified phenotype shows an all-or-none effect of cell cycle block on the emergence of subsequent temporal factors in the NSCs, strongly suggesting that both nuclear division and cytokinesis are required for temporal progression. The authors also extend this phenotype to Type 1 NSCs in the central brain, providing a generalizable characterization of the relationship between cell cycle and temporal patterning.

Weaknesses:

One major weakness of the study is that the authors do not explore the mechanistic relationship between cell cycle and temporal factor expression. Although their results are quite convincing, they do not provide an explanation as to why Cdk1 depletion affects Syp and EcR expression but not the onset of svp. This result suggests that at least a part of the temporal cascade in NSCs is cell-cycle independent which isn't addressed or sufficiently discussed.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Summary:

Drosophila larval type II neuroblasts generate diverse types of neurons by sequentially expressing different temporal identity genes during development. Previous studies have shown that the transition from early temporal identity genes (such as Chinmo and Imp) to late temporal identity genes (such as Syp and Broad) depends on the activation of the expression of EcR by Seven-up (Svp) and progression through the G1/S transition of the cell cycle. In this study, Chaya and Syed examined whether the expression of Syp and EcR is regulated by cell cycle and cytokinesis by knocking down CDK1 or Pav, respectively, throughout development or at specific developmental stages. They find that knocking down CDK1 or Pav either in all type II neuroblasts throughout development or in single-type neuroblast clones after larval hatching consistently leads to failure to activate late temporal identity genes Syp and EcR. To determine whether the failure of the activation of Syp and EcR is due to impaired Svp expression, they also examined Svp expression using a Svp-lacZ reporter line. They find that Svp is expressed normally in CDK1 RNAi neuroblasts. Further, knocking down CDK1 or Pav after Svp activation still leads to loss of Syp and EcR expression. Finally, they also extended their analysis to type I neuroblasts. They find that knocking down CDK1 or Pav, either at 0 hours or at 42 hours after larval hatching, also results in loss of Syp and EcR expression in type I neuroblasts. Based on these findings, the authors conclude that cycle and cytokinesis are required for the transition from early to late temporal identity genes in both types of neuroblasts. These findings add mechanistic details to our understanding of the temporal patterning of Drosophila larval neuroblasts.

Strengths:

The data presented in the paper are solid and largely support their conclusion. Images are of high quality. The manuscript is well-written and clear.

We appreciate the reviewer’s detailed summary and recognition of the study’s strengths.

Weaknesses:

The quantifications of the expression of temporal identity genes and the interpretation of some of the data could be more rigorous.

(1) Expression of temporal identity genes may not be just positive or negative. Therefore, it would be more rigorous to quantify the expression of Imp, Syp, and EcR based on the staining intensity rather than simply counting the number of neuroblasts that are positive for these genes, which can be very subjective. Or the authors should define clearly what qualifies as "positive" (e.g., a staining intensity at least 2x background).

We thank the reviewer for this helpful suggestion. In the new version, we have now clarified how positive expression was defined and added more details of our quantification strategy to the Methods section (page 11, lines 380-388; lines 426-434 in tracked changes file). Fluorescence intensity for each neuroblast was normalized to the mean intensity of neighboring wild-type neuroblasts imaged in the same field. A neuroblast was considered positive for a given factor when its normalized nuclear intensity was at least 2× the local background. This scoring criterion was applied uniformly across all genotypes and time points. All quantifications were performed on the raw LSM files in Fiji prior to assembling the figure panels.

(2) The finding that inhibiting cytokinesis without affecting nuclear divisions by knocking down Pav leads to the loss of expression of Syp and EcR does not support their conclusion that nuclear division is also essential for the early-late gene expression switch in type II NSCs (at the bottom of the left column on page 5). No experiments were done to specifically block the nuclear division in this study specifically. This conclusion should be revised.

We blocked both cell cycle progression and cytokinesis, and both these manipulations affected temporal gene transitions, suggesting that both cell cycle and cytokinesis are essential. To our knowledge, no mechanism/tool exists that selectively blocks nuclear division while leaving cell cycle progression intact. We have added more clarification on page 4, line 123 onwards (lines 126 onwards in tracked changes file).

(3) Knocking down CDK1 in single random neuroblast clones does not make the CDK1 knockdown neuroblast develop in the same environment (except still in the same brain) as wild-type neuroblast lineages. It does not help address the concern whether "type 2 NSCS with cell cycle arrest failed to undergo normal temporal progression is indirectly due to a lack of feedback signaling from their progeny", as discussed (from the bottom of the right column on page 9 to the top of the left column on page 10). The CDK1 knockdown neuroblasts do not divide to produce progeny and thus do not receive a feedback signal from their progeny as wild-type neuroblasts do. Therefore, it cannot be ruled out that the loss of Syp and EcR expression in CDK1 knockdown neuroblasts is due to the lack of the feedback signal from their progeny. This part of the discussion needs to be clarification.

Thanks to the reviewer for raising this critical point. We agree and have added more clarification of our interpretations and limitations to our studies in the revised text on page 8, line 278-282 (lines 296-300 in tracked changes file)

(4) In Figure 2I, there is a clear EcR staining signal in the clone, which contradicts the quantification data in Figure 2J that EcR is absent in Pav RNAi neuroblasts. The authors should verify that the image and quantification data are consistent and correct.

When cytokinesis is blocked using pav-RNAi, the neuroblasts become extremely large and multinucleated. In some large pav RNAi clones, we observed a weak EcR signal near the cell membrane. However, more importantly, none of the nuclear compartments showed detectable EcR staining, where EcR is typically localized. We selected a representative nuclear image for the figure panel. To clarify this observation, we have now added an explanatory note to the discussion section on page 8, lines 283-291 (lines 301-309 in tracked changes file).

Reviewer #2 (Public review):

Summary:

Neural stem cells produce a wide variety of neurons during development. The regulatory mechanisms of neural diversity are based on the spatial and temporal patterning of neural stem cells. Although the molecular basis of spatial patterning is well-understood, the temporal patterning mechanism remains unclear. In this manuscript, the authors focused on the roles of cell cycle progression and cytokinesis in temporal patterning and found that both are involved in this process.

Strengths:

They conducted RNAi-mediated disruption on cell cycle progression and cytokinesis. As they expected, both disruptions affected temporal patterning in NSCs.

We appreciate the reviewer’s positive assessment of our experimental results.

Weaknesses:

Although the authors showed clear results, they needed to provide additional data to support their conclusion sufficiently.

For example, they need to identify type II NSCs using molecular markers (Ase/Dpn).The authors are encouraged to provide a more detailed explanation of each experiment. The current version of the manuscript is difficult for non-expert readers to understand.

Thanks for your feedback. We have now included a detailed description of how we identify type II NSCs in both wild-type and mutant clones. We have also added a representative Asense staining to clearly distinguish type 1 (Ase⁺) from type 2 (Ase^-) NSCs see Figure S1. We have also added a resources table explaining the genotypes associated with each figure, which was omitted due to an error in the previous version of the manuscript. 

Reviewer #3 (Public review):

Summary:

The manuscript by Chaya and Syed focuses on understanding the link between cell cycle and temporal patterning in central brain type II neural stem cells (NSCs). To investigate this, the authors perturb the progression of the cell cycle by delaying the entry into M phase and preventing cytokinesis. Their results convincingly show that temporal factor expression requires progression of the cell cycle in both Type 1 and Type 2 NSCs in the Drosophila central brain. Overall, this study establishes an important link between the two timing mechanisms of neurogenesis.

Strengths:

The authors provide solid experimental evidence for the coupling of cell cycle and temporal factor progression in Type 2 NSCs. The quantified phenotype shows an all-ornone effect of cell cycle block on the emergence of subsequent temporal factors in the NSCs, strongly suggesting that both nuclear division and cytokinesis are required for temporal progression. The authors also extend this phenotype to Type 1 NSCs in the central brain, providing a generalizable characterization of the relationship between cell cycle and temporal patterning.

We thank the reviewer for recognizing the robustness of our data linking the cell cycle to temporal progression.

Weaknesses:

One major weakness of the study is that the authors do not explore the mechanistic relationship between the cell cycle and temporal factor expression. Although their results are quite convincing, they do not provide an explanation as to why Cdk1 depletion affects Syp and EcR expression but not the onset of svp. This result suggests that at least a part of the temporal cascade in NSCs is cell-cycle independent, which isn't addressed or sufficiently discussed.

Thank you for bringing up this important point. We are equally interested in uncovering the mechanism by which the cell cycle regulates temporal gene transitions; however, such mechanistic exploration is beyond the scope of the present study. Interestingly, while the temporal switching factor Svp is expressed independently of the cell cycle, the subsequent temporal transitions are not. We have expanded our discussion on this intriguing finding (page 9, line 307-315; lines 345-355 in tracked changes file). Specifically, we propose that svp activation marks a cell-cycle–independent phase, whereas EcR/Syp induction likely depends on cell-cycle–coupled mechanisms, such as mitosis-dependent chromatin remodeling or daughter-cell feedback. Although further dissection of this mechanism lies beyond the current study, our findings establish a foundation for future work aimed at identifying how developmental timekeeping is molecularly coupled to cell-cycle progression.

Recommendations for the authors:

Reviewer #1 (Recommendations for the authors): 

(1) Figure 1 C and D, it would be better to put a question mark to indicate that these are hypotheses to be tested. 

We appreciate this suggestion and have added question marks in Figure 1C and 1D to clearly indicate that these panels represent hypotheses under investigation clearly.

(2) Figure 2A-I, Figure 4A-I, Figure 5A-I and K-S, in addition to enlarged views of single type II neuroblasts, it would be more convincing to include zoomed-out images of the entire larval brain or at least a portion of the brain to include neighboring wild-type type II neuroblasts as internal controls. Also, it would be ideal to show EcR staining from the same neuroblasts as IMP and Syp staining. 

We thank the reviewer for this valuable input. In our imaging setup, the number of available antibody channels was limited to four (anti-Ase, anti-GFP, anti-Syp, and antiImp). Adding EcR in the same sample was therefore not technically possible, we performed EcR staining separately. 

(3) The authors cited "Syed et al., 2024" (in the middle of the right column on page 5), but this reference is missing in the "References" section and should be added. 

The missing citation has been added to the reference section.  

(4) It would be better to include Ase staining in the relevant figure to indicate neuroblast identity as type I or type II. 

We agree and now include representative Ase staining for both type 1 and type 2 NSC clones in Figure S1, along with corresponding text updates that describe these markers.

Reviewer #2 (Recommendations for the authors): 

Major comments 

(1) The present conclusion relies on the results using Cdk1 RNAi and pav RNAi. It is still possible that Cdk1 and Pav are involved in the regulation of temporal patterning independent of the regulation of cell cycle or cytokinesis, respectively. To avoid this possibility, the authors need to inhibit cell cycle progression or cytokinesis in another alternative manner. 

We thank the reviewer for raising this important point. While we cannot completely exclude gene-specific, cell-cycle-independent roles for Cdk1 or Pav, we observe consistent phenotypes across several independent manipulations that slow or block the cell cycle. Also, earlier studies using orthogonal approaches that delay G1/S (Dacapo/Rbf) or impair mitochondrial OxPhos (which lengthens G1/S; van den Ameele & Brand, 2019) produce similar temporal delays. These concordant phenotypes strongly support the interpretation that altered cell-cycle progression—rather than specific roles of a single gene—is the primary cause of the defect. While we cannot exclude additional, gene-specific effects of Cdk1 or Pav, the concordant phenotypes across independent perturbations make the cell-cycle disruption model the most parsimonious interpretation. We have clarified this reasoning in the discussion section on pages 8-9, lines 293-305 (lines 311-343 in tracked changes file).

(2) To reach the present conclusion, the authors need to address the effects of acceleration of cell cycle progression or cytokinesis on temporal patterning. 

We thank the reviewer for this insightful suggestion. To our knowledge, there are currently no established genetic tools that can specifically accelerate cell-cycle progression in Drosophila neuroblasts. However, our results demonstrate that blocking the cell cycle impairs the transition from early to late temporal gene expression. These findings suggest that proper cell-cycle progression is essential for the transition from early to late temporal identity in neuroblasts.

Minor comments 

(3) P3L2 (right), ... we blocked the NSC cell cycle...

How did they do it? 

Which fly lines were used?

Why did they use the line? 

These details are now included in the Materials and Methods and the Resource Table (pages 11-13). We used Wor-Gal4, Ase-Gal80 to drive UAS-Cdk1RNAi and UASpavRNAi in type 2 NSCs 

(4) P5L1(left), ... we used the flip-out approach...

Why did they conduct it? 

Probably, the authors have reasons other than "to further ensure." 

We have clarified in the text on page 4, lines 137-139, that the flip-out approach was used to generate random single-cell clones, enabling quantitative analysis of type 2 NSCs within an otherwise wild-type brain. 

(5) P5L8(left), ... type 2 hits were confirmed by lack of the type 1 Asense...  The authors must examine Deadpan (Dpn) expression as well. Because there are a lot of Asense (Ase) negative cells in the brain (neurons, glial cell, and neuroepithelial cells). 

Type II NSCs can be identified as Dpn+/Ase- cells.

We agree that Dpn is a helpful marker. However, we reliably distinguished type II NSCs by their lack of Ase and larger cell size relative to surrounding neurons and glia, which are smaller in size and located deeper within the clone. These differences, together with established lineage patterns, allow unambiguous identification of type 2 NSCs across all genotypes. We have now added representative type I and type 2 NSC clones to the supplemental figure S1 (E-G’) with Asense stains to demonstrate how we differentiate type I from type II NSCs. 

(6) P5L32(left), To do this, we induced... 

This sentence should be made more concise.

Please rephrase it. 

The sentence has been rewritten for clarity and concision.

(7)  P5L42(left), ...lack of EcR/Syp expression (Figure 2).  However, EcR expression is still present (Figure 2I). 

In some large pavRNAi clones, a weak EcR signal can be observed near the cell membrane; however, none of the nuclear compartments—where EcR is typically localized—show detectable staining. We selected a representative nuclear image for the figure and addressed this observation on page 8, lines 283-291 (lines 301-309 in tracked changes file).

(8) P7L29(left), ......had persistent Imp expression...

Imp expression is faint compared to that in Figure 2G.

The differences between Figures 2G and 3G should be discussed. 

We thank the reviewer for this comment. We have added a note in the Methods section clarifying that brightness and contrast were adjusted per panel for optimal visualization; thus, apparent differences in signal intensity do not reflect biological variation. Fluorescence intensity for each neuroblast was normalized to the mean intensity of neighboring wild-type neuroblasts imaged in the same field. A neuroblast was considered Imp-positive when its normalized nuclear intensity was at least 2× the local background. This scoring criterion was applied uniformly across all genotypes and time points. All quantifications were performed on the raw LSM files in Fiji prior to assembling the figure panels.

(9) P8 (Figure 5)

The Imp expression is faint compared to that in Figure 5Q.

The difference between Figure 5G and 5Q should be discussed further. 

As mentioned above, we have clarified our image processing approach in the Methods section to explain any differences in signal appearance between these figures.

(10) P10 Materials and Methods

The authors did not mention the fly lines used. This is very important for the readers. 

We thank the reviewer for bringing this oversight to our attention. The Resource Table was inadvertently omitted from the initial submission. The complete list of fly lines and reagents used in this study is now provided in the updated Resource Table.

Reviewer #3 (Recommendations for the authors): 

Major points 

(1) The authors mention that the heat-shock induction at 42ALH is well after svp temporal window and therefore the cell cycle block independently affects Syp and EcR expression. However, Figure 3 shows svp-LacZ expression at 48ALH. If svp expression is indeed transient in Type 2 NSCs, then this must be validated using an immunostaining of the svp-LacZ line with svp antibody. This is crucial as the authors claim that cell cycle block doesn't affect does affect svp expression and is required independently. 

We thank the reviewer for bringing this important issue to our attention. As noted, Svp protein is expressed transiently and stochastically in type 2 NSCs (Syed et al., 2017), making direct antibody quantification challenging upon cell cycle block. Consistent with previous work (Syed et al., 2017), we used the svp-LacZ reporter line to visualize stabilized Svp expression, which reliably captures Svp expression in type 2 NSCs (Syed et al., 2017 https://doi.org/10.7554/eLife.26287, and Dhilon et al., 2024 https://doi.org/10.1242/dev.202504).

(2) The authors have successfully slowed down the cell cycle and showed that it affects temporal progression. However, a converse experiment where the cell cycle is sped up in NSCs would be an important test for the direct coupling of temporal factor expression and cell cycle, wherein the expectation would be the precocious expression of late temporal factors in faster cycle NSCs. 

We agree that such an experiment would be ideal. However, as noted above (Reviewer #2 comment 2), to our knowledge, no suitable tools currently exist to accelerate neuroblast cell-cycle progression without pleiotropic effects.

Minor point 

The authors must include Ray and Li (https://doi.org/10.7554/eLife.75879) in the references when describing that "...cell cycle has been shown to influence temporal patterning in some systems,...".  

We thank the reviewer for this helpful suggestion. The cited reference (Ray and Li, eLife, 2022) has now been included and appropriately referenced in the revised manuscript.

eLife Assessment

The authors investigate arrestin2-mediated CCR5 endocytosis in the context of clathrin and AP2 contributions. Using an extensive set of NMR experiments, and supported by microscopy and other biophysical assays, the authors provide compelling data on the roles of AP2 and clathrin in CCR5 endocytosis. This important work will appeal to an audience beyond those studying chemokine receptors, including those studying GPCR regulation and trafficking. The distinct role of AP2 and not clathrin will be of particular interest to those studying GPCR internalization mechanisms.

Reviewer #1 (Public review):

Petrovic et al. investigate CCR5 endocytosis via arrestin2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data clearly demonstrate chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants. To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization.

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays.

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fits-all model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.

Reviewer #2 (Public review):

Summary:

Based on extensive live cell assays, SEC, and NMR studies of reconstituted complexes, these authors explore the roles of clathrin and the AP2 protein in facilitating clathrin mediated endocytosis via activated arrestin-2. NMR, SEC, proteolysis, and live cell tracking confirm a strong interaction between AP2 and activated arrestin using a phosphorylated C-terminus of CCR5. At the same time a weak interaction between clathrin and arrestin-2 is observed, irrespective of activation.

These results contrast with previous observations of class A GPCRs and the more direct participation by clathrin. The results are discussed in terms of the importance of short and long phosphorylated bar codes in class A and class B endocytosis.

Strengths:

The 15N,1H and 13C,methyl TROSY NMR and assignments represent a monumental amount of work on arrestin-2, clathrin, and AP2. Weak NMR interactions between arrestin-2 and clathrin are observed irrespective of activation of arrestin. A second interface, proposed by crystallography, was suggested to be a possible crystal artifact. NMR establishes realistic information on the clathrin and AP2 affinities to activated arrestin with both kD and description of the interfaces.

Reviewer #3 (Public review):

Summary:

Overall, this is a well-done study, and the conclusions are largely supported by the data, which will be of interest to the field.

Strengths:

Strengths of this study include experiments with solution NMR that can resolve high-resolution interactions of the highly flexible C-terminal tail of arr2 with clathrin and AP2. Although mainly confirmatory in defining the arr2 CBL 376LIELD380 as the clathrin binding site, the use of the NMR is of high interest (Fig. 1). The 15N-labeled CLTC-NTD experiment with arr2 titrations reveals a span from 39-108 that mediates an arr2 interaction, which corroborates previous crystal data, but does not reveal a second area in CLTC-NTD that in previous crystal structures was observed to interact with arr2.

SEC and NMR data suggest that full-length arr2 (1-418) binding with 2-adaptin subunit of AP2 is enhanced in the presence of CCR5 phospho-peptides (Fig. 3). The pp6 peptide shows the highest degree of arr2 activation, and 2-adaptin binding, compared to less phosphorylated peptide or not phosphorylated at all. It is interesting that the arr2 interaction with CLTC NTD and pp6 cannot be detected using the SEC approach, further suggesting that clathrin binding is not dependent on arrestin activation. Overall, the data suggest that receptor activation promotes arrestin binding to AP2, not clathrin, suggesting the AP2 interaction is necessary for CCR5 endocytosis.

To validate the solid biophysical data, the authors pursue validation experiments in a HeLa cell model by confocal microscopy. This requires transient transfection of tagged receptor (CCR5-Flag) and arr2 (arr2-YFP). CCR5 displays a "class B"-like behavior in that arr2 is rapidly recruited to the receptor at the plasma membrane upon agonist activation, which forms a stable complex that internalizes onto endosomes (Fig. 4). The data suggest that complex internalization is dependent on AP2 binding not clathrin (Fig. 5).

The addition of the antagonist experiment/data adds rigor to the study.

Overall, this is a solid study that will be of interest to the field.

Author response:

The following is the authors’ response to the previous reviews

Public Reviews: 

Reviewer #1 (Public review): 

Petrovic et al. investigate CCR5 endocytosis via arrestin 2, with a particular focus on clathrin and AP2 contributions. The study is thorough and methodologically diverse. The NMR titration data clearly demonstrate chemical shift changes at the canonical clathrin-binding site (LIELD), present in both the 2S and 2L arrestin splice variants. 

To assess the effect of arrestin activation on clathrin binding, the authors compare: truncated arrestin (1-393), full-length arrestin, and 1-393 incubated with CCR5 phosphopeptides. All three bind clathrin comparably, whereas controls show no binding. These findings are consistent with prior crystal structures showing peptide-like binding of the LIELD motif, with disordered flanking regions. The manuscript also evaluates a non-canonical clathrin binding site specific to the 2L splice variant. Though this region has been shown to enhance beta2-adrenergic receptor binding, it appears not to affect CCR5 internalization. 

Similar analyses applied to AP2 show a different result. AP2 binding is activation-dependent and influenced by the presence and level of phosphorylation of CCR5-derived phosphopeptides. These findings are reinforced by cellular internalization assays. 

In sum, the results highlight splice-variant-dependent effects and phosphorylation-sensitive arrestin-partner interactions. The data argue against a (rapidly disappearing) one-size-fitsall model for GPCR-arrestin signaling and instead support a nuanced, receptor-specific view, with one example summarized effectively in the mechanistic figure.

We thank the referee for this positive assessment of our manuscript. Indeed, by stepping away from the common receptor models for understanding internalization (b2AR and V2R), we revealed the phosphorylation level of the receptor as a key factor in driving the sequestration of the receptor from the plasma membrane. We hope that the proposed mechanistic model will aid further studies to obtain an even more detailed understanding of forces driving receptor internalization.

Weaknesses: 

Figure 1 shows regions alphaFold model that are intrinsically disordered without making it clear that this is not an expected stable position. The authors NMR titration data are n=1. Many figure panels require that readers pinch and zoom to see the data.

In the “Recommendations for the Authors” section, we addressed the reviewer’s stated weaknesses. In short, for the AlphaFold representation in Figure 1A, we added explicit labeling and revised the legend and main text to clearly state that the depicted loops are intrinsically disordered, absent from crystal structures due to flexibility, and shown only for visualization of their location. We also clarified that the NMR titration experiments inherently have n = 1 due to technical limitations, and that this is standard practice in the field, while ensuring individual data points remain visible. The supplementary NMR figures now have more vibrant coloring, allowing easier data assessment. However, we have not changed the zooming of the microscopy and NMR spectra. We believe that the presentation of microscopy data, which already show zoomed-in regions of interest, follow standard practices in the field. Furthermore, we strongly believe that we should display full NMR spectra in the supplementary figures to allow the reader to assess the overall quality and behavior. As indicated previously, the reader can zoom in to very high resolution, since the spectra are provided by vector graphics. Zoomed regions of the relevant details are provided in the main figures.

Reviewer #2 (Public review): 

Summary: 

Based on extensive live cell assays, SEC, and NMR studies of reconstituted complexes, these authors explore the roles of clathrin and the AP2 protein in facilitating clathrin mediated endocytosis via activated arrestin-2. NMR, SEC, proteolysis, and live cell tracking confirm a strong interaction between AP2 and activated arrestin using a phosphorylated C-terminus of CCR5. At the same time a weak interaction between clathrin and arrestin-2 is observed, irrespective of activation. 

These results contrast with previous observations of class A GPCRs and the more direct participation by clathrin. The results are discussed in terms of the importance of short and long phosphorylated bar codes in class A and class B endocytosis. 

Strengths: 

The 15N,1H and 13C,methyl TROSY NMR and assignments represent a monumental amount of work on arrestin-2, clathrin, and AP2. Weak NMR interactions between arrestin-2 and clathrin are observed irrespective of activation of arrestin. A second interface, proposed by crystallography, was suggested to be a possible crystal artifact. NMR establishes realistic information on the clathrin and AP2 affinities to activated arrestin with both kD and description of the interfaces.

We sincerely thank the referee for this encouraging evaluation of our work and appreciate the recognition of the NMR efforts and insights into the arrestin–clathrin–AP2 interactions.

Weaknesses: 

This reviewer has identified only minor weaknesses with the study. 

(1) I don't observe two overlapping spectra of Arrestin2 (1393) +/- CLTC NTD in Supp Figure 1

We believe the referee is referring to Figure 1 – figure supplement 2. We have now made the colors of the spectra more vibrant and used different contouring to make the differences between the two spectra clearer. The spectra are provided as vector graphics, which allows zooming in to the very fine details.

(2) Arrestin-2 1-418 resonances all but disappear with CCR5pp6 addition. Are they recovered with Ap2Beta2 addition and is this what is shown in Supp Fig 2D

We believe the reviewer is referring to Figure 3 - figure supplement 1. In this figure, the panels E and F show resonances of arrestin2^1-418 (apo state shown with black outline) disappear upon the addition of CCR5pp6 (arrestin2^1-418•CCR5pp6 complex spectrum in red). The panels C and D show resonances of arrestin2^1-418 (apo state shown with black outline), which remain unchanged upon addition of AP2b2 ^701-937 (orange), indicating no complex formation. We also recorded a spectrum of the arrestin2^1-418•CCR5pp6 complex under addition of AP2b2 ^701-937 (not shown), but the arrestin2 resonances in the arrestin2^1-418 •CCR5pp6 complex were already too broad for further analysis. This had been already explained in the text.

“In agreement with the AP2b2 NMR observations, no interaction was observed in the arrestin2 methyl and backbone NMR spectra upon addition of AP2b2 in the absence of phosphopeptide (Figure 3-figure supplement 1C, D). However, the significant line broadening of the arrestin2 resonances upon phosphopeptide addition (Figure 3-figure supplement 1E, F) precluded a meaningful assessment of the effect of the AP2b2 addition on arrestin2 in the presence of phosphopeptide”.

(3) I don't understand how methyl TROSY spectra of arrestin2 with phosphopeptide could look so broadened unless there are sample stability problems?

We thank the referee for this comment. We would like to clarify that in general a broadened spectrum beyond what is expected from the rotational correlation time does not necessarily correlate with sample stability problems. It is rather evidence of conformational intermediate exchange on the micro- to millisecond time scale.

The displayed ¹H-¹⁵N spectra of apo arrestin2 already suffer from line broadening due to such intrinsic mobility of the protein. These spectra were recorded with acquisition times of 50 ms (¹⁵N) and 55 ms (¹H) and resolution-enhanced by a 60˚-shifted sine-bell filter for ¹⁵N and a 60˚-shifted squared sine-bell filter for ¹H, respectively, which leads to the observed resolution with still reasonable sensitivity. The ¹H-¹⁵N resonances in Fig. 1b (arrestin2^1-393) look particularly narrow. However, this region contains a large number of flexible residues. The full spectrum, e.g. Figure 1-figure supplement 2, shows the entire situation with a clear variation of linewidths and intensities. The linewidth variation becomes stronger when omitting the resolution enhancement filters.

The addition of the CCR5pp6 phosphopeptide does not change protein stability, which we assessed by measuring the melting temperature of arrestin2^1-418 and arrestin2^1-418•CCR5pp6 complex (Tm = 57°C in both cases). We believe that the explanation for the increased broadening of the arrestin2 resonances is that addition of the CCR5pp6, possibly due to the release of the arrestin2 strand b20, amplifies the mentioned intermediate timescale protein dynamics. This results in the disappearance of arrestin2 resonances.

We have now included the assessment of arrestin2^1-418 and arrestin2^1-418•CCR5pp6 stability in the manuscript:

“The observed line broadening of arrestin2 in the presence of phosphopeptide must be a result of increased protein motions and is not caused by a decrease in protein stability, since the melting temperature of arrestin2 in the absence and presence of phosphopeptide are identical (56.9 ± 0.1 °C)”.

(4) At one point the authors added excess fully phosphorylated CCR5 phosphopeptide (CCR5pp6). Does the phosphopeptide rescue resolution of arrestin2 (NH or methyl) to the point where interaction dynamics with clathrin (CLTC NTD) are now more evident on the arrestin2 surface?

Unfortunately, when we titrate arrestin2 with CCR5pp6 (please see Isaikina & Petrovic et. al, Mol. Cell, 2023 for more details), the arrestin2 resonances undergo fast-to-intermediate exchange upon binding. In the presence of phosphopeptide excess, very few resonances remain, the majority of which are in the disordered region, including resonances from the clathrin-binding loop. Due to the peak overlap, we could not unambiguously assign arrestin2 resonances in the bound state, which precluded our assessment of the arrestin2-clathrin interaction in the presence of phosphopeptide. We have made this now clearer in the paragraph ‘The arrestin2-clathrin interaction is independent of arrestin2 activation’

“Due to significant line broadening and peak overlap of the arrestin2 resonances upon phosphopeptide addition, the influence of arrestin activation on the clathrin interaction could not be detected on either backbone or methyl resonances “.

(5) Once phosphopeptide activates arrestin-2 and AP2 binds can phosphopeptide be exchanged off? In this case, would it be possible for the activated arrestin-2 AP2 complex to re-engage a new (phosphorylated) receptor?

This would be an interesting mechanism. In principle, this should be possible as long as the other (phosphorylated) receptor outcompetes the initial phosphopeptide with higher affinity towards the binding site. However, we do not have experiments to assess this process directly. Therefore, we rather wish not to further speculate.

(6) I'd be tempted to move the discussion of class A and class B GPCRs and their presumed differences to the intro and then motivate the paper with specific questions. 

We appreciate the referee’s suggestion and had a similar idea previously. However, as we do not have data on other class-A or class-B receptors, we rather don’t want to motivate the entire manuscript by this question.

(7) Did the authors ever try SEC measurements of arrestin-2 + AP2beta2+CCR5pp6 with and without PIP2, and with and without clathrin (CLTC NTD? The question becomes what the active complex is and how PIP2 modulates this cascade of complexation events in class B receptors.

We thank the referee for this question. Indeed, we tested whether PIP2 can stabilize the arrestin2•CCR5pp6•AP2 complex by SEC experiments. Unfortunately, the addition of PIP2 increased the formation of arrestin2 dimers and higher oligomers, presumably due to the presence of additional charges. The resolution of SEC experiments was not sufficient to distinguish arrestin2 in oligomeric form or in arrestin2•CCR5pp6•AP2 complex. We now mention this in the text:

“We also attempted to stabilize the arrestin2-AP2b2-phosphopetide complex through the addition of PIP2, which can stabilize arrestin complexes with the receptor (Janetzko et al., 2022). The addition of PIP2 increased the formation of arrestin2 dimers and higher oligomers, presumably due to the presence of additional charges. Unfortunately, the resolution of the SEC experiments was not sufficient to separate the arrestin2 oligomers from complexes with AP2b2”.

Reviewer #3 (Public review): 

Summary: 

Overall, this is a well-done study, and the conclusions are largely supported by the data, which will be of interest to the field. 

Strengths: 

Strengths of this study include experiments with solution NMR that can resolve high-resolution interactions of the highly flexible C-terminal tail of arr2 with clathrin and AP2. Although mainly confirmatory in defining the arr2 CBL376LIELD380 as the clathrin binding site, the use of the NMR is of high interest (Fig. 1). The 15N-labeled CLTC-NTD experiment with arr2 titrations reveals a span from 39-108 that mediates an arr2 interaction, which corroborates previous crystal data, but does not reveal a second area in CLTC-NTD that in previous crystal structures was observed to interact with arr2. 

SEC and NMR data suggest that full-length arr2 (1-418) binding with 2-adaptin subunit of AP2 is enhanced in the presence of CCR5 phospho-peptides (Fig. 3). The pp6 peptide shows the highest degree of arr2 activation, and 2-adaptin binding, compared to less phosphorylated peptide or not phosphorylated at all. It is interesting that the arr2 interaction with CLTC NTD and pp6 cannot be detected using the SEC approach, further suggesting that clathrin binding is not dependent on arrestin activation. Overall, the data suggest that receptor activation promotes arrestin binding to AP2, not clathrin, suggesting the

AP2 interaction is necessary for CCR5 endocytosis. 

To validate the solid biophysical data, the authors pursue validation experiments in a HeLa cell model by confocal microscopy. This requires transient transfection of tagged receptor (CCR5-Flag) and arr2 (arr2-YFP). CCR5 displays a "class B"-like behavior in that arr2 is rapidly recruited to the receptor at the plasma membrane upon agonist activation, which forms a stable complex that internalizes onto endosomes (Fig. 4). The data suggest that complex internalization is dependent on AP2 binding not clathrin (Fig. 5). 

The addition of the antagonist experiment/data adds rigor to the study. 

Overall, this is a solid study that will be of interest to the field.

We thank the referee for the careful and encouraging evaluation of our work. We appreciate the recognition of the solidity of our data and the support for our conclusions regarding the distinct roles of AP2 and clathrin in arrestin-mediated receptor internalization.

Recommendations for the authors: 

Reviewer #1 (Recommendations for the authors): 

I believe that the authors have made efforts to improve the accessibility to a broader audience. In a few cases, I believe that the authors response either did not truly address the concern or made the problem worse. I am grouping these as 'very strong opinions' and 'sticking point'. 

Very strong opinion 1: 

While data presentation is somewhat at the authors discretion, there were several figures where the presentation did not make the work approachable, including microscopy insets and NMR spectra. A suggestion to 'pinch and zoom' does not really address this. For the overlapping NMR spectra in supporting Figure 1, I actually -can- see this on zooming, but I did not recognize this on first pass because the colors are almost identical for the two spectra. This is an easy fix. Changing the presentation by coloring these distinctly would alleviate this. The Supplemental figure to Fig. 2 looks strange with pinch and zoom. But at the end of the day, data presentation where the reader is to infer that they must zoom in is not very approachable and may prevent readers from being able to independently assess the data. In this case, there doesn't seem to be a strong rationale to not make these panels easier to see at 100% size. 

We appreciate the reviewer’s thoughtful comments regarding figure accessibility and agree that data presentation should be clear and interpretable without requiring readers to zoom in extensively. However, we must note that the presentation of the microscopy data follows standard practices in the field and that the panels already include zoomed-in regions, which enable easier access to key results and observations.

Regarding the NMR data, we have revised Figure 1—figure supplement 2 and Figure 2— figure supplement 1 to match the presentation style of Figure 3—figure supplement 1, which the reviewer apparently found more accessible. We also made the colors of the spectra more vibrant, as the referee suggested. We would like to emphasize that it is absolutely necessary to display the full NMR spectra in order to allow independent assessment of signal assignment, data quality, and overall protein behavior. Zoomed regions of the relevant details are provided in the main figures.

Very strong opinion 2: 

The author's response to lack of individual data points and error bars is that this is an n=1 experiment. I do not believe this meets the minimum standard for best practices in the field.

We respectfully disagree with the reviewer’s assessment. The Figure already displays individual data points, as shown already in the initial submission. Performing NMR titrations with isotopically labeled protein samples is inherently resource-intensive, and single-sample (n = 1) experiments are widely accepted and routinely reported in the field. Numerous studies have used the same approach, including Rosenzweig et al., Science (2013); Nikolaev et al., Nat. Methods (2019); and Hobbs et al., J. Biomol. NMR (2022), as well as our own recent work (Isaikina & Petrovic et al., Mol. Cell, 2023). These studies demonstrate that such NMR-based affinity measurements, even when performed on a single sample, are highly reproducible, precise, and consistent with orthogonal evidence and across different sample conditions.

Sticking point:

Figure 1A - the alphaFold model of arrestin2L depicts the disordered loops as ordered. The depiction is misleading at best, and inaccurate in truth. To use an analogy, what the authors depict is equivalent to publishing an LLM hallucination in the text. Unlike LLMs, alphaFold will actually flag its hallucination with the confidence (pLDDT) in the output. Both for LLMs and for alphaFold, we are spending much time teaching our students in class how to use computation appropriately - both to improve efficiency but also to ensure accuracy by removing hallucinations.

The original review indicated that confidences needed to be shown and that this needed to be depicted in a way that clarifies that this is NOT a structural state of the loops. The newly added description ("The model was used to visualize the clathrin-binding loop and the 344-loop of the arrestin2 Cdomain, which are not detected in the available crystal structures...) worsens the concern because it even more strongly implies that a 0 confidence computational output is a likely structural state. It also indicates that these regions were 'not detected' in crystal structures. These regions of arrestin are intrinsically disordered. AlphaFold (by it's nature) must put out something in terms of coordinates, even if the pLDDT suggests that the region cannot be predicted or is not in a stable position, which is the case here. In crystal structures, these regions are not associated with interpretable electron density, meaning that coordinates are omitted in these regions because adding them would imply that under the conditions used, the protein adopts a low energy structural state in this region. This region is instead intrinsically disordered. 

A good description of why showing disordered loops in a defined position is incorrect and how to instead depict disorder correctly is in Brotzakis et al. Nat communications 16, 1632 (2025) "AlphaFold prediction of structural ensembles of disordered proteins", where figures 3A, 4A, and 5A show one AlphaFold prediction colored by confidence and 3B, 4B and 5B are more accurate depictions of the structural ensemble. 

Coming back to the original comment "The AlphaFold model could benefit from a more transparent discussion of prediction confidence and caveats. The younger crowd (part of the presumed intended readership) tends to be more certain that computational output is 'true'...." Right now, the authors are still showing in Fig 1A a depiction of arrestin with models for the loops that are untrue. They now added text indicating that these loops are visualized in an AlphaFold prediction and 'true' but 'not detected in crystal structures'. There is no indication in the text that these are intrinsically disordered. The lack of showing the pLDDT confidence and the lack of any indication that these are disordered regions is simply incorrect. 

We appreciate the concern of the reviewer towards AlphaFold models. As NMR spectroscopists we are highly aware of intrinsic biomolecular motions. However, our AlphaFold2 model is used as a graphical representation to display the interaction sites of loops; it is not intended to depict the loops as fixed structural states. The flexibility of the loops had been clearly described in the main text before:

“Arrestin2 consists of two consecutive (N- and C-terminal) β-sandwich domains (Figure 1A), followed by the disordered clathrin-binding loop (CBL, residues 353–386), strand b20 (residues 386–390), and a disordered C-terminal tail after residue 393”.

and

“Figure 1B depicts part of a 1H-15N TROSY spectrum (full spectrum in Figure 1-figure supplement 2A) of the truncated 15N-labeled arrestin2 construct arrestin21-393 (residues 1393), which encompasses the C-terminal strand β20, but lacks the disordered C-terminal tail. Due to intrinsic microsecond dynamics, the assignment of the arrestin21-393 1H-15N resonances by triple resonance methods is largely incomplete, but 16 residues (residues 367381, 385-386) within the mobile CBL could be assigned. This region of arrestin is typically not visible in either crystal or cryo-EM structures due to its high flexibility”.

as well as in the legend to Figure 1:

“The model was used to visualize the clathrin-binding loop and the 344-loop of the arrestin2 C-domain, which are not detected in the available crystal structures of apo arrestin2 [bovine: PDB 1G4M (Han et al., 2001), human: PDB 8AS4 (Isaikina et al., 2023)]. In the other structured regions, the model is virtually identical to the crystal structures”.

We have now further added a label ‘AlphaFold2 model’ to Figure 1A and amended the respective Figure legend to

“The model was used to visualize the clathrin-binding loop and the 344-loop of the arrestin2 C-domain, which are not detected in the available crystal structures of apo arrestin2 [bovine: PDB 1G4M (Han et al., 2001), human: PDB 8AS4 (Isaikina et al., 2023)] due to flexibility. In the other structured regions, the model is virtually identical to the crystal structures”.

Reviewer #2 (Recommendations for the authors): 

I appreciated the response by the authors to all of my questions. I have no further comments

We thank the referee for the raised questions, which we believe have improved the quality of the manuscript.

We need a link for details about Hide field ability, same as we have for Searchable or Treandable fields. It will be used in a tooltip in the application, so admin can directly access it. Same for the limitations section.

Spannender Punkt: Der Staat war von Anfang an ein Projekt, das dazu diente, die Privilegien der Mächtigen und REichen zu sichern.

eLife Assessment

This fundamental work by Yamamoto and colleagues advances our understanding of how positional information is coordinated between axes during limb outgrowth and patterning. They provide convincing evidence that the dorsal-ventral axis feeds into anterior-posterior signaling, and identify the responsible molecules by combining transplantations with molecular manipulations. This work will be of broad interest to regeneration, tissue engineering, and evolutionary biologists.

Reviewer #1 (Public review):

Summary:

The manuscript by Yamamoto et al. presents a model by which the four main axes of the limb are required for limb regeneration to occur in the axolotl. A longstanding question in regeneration biology is how existing positional information is used to regenerate the correct missing elements. The limb provides an accessible experimental system by which to study the involvement of the anteroposterior, dorsoventral, and proximodistal axes in the regenerating limb. Extensive experimentation has been performed in this area using grafting experiments. Yamamoto et al. use the accessory limb model and some molecular tools to address this question. There are some interesting observations in the study. In particular, one strength the potent induction of accessory limbs in the dorsal axis with BMP2+Fgf2+Fgf8 is very interesting. Although interesting, the study makes bold claims about determining the molecular basis of DV positional cues, but the experimental evidence is not definitive and does not take into account the previous work on DV patterning in the amniote limb. Also, testing the hypothesis on blastemas after limb amputation would be needed to support the strong claims in the study.

Strengths:

The manuscript presents some novel new phenotypes generated in axolotl limbs due to Wnt signaling. This is generally the first example in which Wnt signaling has provided a gain of function in the axolotl limb model. They also present a potent way of inducing limb patterning in the dorsal axis by the addition of just beads loaded with Bmp2+Fgf8+Fgf2.

Comments on revised version:

Re-evaluation: The authors have significantly improved the manuscript and their conclusions reflect the current state of knowledge in DV patterning of tetrapod limbs. My only point of consideration is their claim of mesenchymal and epithelial expression of Wnt10b and the finding that Fgf2 and Wnt10b are lowly expressed. It is based upon the failed ISH, but this doesn't mean they aren't expressed. In interpreting the Li et al. scRNAseq dataset, conclusions depend heavily on how one analyzes and interprets it. The 7DPA sample shows a very low representation of epithelial cells compared to other time points, but this is likely a technical issue. Even the epithelial marker, Krt17, and the CT/fibroblast marker show some expression elsewhere. If other time points are included in the analysis, Wnt10b, would be interpreted as relatively highly expressed almost exclusively in the epithelium. By selecting the 7dpa timepoint, which may or may not represent the MB stage as it wasn't shown in the paper, the conclusions may be based upon incomplete data. I don't expect the authors to do more work, but it is worth mentioning this possibility. The authors have considered and made efforts to resolve previous concerns.

Reviewer #2 (Public review):

Summary:

This study explores how signals from all sides of a developing limb, front/back and top/bottom, work together to guide the regrowth of a fully patterned limb in axolotls, a type of salamander known for its impressive ability to regenerate limbs. Using a model called the Accessory Limb Model (ALM), the researchers created early staged limb regenerates (called blastemas) with cells from different sides of the limb. They discovered that successful limb regrowth only happens when the blastema contains cells from both the top (dorsal) and bottom (ventral) of the limb. They also found that a key gene involved in front/back limb patterning, called Shh (Sonic hedgehog), is only turned on when cells from both the dorsal and ventral sides come into contact. The study identified two important molecules, Wnt10B and FGF2, that help activate Shh when dorsal and ventral cells interact. Finally, the authors propose a new model that explains how cells from all four sides of a limb, dorsal, ventral, anterior (front), and posterior (back), contribute at both the cellular and molecular level to rebuilding a properly structured limb during regeneration

Strengths:

The techniques used in this study, like delicate surgeries, tissue grafting, and implanting tiny beads soaked with growth factors, are extremely difficult, and only a few research groups in the world can do them successfully. These methods are essential for answering important questions about how animals like axolotls regenerate limbs with the correct structure and orientation. To understand how cells from different sides of the limb communicate during regeneration, the researchers used a technique called in situ hybridization, which lets them see where specific genes are active in the developing limb. They clearly showed that the gene Shh, which helps pattern the front and back of the limb, only turns on when cells from both the top (dorsal) and bottom (ventral) sides are present and interacting. The team also took a broad, unbiased approach to figure out which signaling molecules are unique to dorsal and ventral limb cells. They tested these molecules individually and discovered which could substitute for actual dorsal and ventral cells, providing the same necessary signals for proper limb development. Overall, this study makes a major contribution to our understanding of how complex signals guide limb regeneration, showing how different regions of the limb work together at both the cellular and molecular levels to rebuild a fully patterned structure.

Weaknesses:

Because the expressional analyses are performed on thin sections of regenerating tissue, in the original manuscript, they provided only a limited view of the gene expression patterns in their experiments, opening the possibility that they could be missing some expression in other regions of the blastema. Additionally, the quantification method of the expressional phenotypes in most of the experiments did not appear to be based on a rigorous methodology. The authors' inclusion of an alternate expression analysis, qRT-PCR, on the entire blastema helped validate that the authors are not missing something in the revised manuscript.

Overall, the number of replicates per sample group in the original manuscript was quite low (sometimes as low as 3), which was especially risky with challenging techniques like the ones the authors employ. The authors have improved the rigor of the experiment in the revised manuscript by increasing the number of replicates. The authors have not performed a power analysis to calculate the number of animals used in each experiment that is sufficient to identify possible statistical differences between groups. However, the authors have indicated that there was not sufficient preliminary data to appropriately make these quantifications.

Likewise, in the original manuscript, the authors used an AI-generated algorithm to quantify symmetry on the dorsal/ventral axis, and my concern was that this approach doesn't appear to account for possible biases due to tissue sectioning angles. They also seem to arbitrarily pick locations in each sample group to compare symmetry measurements. There are other methods, which include using specific muscle groups and nerve bundles as dorsal/ventral landmarks, that would more clearly show differences in symmetry. The authors have now sufficiently addressed this concern by including transverse sections of the limbs annd have explained the limitations of using a landmark-based approach in their quantification strategy.

Reviewer #3 (Public review):

Summary:

After salamander limb amputation, the cross-section of the stump has two major axes: anterior-posterior and dorsal-ventral. Cells from all axial positions (anterior, posterior, dorsal, ventral) are necessary for regeneration, yet the molecular basis for this requirement has remained unknown. To address this gap, Yamamoto et al. took advantage of the ALM assay, in which defined positional identities can be combined on demand and their effects assessed through the outgrowth of an ectopic limb. They propose a compelling model in which dorsal and ventral cells communicate by secreting Wnt10b and Fgf2 ligands respectively, with this interaction inducing Shh expression in posterior cells. Shh was previously shown to induce limb outgrowth in collaboration with anterior Fgf8 (PMID: 27120163). Thus, this study completes a concept in which four secreted signals from four axial positions interact for limb patterning. Notably, this work firmly places dorsal-ventral interactions upstream of anterior-posterior, which is striking for a field that has been focussed on anterior-posterior communication. The ligands identified (Wnt10b, Fgf2) are different to those implicated in dorsal-ventral patterning in the non-regenerative mouse and chick models. The strength of this study is in the context of ALM/ectopic limb engineering. Although the authors attempt to assay the expression of Wnt10b and Fgf2 during limb regeneration after amputation, they were unable to pinpoint the precise expression domains of these genes beyond 'dorsal' and 'ventral' blastema. Given that experimental perturbations were not performed in regenerating limbs - almost exclusively under ALM conditions - this author finds the title "Dorsoventral-mediated Shh induction is required for axolotl limb regeneration" a little misleading.

Strengths:

(1) The ALM and use of GFP grafts for lineage tracing (Figures 1-3) take full advantage of the salamander model's unique ability to outgrow patterned limbs under defined conditions. As far as I am aware, the ALM has not been combined with precise grafts that assay 2 axial positions at once, as performed in Figure 3. The number of ALMs performed in this study deserves special mention, considering the challenging surgery involved.

(2) The authors identify that posterior Shh is not expressed unless both dorsal and ventral cells are present. This echoes previous work in mouse limb development models (AER/ectoderm-mesoderm interaction) but this link between axes was not known in salamanders. The authors elegantly reconstitute dorsal-ventral communication by grafting, finding that this is sufficient to trigger Shh expression (Figure 3 - although see also section on Weaknesses).

(3) Impressively, the authors discovered two molecules sufficient to substitute dorsal or ventral cells through electroporation into dorsal- or ventral- depleted ALMs (Figure 5). These molecules did not change the positional identity of target cells. The same group previously identified the ventral factor (Fgf2) to be a nerve-derived factor essential for regeneration. In Figure 6, the authors demonstrate that nerve-derived factors, including Fgf2, are alone sufficient to grow out ectopic limbs from a dorsal wound. Limb induction with a 3-factor cocktail without supplementing with other cells is conceptually important for regenerative engineering.

(4) The writing style and presentation of results is very clear.

Overall appraisal:

This is a logical and well-executed study that creatively uses the axolotl model to advance an important framework for understanding limb patterning. The relevance of the mechanisms to normal limb regeneration are not yet substantiated, in the opinion of this reviewer. Additionally, Wnt10b and Fgf2 should be considered molecules sufficient to substitute dorsal and ventral identity (solely in terms of inducing Shh expression). It is not yet clear whether these molecules are truly necessary (loss of function would address this).

Comments on revisions:

Congratulations - I still find this an elegant and easy-to-read study with significant implications for the field! Linking your mechanisms to normal limb regeneration (i.e. regenerating blastema, not ALM), as well as characterising the cell populations involved, will be interesting directions for the future.

Author response:

The following is the authors’ response to the current reviews.

We sincerely thank all three reviewers for their constructive comments. We deeply appreciate the reviewers’ efforts in summarizing our study, highlighting its strengths, and providing constructive suggestions. To enhance the quality and clarity of our work, we plan to address the concerns raised by the reviewers.

First, as Reviewer #1 suggested, we will note that clearer expression patterns of Wnt10b and Fgf2 may be detectable in scRNA-seq analyses at other stages, and we will also clarify that low-level signals of epithelial and CT/fibroblast markers outside their expected clusters may reflect technical bias. In addition, we agree with the reviewer’s point that our unsuccessful ISH experiments and the low abundance detected by RT-qPCR do not demonstrate absence of expression, and that conclusions from reanalyzing the Li et al. scRNA-seq dataset can depend strongly on analytical choices; therefore, while we focused on the 7 dpa sample because our RT-qPCR data suggested that Wnt10b and Fgf2 may be most enriched around the MB stage (the original study refers to 7 dpa as MB), we will explicitly acknowledge that analyzing a single time point—especially one with a low representation of epithelial cells—may yield incomplete or stage-biased interpretations, and that inclusion of additional time points could reveal clearer and potentially different expression patterns. We will also temper our wording regarding the inferred cellular sources to avoid over-interpretation based on the current data.

Second, to mitigate the concerns raised by Reviewer #3 regarding the generalization of our conclusions to amputation-induced (normal) limb regeneration, we will cite a previous study suggesting that ALM was used as the alternative experimental system for studying limb regeneration (Nacu et al., 2016, Nature, PMID: 27120163; Satoh et al., 2007, Developmental Biology, PMID: 17959163). We are confident that our ALM-based data provide a reasonable basis for understanding limb regeneration. We agree that there are important remaining questions—such as which cell populations express Wnt10b and Fgf2 and how endogenous WNT10B and FGF2 signals induce Shh expression in normal regeneration—which should be investigated in future studies to deepen our understanding of limb regeneration.

We also appreciate Reviewer #2’s careful evaluation of the technical rigor and quantification. We have benefited from the reviewer’s earlier feedback, which guided revisions that improved the manuscript’s rigor and presentation.

We are grateful for the reviewers’ insights and are confident that these revisions will significantly strengthen our manuscript.

The following is the authors’ response to the original reviews.

Recommendations for the authors:

Reviewing Editor Comments:

The authors should be commended for addressing this gap - how cues from the DV axis interact with the AP axis during limb regeneration. Overall, the concept presented in this manuscript is extremely interesting and could be of high value to the field. However, the manuscript in its current form is lacking a few important data and resolution to fully support their conclusions, and the following needs to be addressed before publication:

(1) ISH data on Wnt10b and FGF2 from various regeneration time points are essential to derive the conclusion. Preferably multiplex ISH of Wnt10b/Fgf2/Shh or at least canonical ISH on serial sections to demonstrate their expression in dermis/epidermis and order of gene expression i.e. Shh is only expressed after expression of Wnt10b/FGF2. It would certainly help if this can also be shown in regular blastema.

We are grateful for the constructive suggestion on assessing Wnt10b and Fgf2 expression during regular regeneration, and we agree that clarifying their expression patterns in regular blastemas is important for strengthening the conclusions of our study. Because we cannot currently ensure sufficient sensitivity with multiplex FISH in our laboratory—partly due to high background—, we conducted conventional ISH on serial sections of regular blastemas at several time points (Fig. S5A). However, the expression patterns of Wnt10b and Fgf2 were not clear. To complement the ISH results, we performed RT-qPCR on microdissected dorsal and ventral halves of regular blastemas at the MB stage (Fig. S5B). We found that Wnt10b and Fgf2 were expressed at significantly higher levels in the dorsal and ventral halves, respectively, compared to the opposite half. This dorsal/ventral biased expression of Wnt10b/Fgf2 is consistent with our RNA-seq data. We further quantified expression levels of Wnt10b, Fgf2, and Shh across stages (intact, EB, MB, LB, and ED) and found that Wnt10b and Fgf2 peaked at the MB stage, whereas Shh peaked at the LB stage—consistent with the editor’s request regarding the order of gene expression (Fig. S5C). This temporal offset in upregulation supports our model. These results are now included in the revised manuscript (Line 294‒306).

To identify the cell types expressing Wnt10b or Fgf2, we analyzed published single-cell RNA-seq data (7 dpa blastema (MB), Li et al., 2021). As a result, Fgf2 expression was observed in the mesenchymal cluster, whereas Wnt10b expression was observed in both mesenchymal and epithelial clusters (Fig. S6). However, because only a small fraction of cells expressed Wnt10b, the principal cellular source of WNT10B protein remains unclear. The apparent low abundance likely contributes to the weak ISH signals and reflects current technical limitations. In addition, Wnt10b and Fgf2 expression did not follow Lmx1b expression (Fig. S6J, K), and Wnt10b and Fgf2 themselves were not exclusive (Fig. S6L). These results are now included in the revised manuscript (Line 307‒321). Together with the RT-qPCR data (Fig. S5B), these results suggest that Wnt10b and Fgf2 are not exclusively confined to purely dorsal or ventral cells at the single-cell level, even though they show dorsoventral bias when assessed in bulk tissue. These results suggest that Wnt10b/Fgf2 expression is not restricted to dorsal/ventral cells but mediated by dorsal/ventral cells, and co-existence of both signals should provide a permissive environment for Shh induction. Defining the precise spatial patterns of Wnt10b and Fgf2 in regular regeneration will therefore be an important goal for future work.  

(2) Validation of the absence of gene expression via qRT PCR in the given sample will increase the rigor, as suggested by reviewers.

We thank for this important suggestion and agree that validation by qRT-PCR increases the rigor of our study. Accordingly, we performed RT-qPCR on AntBL, PostBL, DorBL, and VentBL to corroborate the ISH results. The results are now included in Fig. 2. We also verified by RT-qPCR that Shh expression following electroporation and the quantitative results are now provided in Fig. 5.

(3) Please increase n for experiments where necessary and mention n values in the figures.

We thank for this helpful comment and agree on the importance of providing sufficient sample sizes. Accordingly, we increased the n for the relevant experiments and have indicated the n values in the corresponding figure legends.

(4) Most comments by all three reviewers are constructive and largely focus on improving the tone and language of the manuscript, and I expect that the authors should take care of them.

We thank the reviewers for their constructive feedback on the tone and language of the manuscript. We have carefully revised the text according to each comment, and we hope these modifications have improved both clarity and readability.

In addition, in revising the manuscript we also refined the conceptual framework. Our new analysis of Wnt10b and Fgf2 expression during normal regeneration suggests that these genes are not expressed in a strictly dorsal- or ventral-specific manner at the single-cell level. When these observations are considered together with (i) the RNA-seq comparison of dorsally and ventrally induced ALM blastemas, (ii) RT-qPCR of microdissected dorsal and ventral halves of regenerating blastemas, and (iii) the functional electroporation experiments, our interpretation is that Wnt10b and Fgf2 act as dorsal- and ventral-mediated signals, respectively: their production is regulated by dorsal or ventral cells, and the presence of both signals is required to induce Shh expression. Given those, we now think our conclusion might be explained without using the confusing term, “positional cue”. Because the distinction between “positional cue” and “positional information” could be confusing as noted by the reviewers, we rewrote our manuscript without using “positional cue.”

Reviewer #1 (Recommendations for the authors):

(1) Line 61: More explanation for what a double-half limb means is needed.

We thank the reviewer for this suggestion. We have revised the manuscript (Line 73‒76). Specifically, we now explain that a double-dorsal limb, for example, is a chimeric limb generated by excising the ventral half and replacing it with a dorsal half from the contralateral limb while preserving the anteroposterior orientation.

(2) Line 63-65: "Such blastemas form hypomorphic, spike-like structures or fail to regenerate entirely." This statement does not represent the breadth of work on the APDV axis in limb regeneration. The cited Bryant 1976 reference tested only double-posterior and double-anterior newt limbs, demonstrating the importance of disposition along the AP axis, not DV. Others have shown that the regeneration of double-half limbs depends upon the age of the animal and the length of time between the grafting of double-half limbs and amputation. Also, some double-dorsal or double-ventral limbs will regenerate complete AP axes with symmetrical DV duplications (Burton, Holder, and Jesani, 1986). Also, sometimes half dorsal stylopods regenerate half dorsal and half ventral, or regenerate only half ventral, suggesting there are no inductive cues across the DV axis as there are along the AP axis. Considering this is the basis of the study under question, more is needed to convince that the DV axis is necessary for the generation of the AP axis.

We thank the reviewer for this detailed and constructive comment. We acknowledge that previous studies have reported a range of outcomes for double-half limbs. For example, Burton et al. (1986) described regeneration defects in double-dorsal (DD) and double-ventral (VV) limbs, although limb patterning did occur in some cases (Burton et al., 1986, Table 1). As the reviewer notes, regenerative outcomes depend on variables such as animal age and the interval between construction of the double-half limb and amputation, sometimes called the effect of healing time (Tank and Holder, 1978). Moreover, variability has been reported not only in DD/VV limbs but also in double-anterior (AA) and double-posterior (PP) limbs (e.g., Bryant, 1976; Bryant and Baca, 1978; Burton et al., 1986). In the revised manuscript, we have therefore modified the statement to avoid over-generalization and to emphasize that regeneration can be incomplete under these conditions (Line 76‒82). Importantly, in order to provide the additional evidence requested and to directly re-evaluate whether dorsal and ventral cells are required for limb patterning, we performed the ALM experiments shown in Fig. 1. The ALM system allows us to assess this question in a binary manner (regeneration vs. non-regeneration), thereby strengthening the rationale for our conclusions regarding the necessity of the APDV orientations. We also revised a sentence at the beginning of the Results section to emphasize this point (Line 139‒140).

(3) Line 71: These findings suggest that specific signals from all four positional domains must be integrated for successful limb patterning, such that the absence of any one of them leads to failure." I was under the impression that half posterior limbs can grow all elements, but half anterior can only grow anterior elements.

We thank the reviewer for this helpful clarification. As summarized by Stocum, half-limb experiments show that while some digit formation can occur, limb patterning remains incomplete in both anterior-half and posterior-half limbs in some cases (Stocum, 2017). We see this point as closely related to the broader question of whether proper limb patterning requires the integration of signals from all four positional domains. As noted in our response above, our ALM experiments in Fig. 1 were designed to test this point directly, and our data support the interpretation that cells from all four orientations are necessary for correct limb patterning.

(4) Line 79-81: This is stated later in lines 98-105. I suggest expanding here or removing it here.

We thank the reviewer for this suggestion. In the original version, lines 79–81 introduced our use of the terms “positional cue” and “positional information,” and this content partially overlapped with what later appeared in lines 98–105. In the revised manuscript, we have substantially rewritten this section (Line 82‒84), including the sentences corresponding to lines 79–81 in the original version, to remove the term “positional cue,” as explained in our response to the Editor’s comment (4); our revision reflects new analyses indicating that Wnt10b and Fgf2 appear not be strictly restricted to dorsal or ventral cell populations, and we now describe these factors as dorsal- or ventral-mediated signals that act across dorsoventral domains to induce Shh expression. Accordingly, we no longer maintain the original use of “positional cue” and “positional information.”

(5) Line 92 - 93: "Similarly, an ALM blastema can be induced in a position-specific manner along the limb axes. In this case, the induced ALM blastema will lack cells from the opposite side." This sentence is difficult to follow. Isn't it the same thing stated in lines 88-90?

We thank the reviewer for this comment. We revised the sentence to improve readability and to avoid redundancy with original Lines 88–90 (Line 104‒106).

(6) Line 107: I think the appropriate reference is McCusker et al., 2014 (Position-specific induction of ectopic limbs in non-regenerating blastemas on axolotl forelimbs), although Vieira et al., 2019 can be included here. In addition, Ludolph et al 1990 should be cited.

We thank the reviewer for this suggestion. We have added McCusker et al. (2014) and Ludolph et al. (1990) as references in the revised manuscript (Line 120‒121).

(7) Line 107-109: A missing point is how the ventral information is established in the amniote limb. From what I remember, it is the expression of Engrailed 1, which inhibits the ventral expression of Wnt7a, and hence Lmx1b. This would suggest that there is no secreted ventral cue. This is a relatively large omission in the manuscript.

We thank the reviewer for this comment. We agree that ventral fate in amniotes is specified by En1 in the ventral ectoderm, which represses Wnt7a and thereby prevents induction of Lmx1b; accordingly, a secreted ventral morphogen analogous to dorsal Wnt7a has not been established. We added this point to the revised Introduction (Line 61‒64).

By contrast, in axolotl limb regeneration, our previous work on Lmx1b expression suggests that DV identities reflect the original positional identity rather than being re-specified during regeneration (Yamamoto et al., 2022). Within this framework, our original use of the term “ventral positional cue” does not imply a ventral patterning morphogen in the amniote sense; rather, it denotes downstream signals induced by cells bearing ventral identity that are required for the blastema to form a patterned limb. This interpretation is consistent with classic studies on double-half chimeras and ectopic contacts between opposite regions (Iten & Bryant, 1975; Bryant & Iten, 1976; Maden, 1980; Stocum, 1982) as well as with our ALM data (Fig. 1). For this reason, we intentionally used the term “positional cues” to refer to signals provided by cells bearing ventral identity, which can be considered separable from the DV patterning mechanism itself, in the original text. As explained in our response to the Editor’s comment (4), we describe these signals as “signals mediated by dorsal/ventral cells,” rather than “positional cues” in the revised manuscript.

The necessity of dorsal- and ventral-mediated signals is supported by classic studies on the double-half experiment. In the non-regenerating cases, structural patterns along the anteroposterior axis appear to be lost even though both anterior and posterior cells should, in principle, be present in a blastema induced from a double-dorsal or double-ventral limbs. In limb development of amniotes, Wnt7a/Lmx1b or En-1 mutants show that limbs can exhibit anteroposterior patterning even when tissues are dorsalized or ventralized—that is, in the relative absence of ventral or dorsal cells, respectively (Riddle et al., 1995; Chen et al., 1998; Loomis et al., 1996). Taken together, axolotl limb regeneration, in which the presence of both dorsal and ventral cells plays a role in anteroposterior patterning, should differ from other model organisms. It is reasonable to predict the dorsal- and ventral-mediated signals in axolotl limb regeneration. We included this point in the revised manuscript (Line 82‒89). However, there is no evidence that these signals are secreted molecules. For this reason, we have carefully used the term “dorsal-/ventral-mediated signals” in the Introduction without implying secretion.

(8) Introduction - In general, the argument is a bit misleading. It is written as if it is known that a ventral cue is necessary, but the evidence from other animal models is lacking, from what I know. I may be wrong, but further argument would strengthen the reasoning for the study.

We thank the reviewer for this thoughtful comment. We agree that it should not read as if it is known that a ventral cue is necessary. In the revised Introduction, we have addressed this in several ways. First, as described in our response to comment (7), we now explicitly note that in amniote limb development ventral identity is specified by En1-mediated repression of Wnt7a, and that a secreted ventral morphogen equivalent to dorsal Wnt7a has not been established. Second, we removed the term “positional cue” and no longer present “ventral positional cue” as a defined entity. Instead, we use mechanistic phrasing such as “signals mediated by ventral cells” and “signals mediated by dorsal cells,” which does not assume that such signals are secreted morphogens or universally conserved. Third, we have reframed the role of dorsal- and ventral-mediated signals as a working hypothesis specific to axolotl limb regeneration, rather than as a general conclusion across model systems.

(9) Line 129: Remove "As mentioned before".

We thank the reviewer for this suggestion. We have removed the phrase “As mentioned before” in the revised manuscript (Line 143).

(10) Figure 1: Are Lmx1, Fgf8, and Shh mutually exclusive? Multiplexed FISH would provide this information, and is a relatively important question considering the strong claims in the study.

We thank the reviewer for raising this important point. As noted in our response to the editor’s comment, we cannot currently ensure sufficiently high detection sensitivity with multiplex FISH in our laboratory. However, based on previous reports (Nacu et al., 2016), Fgf8 and Shh should be mutually exclusive. In contrast, with respect to Lmx1b, our analysis suggests that its expression is not mutually exclusive with either Fgf8 or Shh, at least their expression domains. To confirm this, we analyzed the published scRNA-seq data and the results were added to the supplemental figure 6. Fgf8 and Shh were expressed in both Lmx1b-positive and Lmx1b-negative cells (Fig. S6H, I), but Fgf8 and Shh themselves were mutually exclusive (Fig. S6M). This point is now included in the revised manuscript (Line 314‒317).

(11) Results section and Figure 2: More evidence is needed for the lack of Shh expression ISH in tissue sections. Demonstrating the absence of something needs some qPCR or other validation to make such a claim.

We thank the reviewer for this suggestion. We performed qRT-PCR on ALM blastemas to complement the ISH data (Fig. 2).

(12) Line 179: I think they are likely leucistic d/d animals and not wild-type animals based upon the images.

We thank the reviewer for this observation. In the revised manuscript, we have corrected the description to “leucistic animals” (Line 194).

(13) Line 183-186: I'm a bit confused about this interpretation. If Shh turns on in just a posterior blastema, wouldn't it turn on in a grafted posterior tissue into a dorsal or ventral region? Isn't this independent of environment, meaning Shh turns on if the cells are posterior, regardless of environment?

Our interpretation is that only posterior-derived cells possess the competency to express Shh. In other words, whether a cell is capable of expressing Shh depends on its original positional identity (Iwata et al., 2020), but whether it actually expresses Shh depends on the environment in which the cell is placed. The results of Fig. 3E and G indicate that Shh activation is dependent on environment and that the posterior identity is not sufficient to activate Shh expression. We have revised the manuscript to emphasize this distinction more clearly (Line 198‒203).

(14) Figure 4: Do the limbs have an elbow, or is it just a hand?

We thank the reviewer for this thoughtful question. From the appearance, an elbow-like structure can occasionally be seen; however, we did not examine the skeletal pattern in detail because all regenerated limbs used for this analysis were sectioned for the purpose of symmetry evaluation, and we therefore cannot state this conclusively. While this is indeed an important point, analyzing proximodistal patterning would require a very large number of additional experiments, which falls outside the main focus of the present study. For this reason, and also to minimize animal use in accordance with ethical considerations, we did not pursue further experiments here. In response to this point, we have added a description of the skeletal morphology of ectopic limbs induced by BMP2+FGF2+FGF8 bead implantation (Fig. 6). In these experiments, multiple ectopic limbs were induced along the same host limb. In most cases, these ectopic limbs did not show fusion with the proximal host skeleton, similar to standard ALM-induced limbs, although in one case we observed fusion at the stylopod level. We now note this observation in the revised manuscript (Line 347‒354).

We regard the relationship between APDV positional information and proximodistal patterning as an important subject for future investigation.

(15) Line 203 - 237: I appreciate the symmetry score to estimate the DV axis. Are there landmarks that would better suggest a double-dorsal or double-ventral phenotype, like was done in the original double-half limb papers?

We thank the reviewer for this thoughtful comment. In most cases, the limbs induced by the ALM exhibit abnormal and highly variable morphologies compared to normal limbs, making it difficult to apply consistent morphological landmarks as used in the original double-half limb studies. For this reason, we focused our analysis on “morphological symmetry” as a quantitative measure of DV axis patterning, and we have added this explanation to the manuscript (Line 232‒235). Additionally, we provided transverse sections along the proximodistal axis as supplemental figures (Figs. S2 and S4). In addition to reporting the symmetry score, we have explicitly stated in the text that symmetry was also assessed by visual inspection of these sections.

(16) Line 245-247: The experiment was done using bulk sequencing, so both the epithelium and mesenchyme were included in the sample. The posterior (Shh) and anterior (Fgf8) patterning cues are mesenchymally expressed. In amniotes, the dorsal cue has been thought to be Wnt7a from the epithelium. Can ISH, FISH, or previous scRNAseq data be used to identify genes expressed in the mesenchyme versus epithelium? This is very important if the authors want to make the claim for defining "The molecular basis of the dorsal and ventral positional cues" as was stated by the authors.

We thank the reviewer for highlighting this important point. As the reviewer notes, our bulk RNA-seq data do not distinguish between epithelial and mesenchymal expression domains. As noted in our response to the editor’s comment, we performed ISH and qPCR on regular blastemas. However, these approaches did not provide definitive information regarding the specific cell types expressing Wnt10b and Fgf2. To complement this, we re-analyzed publicly available single-cell RNA-seq data (from Li et al., 2021). As a results, Fgf2 was expressed mainly by the mesenchymal cells, and Wnt10b expression was observed in both mesenchymal and epithelial cells. These results are now included in the revised manuscript (Line 294‒321) and in supplemental figures (Fig. S6, S7).

(17) Was engrailed 1, lmx1b, or Wnt7a differentially expressed along the DV axis, suggesting similar signaling between? Are these expressed in mesenchyme? Previous work suggests Wnt7a is expressed throughout the mesenchyme, but publicly available scRNAseq suggests that it is expressed in the epithelium.

We thank the reviewer for this important comment. As noted, the reported expression patterns of DV-related genes are not consistent across studies, which likely reflects the technical difficulty of detecting these genes with high sensitivity. In our own experiments, expression of DV markers other than Lmx1b has been very weak or unclear by ISH. Whether these genes are expressed in the epithelium or mesenchyme also appears to vary depending on the detection method used. In our RNA-seq dataset, Wnt7a expression was detected at very low levels and showed no significant difference along the DV axis, while En1 expression was nearly absent. We have clarified these results in the revised manuscript (Line 437‒441). Our reanalysis of the published scRNA-seq likewise detected Wnt7a in only a very small fraction of cells. Accordingly, we consider it premature to reach a definitive conclusion—such as whether Wnt7a is broadly mesenchymal or restricted to epithelium—as suggested in prior reports. We also note that whether Wnt7a is epithelial or mesenchymal does not affect the conclusions or arguments of the present study. Although the roles of Wnt7a and En1 in axolotl DV patterning are certainly important, we feel that drawing a definitive conclusion on this issue lies beyond the scope of the present study, and we have therefore limited our description to a straightforward presentation of the data.

(18) Line 247-249: The sentence suggests that all the ligands were tried. This should be included in the supplemental data.

We thank the reviewer for this clarification. In fact, we tested only Wnt4, Wnt10b, Fgf2, Fgf7, and Tgfb2, and all of these results are presented in the figures. To avoid misunderstanding, we have revised the text to explicitly state that our analysis focused on these five genes (Line 272‒274).

(19) Line 249: An n =3 seems low and qPCR would be a more sensitive means of measuring gene induction compared to ISH. The ISH would confirm the qPCR results. Figure 5C is also not the most convincing image of Shh induction without support from a secondary method.

We have increased the sample size for these experiments (Line 277‒280). In addition, to complement the ISH results, we confirmed Shh induction by qPCR following electroporation of Wnt10b and Fgf2 (Fig. 5D, E). In addition, because Shh signal in the Wnt10b-electroporated VentBL images was particularly weak and difficult to discern, we replaced that panel with a representative example in which Shh signal is more clearly visible. These data are now included in the revised manuscript (Line 280‒282).

(20) Line 253: It is confusing why Wnt10b, but not Wnt4 would work? As far as I know, both are canonical Wnt ligands. Was Wnt7a identified as expressed in the RNAseq, but not dorsally localized? Would electroporation of Wnt7a do the same thing as Wnt10b and hence have the same dorsalizing patterning mechanisms as amniotes?

We thank the reviewer for raising this challenging but important question. Wnt10b was identified directly from our bulk RNA-seq analysis, as was Wnt4. The difference in the ability of Wnt10b and Wnt4 to induce Shh expression in VentBL may reflect differences in how these ligands activate downstream WNT signaling programs. WNT10B is a potent activator of the canonical WNT/β-catenin pathway (Bennett et al., 2005), although WNT10B has also been reported to trigger a β-catenin–independent pathway (Lin et al., 2021). By contrast, WNT4 can signal through both canonical and non-canonical (β-catenin–independent) pathways, and the balance between these outputs is known to depend on cellular context (Li et al., 2013; Li et al., 2019). Consistent with a requirement for canonical WNT signaling, we found that pharmacological activation of canonical WNT signaling with BIO (a GSK3 inhibitor) was also sufficient to induce Shh expression in VentBL. However, despite this, it is still unclear why Wnt10b, but not Wnt4, was able to induce Shh under our experimental conditions. One possible explanation is that different WNT ligands can engage the same receptors (e.g., Frizzled/LRP6) yet can drive distinct downstream transcriptional programs (This may depend on the state of the responding cells, as Voss et al. predicted), resulting in ligand-specific outputs (Voss et al., 2025). This point is now included in the revised discussion section (Line 402‒412). At present, we cannot distinguish between these possibilities experimentally, and we therefore refrain from making a stronger mechanistic claim.

With respect to Wnt7a, we detected Wnt7a expression at very low levels, and without a clear dorsoventral bias, in our RNA-seq analysis of ALM blastemas (we describe this point in Line 437‒440). This is consistent with previous work suggesting that axolotl Wnt7a is not restricted to the dorsal region in regeneration. Because of this low and unbiased expression, and because our data already implicated Wnt10b as a dorsal-mediated signal that can act across dorsoventral domains to permit Shh induction, we did not prioritize Wnt7a electroporation in the present study. We therefore cannot conclude whether Wnt7a would behave similarly to Wnt10b in this context.

Importantly, these uncertainties about ligand-specific mechanisms do not alter our main conclusion. Our data support the idea that a dorsal-mediated WNT signal (represented here by WNT10B and canonical WNT activation) and a ventral-mediated FGF signal (FGF2) must act together to permit Shh induction, and that the coexistence of these dorsal- and ventral-mediated signals is required for patterned limb formation in axolotl limb regeneration.

(21) Is canonical Wnt signaling induced after electroporation of Wnt10b or Wnt4? qPCR of Lef1 and axin is the most common way of showing this.

We thank the reviewer for this helpful suggestion. In addition to examining Shh expression, we also assessed canonical WNT signaling by qPCR analysis of Axin2 and Lef1 following Wnt10b electroporation. The data is now included in Fig. 5.

(22) Line 255-256: qPCR was presented for Figure 5D, but ISH was used for everything else. Is there a technical reason that just qPCR was used for the bead experiments?

We thank the reviewer for this helpful comment. In the original submission, our goal was to test whether treatment with commercial FGF2 protein or BIO could reproduce the results obtained by electroporation. In the revised manuscript, to avoid confusion between distinct experimental aims, we removed the FGF2–bead data from this section and instead used RT-qPCR to quantitatively corroborate Shh induction after electroporation (Fig. 5D–E). RT-qPCR provided a sensitive, whole-blastema readout and allowed a paired design (left limb: factor; right limb: GFP control) that increased statistical power while minimizing animal use. To address the reviewer’s point more directly, we additionally performed ISH for the BIO treatment and now include those results in Supplementary Figure 3 (Line 287‒288).

(23) Line 261-263: The authors did not show where Wnt10B or Fgf2 is expressed in the limb as claimed. The RNAseq was bulk, so ISH of these genes is needed to make this claim. Where are Wnt10b and Fgf2 expressed in the amputated limb? Do they show a dorsal (Wnt10b) and ventral (Fgf2) expression pattern?

We thank the reviewer for raising this important point. As noted in our response to the editor’s comment, we performed ISH on serial sections of regular blastemas at several time points (Fig. S5A). However, the expression patterns of Wnt10b and Fgf2 along the dorsoventral axis were not clear. To complement the ISH results, we performed RT-qPCR on microdissected dorsal and ventral halves of regular blastemas at the MB stage (Fig. S5B). We found that Wnt10b and Fgf2 were expressed at significantly higher levels in the dorsal and ventral halves, respectively, compared to the opposite half. This dorsal/ventral biased expression of Wnt10b/Fgf2 is consistent with our RNA-seq data. To identify the cell types expressing Wnt10b or Fgf2, we analyzed published single-cell RNA-seq data (7 dpa blastema (MB), Li et al., 2021). As a result, Fgf2 expression was observed in the mesenchymal cluster, whereas Wnt10b expression was observed in both mesenchymal and epithelial clusters (Fig. S6). However, because only a small fraction of cells expressed Wnt10b, the principal cellular source of WNT10B protein remains unclear. The apparent low abundance likely contributes to the weak ISH signals and reflects current technical limitations. In addition, Wnt10b and Fgf2 expression did not follow Lmx1b expression (Fig. S6J, K), and Wnt10b and Fgf2 themselves were not exclusive (Fig. S6L). Together with the RT-qPCR data (Fig. S5B), these results suggest that Wnt10b and Fgf2 are not exclusively confined to purely dorsal or ventral cells at the single-cell level, even though they show dorsoventral bias when assessed in bulk tissue, suggesting that Wnt10b/Fgf2 expression is not dorsal-/ventral-specific but mediated by dorsal/ventral cells. Defining the precise spatial patterns of Wnt10b and Fgf2 in regular regeneration will therefore be an important goal for future work. These points are now included in the revised manuscript (Line 485‒501).

(24) Line 266-288: The formation of multiple limbs is impressive. Do these new limbs correspond to the PD location they are generated?

We thank the reviewer for this interesting question. Interestingly, from our observations, there does appear to be a tendency for the induced limbs to vary in length depending on their PD location. The skeletal patterns of the induced multiple limbs are now included in Fig. 6. However, as noted earlier, the supernumerary limbs exhibit highly variable morphologies, and a rigorous analysis of PD correlation would require a large number of induced limbs. Since this lies outside the main focus of the present study, we have not pursued this point further in the manuscript.

(25) Line 288: The minimal requirement for claiming the molecular basis for DV signaling was identified is to ISH or multiplexed FISH for Wnt10b and Fgf2 in amputated limb blastemas to show they are expressed in the mesenchyme or epithelium and are dorsally and ventrally expressed, respectively. In addition, the current understanding of DV patterning through Wnt7a, Lmx1b, and En1 shown not to be important in this model.

We thank the reviewer for this comment and fully agree with the point raised. We would like to clarify that we are not claiming to have identified the molecular basis of DV patterning. As the reviewer notes, molecules such as Lmx1b, Wnt7a, and En1 are well identified in other animal models as key regulators of DV positional identity. There is no doubt that these molecules play central roles in DV patterning. However, in axolotl limb regeneration, clear DV-specific expression has not been demonstrated for these genes except for Lmx1b. Therefore, further studies will be required to elucidate the molecular basis of DV patterning in axolotls.

Our focus here is more limited: we aim to identify the molecular basis for the mechanisms in which positional domain-mediated signals (FGF8, SHH, WNT10B, and FGF2) regulate the limb patterning process, rather than the molecular basis of DV patterning. In fact, our results on Wnt10b and Fgf2 suggest that these genes did not affect dorsoventral identities.

We recognize that this distinction was not sufficiently clear in the original text, and we have revised the manuscript to describe DV patterning mechanisms in other animals and clarify that the dorsal- and ventral-mediated signals are distinct from DV patterning (Line 444‒450). At least, we avoid claiming that the molecular basis for DV signaling was identified.

(26) Line 335: References are needed for this statement. From what I found, Wnt4 can be canonical or non-canonical.

We thank the reviewer for this helpful comment. We have revised the manuscript (Line 404‒407). We added these citations at the relevant location and adjusted nearby wording to avoid implying pathway exclusivity, in alignment with our response to comment (20).

(27) Line 337-338: The authors cannot claim "that canonical, but not non-canonical, WNT signaling contributes to Shh induction" as this was not thoroughly tested is based upon the negative result that Wnt4 electroporation did not induce Shh expression.

We thank the reviewer for this important clarification. We agree that our data do not allow us to conclude that non-canonical WNT signaling in general does not contribute to Shh induction. Accordingly, we have removed the phrase “but not non-canonical” and revised the text to emphasize that, within the scope of our experiments, Shh induction was not observed following Wnt4 electroporation, whereas it was observed with Wnt10b.

(28) Line 345: In order to claim "WNT10B via the canonical WNT pathway...appears to regulate Shh expression" needs at least qPCR to show WNT10B induces canonical signaling.

We thank the reviewer for this comment. As noted in our response to comment (21), we also assessed canonical WNT signaling by qPCR analysis of Axin2 and Lef1 following Wnt10b electroporation (Line 282‒285).

(29) Lines 361-372: A few studies have been performed on DV patterning of the mouse digit regeneration in regards to Lmx1b and En1. It may be good to discuss how the current study aligns with these findings.

We appreciate the reviewer’s suggestion. As the reviewer refers, several studies have been performed on dorsoventral (DV) patterning in mouse digit tip regeneration in relation to Lmx1b and En1 (e.g., Johnson et al., 2022; Castilla-Ibeas et al., 2023). In the present study, however, our main conclusion is different in the scope of studies on mouse digit tip regeneration. We show that, in the axolotl, pre-existing dorsal and ventral identities (as reflected by dorsally derived and ventrally derived cells in the ALM blastema) are required together to induce Shh expression, and that this Shh induction in turn supports anteroposterior interaction at the limb level. This mechanism—dorsal-mediated and ventral-mediated signals acting in combination to permit Shh expression—does not have a clear direct counterpart in the mouse digit tip literature. Moreover, even with respect to Lmx1b, the two systems behave differently. In mouse digit tip regeneration, loss of Lmx1b during regeneration does not grossly affect DV morphology of the regenerate (Johnson et al., 2022). By contrast, in our axolotl ALM system, the presence or absence of Lmx1b-positive dorsal tissue correlates with the final dorsoventral organization of the induced limb-like structures (e.g., production of double-dorsal or double-ventral symmetric structures in the absence of appropriate dorsoventral contact). Thus, the role of dorsoventral identity in our model is directly tied to patterned limb outgrowth at the whole-limb scale, whereas in the mouse digit tip it has been reported primarily in the context of digit tip regrowth and bone regeneration competence, not robust DV repatterning (Johnson et al., 2022).

For these reasons, we believe that an extended discussion of mouse digit tip regeneration would risk implying a mechanistic equivalence between axolotl limb regeneration and mouse digit tip regeneration that is not supported by current data. Because the regenerative contexts differ, and because Lmx1b does not appear to re-establish DV patterning in the mouse regenerates (Johnson et al., 2022), we have chosen not to include an explicit discussion of mouse digit tip regeneration in the main text.

(30) Line 408-433: Although I appreciate generating a model, this section takes some liberties to tell a narrative that is not entirely supported by previous literature or this study. For example, lines 415-416 state "Wnt10b and Fgf2 are expressed at higher levels in dorsal and the ventral blastemal cells, respectively" which were not shown in the study or other studies.

We thank the reviewer for this important comment. We agree that the original model based on RNA-seq data overstated the evidence. To address this point experimentally, we examined Wnt10b and Fgf2 expression in regular blastemas (Supplemental Figure 5 and 6). Accordingly, our model is now framed as an inductive mechanism for Shh expression—supported by results in ALM (WNT10B in VentBL; FGF2 in DorBL) and by DV-biased expression. Concretely, the sentence previously paraphrased as “Wnt10b and Fgf2 are expressed at higher levels in dorsal and ventral blastemal cells, respectively” has been replaced with wording that (i) avoids single-cell DV specificity and (ii) emphasizes dorsal-/ventral-mediated regulation and the requirement for both signals to allow Shh induction (Line 510‒511).

Reviewer #2 (Recommendations for the authors):

(1) Introduction:

The authors' definitions of positional cues vs positional information are a little hard to follow, and do not appear to be completely accurate. From my understanding of what the authors explain, "positional information" is defined as a signal that generates positional identities in the regenerating tissue. This is a somewhat different definition than what I previously understood, which is the intrinsic (likely epigenetic) cellular identity associated with specific positional coordinates. On the other hand, the authors define "positional cues" as signals that help organize the cells according to the different axes, but don't actually generate positional identities in the regenerating cells. The authors provide two examples: Wnt7a as an example of positional information, and FGF8 as a positional cue. I think that coording to the authors definitions, FGF8 (and probobly Shh) are bone fide positional cues, since both signals work together to organize the regenerating limb cells - yet do not generate positional identities, because ectopic limbs formed from blastemas where these pathways have been activated do not regenerate (Nacu et al 2016). However, I am not sure Wnt7a constitutes an example of a "positional information" signal, since as far as I know, it has not been shown to generate stable dorsal limb identities (that remain after the signal has stopped) - at least yet. If it has, the authors should cite the paper that showed this. I think that some sort of diagram to help define these visually will be really helpful, especially to people who do not study regenerative patterning.

We thank the reviewer for this thoughtful comment. We now agree with the reviewer that our use of “positional cue” and “positional information” may have been confusing. In the revision—and as noted in our response to the Editor’s comment (4)—we have removed the term “positional cue” and no longer attempt to contrast it with “positional information.” Instead, we adopt phrasing that reflects our data and hypothesis: during limb patterning, dorsal-mediated signals act on ventral cells and ventral-mediated signals act on dorsal cells to induce Shh expression. This wording avoids implying that these signals specify dorsoventral identity.

Regarding WNT7A, we agree it has not been shown to generate a stable dorsal identity after signal withdrawal. In the revised Introduction we therefore describe WNT7A in amniote limb development as an extracellular regulator that induces Lmx1b in dorsal mesenchyme (with En1 repressing Wnt7a ventrally), rather than labeling it as “positional information” in a strict, identity-imprinting sense. We highlight this contrast because, in our axolotl experiments, WNT10B and FGF2 did not alter Lmx1b expression or dorsal–ventral limb characteristics when overexpressed, consistent with the idea that they act downstream of DV identity to enable Shh induction, not to establish DV identity.

(2) Results:

It would be helpful if the number of replicates per sample group were reported in the figure legends.

We thank the reviewer for this suggestion. In accordance with the comment, we have added the number of replicates (n) for each sample group in the figure legends.

Figure 2 shows ISH for A/P and D/V transcripts in different-positioned blastemas without tissue grafts. The images show interesting patterns, including the lack of Shh expression in all blastemas except in posterior-located blastemas, and localization of the dorsal transcript (Lmx1b) to the dorsal half of A or P located blastemas. My only concern about this data is that the expression patterns are described in only a small part of the ectopic blastema (how representative is it?) and the diagrams infer that these expression patterns are reflective of the entire blastema, which can't be determined by the limited field of view. It is okay if the expression patterns are not present in the entire blastema -in fact, that might be an important observation in terms of who is generating (and might be receiving) these signals.

We thank the reviewer for this insightful comment. Because Fgf8 and Shh expression was detectable only in a limited subset of cells, the original submission included only high-magnification images. In response to the reviewer’s valid concern about representativeness, we have now added low-magnification overviews of the entire blastema as a supplemental figure (Fig. S1) and clarified in the figure legend that these expression patterns can be focal rather than pan-blastemal (Line 795‒796).

In Figure 3, they look at all of these expression patterns in the grafted blastemas, showing that Shh expression is only visible when both D and V cells are present in the blastema. My only concern about this data is that the number of replicates is very low (some groups having only an N=3), and it is unclear how many sections the authors visualized for each replicate. This is especially important for the sample groups where they report no Shh expression -I agree that it is not observable in the single example sections they provide, but it is uncertain what is happening in other regions of the blastema.

We thank the reviewer for this important comment. To increase the reliability of the results, we have increased the number of biological replicates in groups where n was previously low. For all samples, we collected serial sections spanning the entire blastema. For blastemas in which Shh expression was observed, we present representative sections showing the signal. For blastemas without detectable Shh expression, we selected a section from the central region that contains GFP-positive cells for the Figure. To make these points explicit, we have added the following clarification to the Fig. 3 legend (Line 811‒815).

Figure 4: Shh overexpression in A/P/D/V blastemas - expression induces ectopic limbs in A/D/V locations. They analyzed the symmetry of these regenerates (assuming that Do and V located blastemas will exhibit D/V symmetry because they only contain cells from one side of that axis. I am a little concerned about how the symmetry assay is performed, since oblique sections through the digits could look asymmetric, while they are actually symmetric. It is also unclear how the angle of the boxes that the symmetry scores were based on was decided - I imagine that the score would change depending on the angle. It also appears that the authors picked different digits to perform this analysis on the different sample groups. I also admit that the logic of classification scheme that the authors used AI to perform their symmetry scoring analysis (both in Figures 4 and 5) is elusive to me. I think it would have been more informative if the authors leveraged the structural landmarks, like the localization of specific muscle groups. (If this experiment were performed in WT animals, the authors could have used pigment cell localization)... or generate more proximal sections to look at landmarks in the zeugopod.

We thank the reviewer for these detailed comments regarding the symmetry analysis. Because reliance on a computed symmetry score alone could raise the concerns noted by the reviewer, we now provide transverse sections along the proximodistal axis as supplemental figures (Figs. S2 and S4). These include levels corresponding to the distal end of the zeugopod and the proximal end of the autopod. In addition to reporting the symmetry score, we have explicitly stated in the text that symmetry was also assessed by visual inspection of these sections.

As also noted in our response to Reviewer #1 (comment 15), ALM-induced limbs frequently exhibit abnormal and highly variable morphologies, which makes it difficult to use consistent anatomical landmarks such as particular digits or muscle groups. For this reason, we focused our analysis on morphological symmetry rather than landmark-based metrics, and we emphasize this rationale in the revised text (Line 232‒235).

Regarding the use of bounding boxes, this procedure was chosen to minimize the effects of curvature or fixation-induced distortion. For each section, the box angle was adjusted so that the outer contour (epidermal surface) was aligned symmetrically; this procedure was applied uniformly across all conditions to avoid bias. We analyzed multiple biological replicates in each group, which helps mitigate potential artifacts due to oblique sectioning. To further reduce bias, we increased the number of fields included in the analysis to n = 24 per group in the revised version.

In addition, staining intensity varied among samples, such that a region identified as “muscle” in one sample could be assigned differently in another if classification were based solely on color. To avoid this problem, we used a machine-learning classifier trained separately for each sample, allowing us to group the same tissues consistently within that sample irrespective of intensity differences. In the context of ALM-induced limbs, where stable anatomical landmarks are not available, we consider this strategy the most appropriate. We have added this rationale to the revised manuscript for clarity (Line 239‒247).

Figure 5: The number of replicates in sample groups is relatively low and is quite variable between groups (ranging between 3 and 7 replicates). Zoom in to visualize Shh expression is small relative to the blastema, and it is difficult to discern why the authors positioned the window where they did, and how they maintained consistency among their different sample groups. In the examples of positive Shh expression - the signal is low and hard to see. Validating these expression patterns using some sort of quantitative transcriptional assay (like qRTPCR) would increase the rigor of this experiment ... especially given that they will be able to analyze gene expression in the entire blastema as opposed to sections that might not capture localized expression.

We thank the reviewer for this important comment. To increase the rigor of these experiments, we have increased the number of biological replicates in groups where n was previously low. In addition, because Shh signal in the Wnt10b-electroporated VentBL images was particularly weak and difficult to discern, we replaced that panel with a representative example in which Shh signal is more clearly visible. We also validated the Shh expression for Wnt10b–electroporated VentBL and Fgf2–electroporated DorBL by RT-qPCR, which assesses gene expression across the entire blastema. These results are now included in Fig. 5 and Line 280‒282. Finally, we clarified in the figure legend how the “window” for imaging was chosen: for samples with detectable Shh expression, the window was placed in the region where the signal was observed; for conditions without detectable Shh expression, the window was positioned in a comparable region containing GFP-positive cells (Line 836‒839). These revisions are included in the revised manuscript.

Figure 6: They treat dorsal and ventral wounds with gelatin beads soaked in a combination of BMP2+FGF8 (nerve factors) and FGF2 proposed ventral factor). Remarkably, they observe ectopic limb expression in only dorsal wounds, further supporting the idea that FGF2 provides the "ventral" signal. They show examples of this impressive phenotype on limbs with multiple ectopic structures that formed along the Pr/Di axis. Including images of tubulin staining (as they have in Figures 1 and 2) to ensure that the blastemas (or final regenerates) are devoid of nerves. The authors' whole-mount skeletal staining which shows fusion of the ectopic humerus with the host humerus, is a phenotype associated with deep wounding, which could provide an opportunity for more cellular contribution from different limb axes.

We thank the reviewer for these constructive comments. As noted in the prior study, when beads are used to induce blastemas without surgical nerve orientation, fine nerve ingrowth can still occur (Makanae et al., 2014), and the induced blastemas are not completely devoid of nerves. While it is still uncertain whether these recruited nerves are functional after blastema induction, it is an important point, and we added sentences about this in the revised manuscript (Line 341‒345).

Regarding the skeletal phenotype, despite careful implantation to avoid injuring deep tissues, bead-induced ectopic limbs on the dorsal side occasionally displayed fusion of the stylopod with the host humerus—a phenotype associated with deep wounding, as the reviewer notes. This observation suggests that contributions from a broader cellular population cannot be excluded. However, because fusion was observed in only 1 of 16 induced limbs analyzed, and because ectopic limbs induced at the forearm (zeugopod) level did not exhibit such fusion (n=1/6 for stylopod-level inductions; n=0/10 for zeugopod-level inductions), we believe that our main conclusion remains valid. Because fusion is not a typical outcome, we now present representative non-fusion cases—including zeugopod-origin examples—in the figure (Fig. 6L1, L2), and we report the fusion incidence explicitly in the text (Line 350‒354). We also note in the revised manuscript that stylopod fusion can occur in a minority of cases (Line 347‒349).

Figure 7 nicely summarizes their findings and model for patterning.

We thank the reviewer for this positive comment.

The table is cut off in the PDF, so it cannot be evaluated at this time.

In our copy of the PDF, the table appears in full, so this may have been a formatting issue. We have carefully checked the file and ensured that the table is completely included in the revised submission.

There is a supplemental figure that doesn't seem to be referenced in the text.

The supplemental figure (Fig. S1 of the original manuscript) is referenced in the text, but it may have been overlooked. To improve clarity, we have expanded the description in the manuscript so that the supplemental figure is more clearly referenced (Line 285‒291).

(3) Materials and Methods:

No power analysis was performed to calculate sample group sizes. The authors have used these experimental techniques in the past and could have easily used past data to inform these calculations.

We thank the reviewer for this important comment. We did not include a power analysis in the manuscript because this was the first time we compared Shh and other gene expression levels among ALM blastemas of different positional origins using RT-qPCR in our experimental system. As we did not have prior knowledge of the expected variability under these specific conditions, it was difficult to predetermine appropriate sample sizes.

Reviewer #3 (Recommendations for the authors):

General:

Congratulations - I found this an elegant and easy-to-read study with significant implications for the field! If possible, I would urge you to consider adding some more characterisation of Wnt10b and Fgf2- which cell types are they expressed in? If you can link your mechanisms to normal limb regeneration too (i.e., regenerating blastema, not ALM), this would significantly elevate the interest in your study.

We sincerely thank the reviewer for these encouraging comments. As also noted in our response to the editor’s comment, we have analyzed the expression patterns of Wnt10b and Fgf2 in regular blastemas (Line 294‒306). Although clear specific expression patterns along dorsoventral axis were not detected by ISH, likely due to technical limitations of sensitivity, RT-qPCR revealed significantly higher expression levels of Wnt10b in the dorsal half and Fgf2 in the ventral half of a regular blastema (Fig. S5). In addition, we analyzed published single-cell RNA-seq data (7 dpa blastema, Li et al., 2021) (Line 307‒321). As a result, Fgf2 expression was observed in the mesenchymal clusters, whereasWnt10b expression was observed in both mesenchymal and epithelial clusters (Fig. S6). However, because only a small fraction of cells expressed Wnt10b, the principal cellular source of WNT10B protein remains unclear. Therefore, defining the precise spatial patterns of Wnt10b and Fgf2 in regular regeneration will be an important goal for future work.

Data availability:

I assume that the RNA-sequencing data will be deposited at a public repository.

RNA-seq FASTQ files have been deposited in the DNA Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/) under BioProject accession PRJDB38065. We have added a Data availability section to the revised manuscript.

References

Castilla-Ibeas, A., Zdral, S., Oberg, K. C., & Ros, M. A. (2024). The limb dorsoventral axis: Lmx1b’s role in development, pathology, evolution, and regeneration. Developmental Dynamics, 253(9), 798–814. https://doi.org/10.1002/dvdy.695

Johnson, G. L., Glasser, M. B., Charles, J. F., Duryea, J., & Lehoczky, J. A. (2022). En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration. Cell Reports, 41(8), 111701. https://doi.org/10.1016/j.celrep.2022.111701

Stocum, D. (2017). Mechanisms of urodele limb regeneration. Regeneration, 4. https://doi.org/10.1002/reg2.92

Tank, P. W., & Holder, N. (1978). The effect of healing time on the proximodistal organization of double-half forelimb regenerates in the axolotl, Ambystoma mexicanum. Developmental Biology, 66(1), 72–85. https://doi.org/10.1016/0012-1606(78)90274-9

spot on

forget blockchain use timechain as the permanent anchor

eLife Assessment

This study examines an important question regarding the developmental trajectory of neural mechanisms supporting facial expression processing. Leveraging a rare intracranial EEG (iEEG) dataset including both children and adults, the authors reported that facial expression recognition mainly engaged the posterior superior temporal cortex (pSTC) among children, while both pSTC and the prefrontal cortex were engaged among adults. In terms of strength of evidence, the solid methods, data and analyses broadly support the claims with minor weaknesses.

28 января 1986 года. Взрыв космическог корабля 11:30 утра. полаю вот с какого события взяли сюжетку для the alters.

они 5 дней пытались взлететь. то погода не та, то холодно, то вся платформа замёрзла за ночь.

одна из участников была простая учительница, Которая выиграла конкурс. хотя недавно он ои снизил фенансирвоания.

Шатлы начали строить по причине, того, что никсон захотел шатл, который даст возможность перелетать с одного место в другое, без ракет и сложных платфор для старта.

в тоже время военные хотели, чтоб он был транспортом с ангарам, планы конечно космические, но выбора нету - им за это деньги дают.

с другой стороны экономисты вкинули, что эти шатлы должны окупиться за 20 лет с 300 полётами.

думали, что какая же это выгодная штука. другие страны могли бы с ними договариваться и вот деньги.

кароче, чистая фантазия

никто кроме наса не понимал, что это не возможно, но выбора не было. надо делать.

в последствие этого им приходилось экономить на деталях. не добавлять двигатель к шатлу. Ракеты, были сделаны не литтые, а фрагментированные

у них были кресла катапультирования. летишь такой на скорости звука и такой, а вот бы сейчас катапультироваться.

по итогу это были ваще не выгодные штуки, и никому это нафиг не сдалось, но их дальше запускали.

но в какой-то момент, когда они уже на расслабоне делали пошло всё на смарку и произошле взрыв. мгновенно всё испралиось, и ракеты, который должны были отцыпиться от шатла, когда кончаться просто летели дальше одни.

по итогу Около 4 месяцев один доставали из дна окена кусочки людей в костюмах, используя самые новороченные средства для погружения. даже подводные лодки были использованы. и около 10000 человек.

31:47 - картинки.

кароче, Фейман в своей манере погнал без просу в наса исселедовать что вообще как работает.

ну и оказалось, что там резинки между фрагментами ракеты подозрительно не надежные. то поттекает, что-то.

по итогу долгих бесед, встреч и т.д

фейман показал, на примере резины из модели корабля, что резина при нулевой температуре уже может терять свою упругость, что критично. и другие не дочёты строения корабля

по итогу феймана и его компания сказали, что виноватые есть.

фейман искал причинность всё глубже и глубже. но мы забываем, что НАСА создала буквально эксперементабельный апарат с бесконечнымм количеством переменных, свойств. их все просто не возможно было учесть.

в системе было много не доработок, не дочётов, и это было окей, учитвая, что бюжет для постройки был не бесконечный, были планы, ограничения.

в любой момент подобная ситуация должна была случиться просто из-за своей природы. аппарт не был идеальным.

это бы всё равно случилось бы рано или поздно, но это или другой причине

eLife Assessment

This revised paper provides a valuable and novel neural network-based framework for parameterizing individual differences and predicting individual decision-making across task conditions. The methods and analyses are solid yet could benefit from further validation of the superiority of the proposed framework against other baseline models. With these concerns addressed, this study would offer a proof-of-concept neural network approach to scientists working on the generalization of cognitive skills across contexts.

Reviewer #1 (Public review):

Summary

The manuscript presents EIDT, a framework that extracts an "individuality index" from a source task to predict a participant's behaviour in a related target task under different conditions. However, the evidence that it truly enables cross-task individuality transfer is not convincing.

Strengths

The EIDT framework is clearly explained, and the experimental design and results are generally well-described. The performance of the proposed method is tested on two distinct paradigms: a Markov Decision Process (MDP) task (comparing 2-step and 3-step versions) and a handwritten digit recognition (MNIST) task under various conditions of difficulty and speed pressure. The results indicate that the EIDT framework generally achieved lower prediction error compared to baseline models and that it was better at predicting a specific individual's behaviour when using their own individuality index compared to using indices from others.

Furthermore, the individuality index appeared to form distinct clusters for different individuals, and the framework was better at predicting a specific individual's behaviour when using their own derived index compared to using indices from other individuals.

Comments on revisions:

I thank the author for the additional analyses. They have fully addressed all of my previous concerns, and I have no further recommendations.

Reviewer #2 (Public review):

This paper introduces a framework for modeling individual differences in decision-making by learning a low-dimensional representation (the "individuality index") from one task and using it to predict behaviour in a different task. The approach is evaluated on two types of tasks: a sequential value-based decision-making task and a perceptual decision task (MNIST). The model shows improved prediction accuracy when incorporating this learned representation compared to baseline models.

The motivation is solid, and the modelling approach is interesting, especially the use of individual embeddings to enable cross-task generalization. That said, several aspects of the evaluation and analysis could be strengthened.

(1) The MNIST SX baseline appears weak. RTNet isn't directly comparable in structure or training. A stronger baseline would involve training the GRU directly on the task without using the individuality index-e.g., by fixing the decoder head. This would provide a clearer picture of what the index contributes.

(2) Although the focus is on prediction, the framework could offer more insight into how behaviour in one task generalizes to another. For example, simulating predicted behaviours while varying the individuality index might help reveal what behavioural traits it encodes.

(3) It's not clear whether the model can reproduce human behaviour when acting on-policy. Simulating behaviour using the trained task solver and comparing it with actual participant data would help assess how well the model captures individual decision tendencies.

(4) Figures 3 and S1 aim to show that individuality indices from the same participant are closer together than those from different participants. However, this isn't fully convincing from the visualizations alone. Including a quantitative presentation would help support the claim.

(5) The transfer scenarios are often between very similar task conditions (e.g., different versions of MNIST or two-step vs three-step MDP). This limits the strength of the generalization claims. In particular, the effects in the MNIST experiment appear relatively modest, and the transfer is between experimental conditions within the same perceptual task. To better support the idea of generalizing behavioural traits across tasks, it would be valuable to include transfers across more structurally distinct tasks.

(6) For both experiments, it would help to show basic summaries of participants' behavioural performance. For example, in the MDP task, first-stage choice proportions based on transition types are commonly reported. These kinds of benchmarks provide useful context.

(7) For the MDP task, consider reporting the number or proportion of correct choices in addition to negative log-likelihood. This would make the results more interpretable.

(8) In Figure 5, what is the difference between the "% correct" and "% match to behaviour"? If so, it would help to clarify the distinction in the text or figure captions.

(9) For the cognitive model, it would be useful to report the fitted parameters (e.g., learning rate, inverse temperature) per individual. This can offer insight into what kinds of behavioural variability the individuality index might be capturing.

(10) A few of the terms and labels in the paper could be made more intuitive. For example, the name "individuality index" might give the impression of a scalar value rather than a latent vector, and the labels "SX" and "SY" are somewhat arbitrary. You might consider whether clearer or more descriptive alternatives would help readers follow the paper more easily.

(11) Please consider including training and validation curves for your models. These would help readers assess convergence, overfitting, and general training stability, especially given the complexity of the encoder-decoder architecture.

Comments on revisions:

Thank you to the authors for the updated manuscript. The authors have addressed the majority of my concerns, and the paper is now in a much better form.

Regarding my previous Comment 6, I still believe it would be helpful to include a graph similar to what is typically reported for these tasks-specifically, a breakdown of choices based on rare versus common transitions (see Model-Based Influences on Humans' Choices and Striatal Prediction Errors, Figure 2). Presenting this for both the actual behaviour and the simulated data would strengthen the paper and allow for clearer comparison.

Reviewer #3 (Public review):

Summary:

This work presents a novel neural network-based framework for parameterizing individual differences in human behavior. Using two distinct decision-making experiments, the author demonstrates the approach's potential and claims it can predict individual behavior (1) within the same task, (2) across different tasks, and (3) across individuals. While the goal of capturing individual variability is compelling and the potential applications are promising, the claims are weakly supported, and I find that the underlying problem is conceptually ill-defined.

Strengths:

The idea of using neural networks for parameterizing individual differences in human behavior is novel, and the potential applications can be impactful.

Weaknesses:

(1) To demonstrate the effectiveness of the approach, the authors compare a Q-learning cognitive model (for the MDP task) and RTNet (for the MNIST task) against the proposed framework. However, as I understand it, neither the cognitive model nor RTNet is designed to fit or account for individual variability. If that is the case, it is unclear why these models serve as appropriate baselines. Isn't it expected that a model explicitly fitted to individual data would outperform models that do not? If so, does the observed superiority of the proposed framework simply reflect the unsurprising benefit of fitting individual variability? I think the authors should either clarify why these models constitute fair control or validate the proposed approach against stronger and more appropriate baselines.

(2) It's not very clear in the results section what it means by having a shorter within-individual distance than between-individual distances. Related to the comment above, is there any control analysis performed for this? Also, this analysis appears to have nothing to do with predicting individual behavior. Is this evidence toward successfully parameterizing individual differences? Could this be task-dependent, especially since the transfer is evaluated on exceedingly similar tasks in both experiments? I think a bit more discussion of the motivation and implications of these results will help the reader in making sense of this analysis.

(3) The authors have to better define what exactly he meant by transferring across different "tasks" and testing the framework in "more distinctive tasks". All presented evidence, taken at face value, demonstrated transferring across different "conditions" of the same task within the same experiment. It is unclear to me how generalizable the framework will be when applied to different tasks.

(4) Conceptually, it is also unclear to me how plausible it is that the framework could generalize across tasks spanning multiple cognitive domains (if that's what is meant by more distinctive). For instance, how can an individual's task performance on a Posner task predict task performance on the Cambridge face memory test? Which part of the framework could have enabled such a cross-domain prediction of task performance? I think these have to be at least discussed to some extent, since without it the future direction is meaningless.

(5) How is the negative log-likelihood, which seems to be the main metric for comparison, computed? Is this based on trial-by-trial response prediction or probability of responses, as what usually performed in cognitive modelling?

(6) None of the presented evidence is cross-validated. The authors should consider performing K-fold cross-validation on the train, test, and evaluation split of subjects to ensure robustness of the findings.

(7) The authors excluded 25 subjects (20% of the data) for different reasons. This is a substantial proportion, especially by the standards of what is typically observed in behavioral experiments. The authors should provide a clear justification for these exclusion criteria and, if possible, cite relevant studies that support the use of such stringent thresholds.

(8) The authors should do a better job of creating the figures and writing the figure captions. It is unclear which specific claim the authors are addressing with the figure. For example, what is the key message of Figure 2C regarding transfer within and across participants? Why are the stats presentation different between the Cognitive model and the EIDT framework plots? In Figure 3, it's unclear what these dots and clusters represent and how they support the authors' claim that the same individual forms clusters. And isn't this experiment have 98 subjects after exclusion, this plot has way less than 98 dots as far as I can tell. Furthermore, I find Figure 5 particularly confusing, as the underlying claim it is meant to illustrate is unclear. Clearer figures and more informative captions are needed to guide the reader effectively.

(9) I also find the writing somewhat difficult to follow. The subheadings are confusing, and it's often unclear which specific claim the authors are addressing. The presentation of results feels disorganized, making it hard to trace the evidence supporting each claim. Also, the excessive use of acronyms (e.g., SX, SY, CG, EA, ES, DA, DS) makes the text harder to parse. I recommend restructuring the results section to be clearer and significantly reducing the use of unnecessary acronyms.

Comments on revisions:

The authors have addressed my previous comments with great care and detail. I appreciate the additional analyses and edits. I have no further comments.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

Because the "source" and "target" tasks are merely parameter variations of the same paradigm, it is unclear whether EIDT achieves true crosstask transfer. The manuscript provides no measure of how consistent each participant's behaviour is across these variants (e.g., two- vs threestep MDP; easy vs difficult MNIST). Without this measure, the transfer results are hard to interpret. In fact, Figure 5 shows a notable drop in accuracy when transferring between the easy and difficult MNIST conditions, compared to transfers between accuracy-focused and speedfocused conditions. Does this discrepancy simply reflect larger withinparticipant behavioural differences between the easy and difficult settings? A direct analysis of intra-individual similarity for each task pair and how that similarity is related to EIDT's transfer performance is needed.

Thank you for your insightful comment. We agree that the tasks used in our study are variations of the same paradigm. Accordingly, we have revised the manuscript to consistently frame our findings as demonstrating individuality transfer "across task conditions" rather than "across distinct tasks."

In response to your suggestion, we have conducted a new analysis to directly investigate the relationship between individual behavioural patterns and transfer performance. As show in the new Figures 4, 11, S8, and S9, we found a clear relationship between the distance in the space of individual latent representation (called individuality index in the previous manuscript) and prediction performance. Specifically, prediction accuracy for a given individual's behaviour degrades as the latent representation of the model's source individual becomes more distant. This result directly demonstrates that our framework captures meaningful individual differences that are predictive of transfer performance across conditions.

We have also expanded the Discussion (Lines 332--343) to address the potential for applying this framework to more structurally distinct tasks, hypothesizing that this would rely on shared underlying cognitive functions.

Related to the previous comment, the individuality index is central to the framework, yet remains hard to interpret. It shows much greater within-participant variability in the MNIST experiment (Figure S1) than in the MDP experiment (Figure 3). Is such a difference meaningful? It is hard to know whether it reflects noisier data, greater behavioural flexibility, or limitations of the model.

Thank you for raising this important point about interpretability. To enhance the interpretability of the individual latent representation, we have added a new analysis for the MDP task (see Figures 6 and S4). By applying our trained encoder to data from simulated Q-learning agents with known parameters, we demonstrate that the dimensions of the latent space systematically map onto the agents' underlying cognitive parameters (learning rate and inverse temperature). This analysis provides a clearer interpretation by linking our model's data-driven representation to established theoretical constructs.

Regarding the greater within-participant variability observed in the MNIST task (visualized now in Figure S7), this could be attributed to several factors, such as greater behavioural flexibility in the perceptual task. However, disentangling these potential factors is complex and falls outside the primary scope of the current study, which prioritizes demonstrating robust prediction accuracy across different task conditions.

The authors suggests that the model's ability to generalize to new participants "likely relies on the fact that individuality indices form clusters and individuals similar to new participants exist in the training participant pool". It would be helpful to directly test this hypothesis by quantifying the similarity (or distance) of each test participant's individuality index to the individuals or identified clusters within the training set, and assessing whether greater similarity (or closer proximity) to the clusters in the training set is associated with higher prediction accuracy for those individuals in the test set.

Thank you for this excellent suggestion. We have performed the analysis you proposed to directly test this hypothesis. Our new results, presented in Figures 4, 11, S5, S8, and S9, quantify the distance between the latent representation of a test participant and that of the source participant used to generate the prediction model.

The results show a significant negative correlation: prediction accuracy consistently decreases as the distance in the latent space increases. This confirms that generalization performance is directly tied to the similarity of behavioural patterns as captured by our latent representation, strongly supporting our hypothesis.

Reviewer #2 (Public review):

The MNIST SX baseline appears weak. RTNet isn't directly comparable in structure or training. A stronger baseline would involve training the GRU directly on the task without using the individuality index-e.g., by fixing the decoder head. This would provide a clearer picture of what the index contributes.

We agree that a more direct baseline is crucial for evaluating the contribution of our transfer mechanism. For the Within-Condition Prediction scenario, the comparison with RTNet was intended only to validate that our task solver architecture could achieve average humanlevel task performance (Figure 7).

For the critical Cross-Condition Transfer scenario, we have now implemented a stronger and more appropriate baseline, which we call ``task solver (source).'' This model has the same architecture as our EIDT task solver but is trained directly on the source task data of the specific test participant. As shown in revised Figure 9, our EIDT framework significantly outperforms this direct-training approach, clearly demonstrating the benefit of the individuality transfer mechanism.

Although the focus is on prediction, the framework could offer more insight into how behaviour in one task generalizes to another. For example, simulating predicted behaviours while varying the individuality index might help reveal what behavioural traits it encodes.

Thank you for this valuable suggestion. To provide more insight into the encoded behavioural traits, we have conducted a new analysis linking the individual latent representation to a theoretical cognitive model. As detailed in the revised manuscript (Figures 6 and S4), we applied our encoder to simulated data from Q-learning agents with varying parameters. The results show a systematic relationship between the latent space coordinates and the agents' learning rates and inverse temperatures, providing a clearer interpretation of what the representation captures.

It's not clear whether the model can reproduce human behaviour when acting on-policy. Simulating behaviour using the trained task solver and comparing it with actual participant data would help assess how well the model captures individual decision tendencies.

We have added the suggested on-policy evaluation (Lines 195--207). In the revised manuscript (Figure 5), we present results from simulations where the trained task solvers performed the MDP task. We compared their performance (total reward and rate of the highly-rewarding action selected) against their corresponding human participants. The strong correlations observed demonstrate that our model successfully captures and reproduces individual-specific behavioural tendencies in an onpolicy setting.

Figures 3 and S1 aim to show that individuality indices from the same participant are closer together than those from different participants. However, this isn't fully convincing from the visualizations alone. Including a quantitative presentation would help support the claim.

We agree that the original visualizations of inter- and intraparticipant distances was not sufficiently convincing. We have therefore removed that analysis. In its place, we have introduced a more direct and quantitative analysis that explicitly links the individual latent representation to prediction performance (see Figures 4, 11, S5, S8, and S9). This new analysis demonstrates that prediction error for an individual is a function of distance in the latent space, providing stronger evidence that the representation captures meaningful, individual-specific information.

The transfer scenarios are often between very similar task conditions (e.g., different versions of MNIST or two-step vs three-step MDP). This limits the strength of the generalization claims. In particular, the effects in the MNIST experiment appear relatively modest, and the transfer is between experimental conditions within the same perceptual task. To better support the idea of generalizing behavioural traits across tasks, it would be valuable to include transfers across more structurally distinct tasks.

We agree with this limitation and have revised the manuscript to be more precise. We now frame our contribution as "individuality transfer across task conditions" rather than "across tasks" to accurately reflect the scope of our experiments. We have also expanded the Discussion section (Line 332-343) to address the potential and challenges of applying this framework to more structurally distinct tasks, noting that it would likely depend on shared underlying cognitive functions.

For both experiments, it would help to show basic summaries of participants' behavioural performance. For example, in the MDP task, first-stage choice proportions based on transition types are commonly reported. These kinds of benchmarks provide useful context.

We have added behavioral performance summaries as requested. For the MDP task, Figure 5 now compares the total reward and rate of highlyrewarding action selected between humans and our model. For the MNIST task, Figure 7 shows the rate of correct responses for humans, RTNet, and our task solver across all conditions. These additions provide better context for the model's performance.

For the MDP task, consider reporting the number or proportion of correct choices in addition to negative log-likelihood. This would make the results more interpretable.

Thank you for the suggestion. To make the results more interpretable, we have added a new prediction performance metric: the rate for behaviour matched. This metric measures the proportion of trials where the model's predicted action matches the human's actual choice. This is now included alongside the negative log-likelihood in Figures 2, 3, 4, 8, 9, and 11.

In Figure 5, what is the difference between the "% correct" and "% match to behaviour"? If so, it would help to clarify the distinction in the text or figure captions.

We have clarified these terms in the revised manuscript. As defined in the Result section (Lines 116--122, 231), "%correct" (now "rate of correct responses") is a measure of task performance, whereas "%match to behaviour" (now "rate for behaviour matched") is a measure of prediction accuracy.

For the cognitive model, it would be useful to report the fitted parameters (e.g., learning rate, inverse temperature) per individual. This can offer insight into what kinds of behavioural variability the individual latent representation might be capturing.

We have added histograms of the fitted Q-learning parameters for the human participants in Supplementary Materials (Figure S1). This analysis revealed which parameters varied most across the population and directly informed the design of our subsequent simulation study with Q-learning agents (see response to Comment 2-2), where we linked these parameters to the individual latent representation (Lines 208--223).

A few of the terms and labels in the paper could be made more intuitive. For example, the name "individuality index" might give the impression of a scalar value rather than a latent vector, and the labels "SX" and "SY" are somewhat arbitrary. You might consider whether clearer or more descriptive alternatives would help readers follow the paper more easily.

We have adopted the suggested changes for clarity.

"Individuality index" has been changed to "individual latent representation".

"Situation SX" and "Situation SY" have been renamed to the more descriptive "Within-Condition Prediction" and "Cross-Condition Transfer", respectively.

We have also added a table in Figure 7 to clarify the MNIST condition acronyms (EA/ES/DA/DS).

Please consider including training and validation curves for your models. These would help readers assess convergence, overfitting, and general training stability, especially given the complexity of the encoder-decoder architecture.

Training and validation curves for both the MDP and MNIST tasks have been added to Supplementary Materials (Figure S2 and S6) to show model convergence and stability.

Reviewer #3 (Public review):

To demonstrate the effectiveness of the approach, the authors compare a Q-learning cognitive model (for the MDP task) and RTNet (for the MNIST task) against the proposed framework. However, as I understand it, neither the cognitive model nor RTNet is designed to fit or account for individual variability. If that is the case, it is unclear why these models serve as appropriate baselines. Isn't it expected that a model explicitly fitted to individual data would outperform models that do not? If so, does the observed superiority of the proposed framework simply reflect the unsurprising benefit of fitting individual variability? I think the authors should either clarify why these models constitute fair control or validate the proposed approach against stronger and more appropriate baselines.

Thank you for raising this critical point. We wish to clarify the nature of our baselines:

For the MDP task, the cognitive model baseline was indeed designed to account for individual variability. We estimated its parameters (e.g., learning rate) from each individual's source task behaviour and then used those specific parameters to predict their behaviour in the target task. This makes it a direct, parameter-based transfer model and thus a fair and appropriate baseline for individuality transfer.

For the MNIST task, we agree that the RTNet baseline was insufficient for evaluating individual-level transfer in the "Cross-Condition Transfer" scenario. We have now introduced a much stronger baseline, the "task solver (source)," which is trained specifically on the source task data of each test participant. Our results (Figure 9) show that the EIDT framework significantly outperforms this more appropriate, individualized baseline, highlighting the value of our transfer method over direct, within-condition fitting.

It's not very clear in the results section what it means by having a shorter within-individual distance than between-individual distances. Related to the comment above, is there any control analysis performed for this? Also, this analysis appears to have nothing to do with predicting individual behavior. Is this evidence toward successfully parameterizing individual differences? Could this be task-dependent, especially since the transfer is evaluated on exceedingly similar tasks in both experiments? I think a bit more discussion of the motivation and implications of these results will help the reader in making sense of this analysis.

We agree that the previous analysis on inter- and intra-participant distances was not sufficiently clear or directly linked to the model's predictive power. We have removed this analysis from the manuscript. In its place, we have introduced a new, more direct analysis (Figures 4, 11, S5, S8, and S9) that demonstrates a quantitative relationship between the distance in the latent space and prediction accuracy. This new analysis shows that prediction error for an individual increases as a function of this distance, providing much stronger and clearer evidence that our framework successfully parameterizes meaningful individual differences.

The authors have to better define what exactly he meant by transferring across different "tasks" and testing the framework in "more distinctive tasks". All presented evidence, taken at face value, demonstrated transferring across different "conditions" of the same task within the same experiment. It is unclear to me how generalizable the framework will be when applied to different tasks.

Conceptually, it is also unclear to me how plausible it is that the framework could generalize across tasks spanning multiple cognitive domains (if that's what is meant by more distinctive). For instance, how can an individual's task performance on a Posner task predict task performance on the Cambridge face memory test? Which part of the framework could have enabled such a cross-domain prediction of task performance? I think these have to be at least discussed to some extent, since without it the future direction is meaningless.

We agree with your assessment and have corrected our terminology throughout the manuscript. We now consistently refer to the transfer as being "across task conditions" to accurately describe the scope of our findings.

We have also expanded our Discussion (Line 332-343) to address the important conceptual point about cross-domain transfer. We hypothesize that such transfer would be possible if the tasks, even if structurally different, rely on partially shared underlying cognitive functions (e.g., working memory). In such a scenario, the individual latent representation would capture an individual's specific characteristics related to that shared function, enabling transfer. Conversely, we state that transfer between tasks with no shared cognitive basis would not be expected to succeed with our current framework.

How is the negative log-likelihood, which seems to be the main metric for comparison, computed? Is this based on trial-by-trial response prediction or probability of responses, as what usually performed in cognitive modelling?

The negative log-likelihood is computed on a trial-by-trial basis. It is based on the probability the model assigned to the specific action that the human participant actually took on that trial. This calculation is applied consistently across all models (cognitive models, RTNet, and EIDT). We have added sentences to the Results section to clarify this point (Lines 116--122).

None of the presented evidence is cross-validated. The authors should consider performing K-fold cross-validation on the train, test, and evaluation split of subjects to ensure robustness of the findings.

All prediction performance results reported in the revised manuscript are now based on a rigorous leave-one-participant-out cross-validation procedure to ensure the robustness of our findings. We have updated the

Methods section to reflect this (Lines 127--129 and 229).

For some purely illustrative visualizations (e.g., plotting the entire latent space in Figures S3 and S7), we used a model trained on all participants' data to provide a single, representative example and avoid clutter. We have explicitly noted this in the relevant figure captions.

The authors excluded 25 subjects (20% of the data) for different reasons. This is a substantial proportion, especially by the standards of what is typically observed in behavioral experiments. The authors should provide a clear justification for these exclusion criteria and, if possible, cite relevant studies that support the use of such stringent thresholds.

We acknowledge the concern regarding the exclusion rate. The previous criteria were indeed empirical. We have now implemented more systematic exclusion procedure based on the interquartile range of performance metrics, which is detailed in Section 4.2.2 (Lines 489--498). This revised, objective criterion resulted in the exclusion of 42 participants (34% of the initial sample). While this rate is high, we attribute it to the online nature of the data collection, where participant engagement can be more variable. We believe applying these strict criteria was necessary to ensure the quality and reliability of the behavioural data used for modeling.

The authors should do a better job of creating the figures and writing the figure captions. It is unclear which specific claim the authors are addressing with the figure. For example, what is the key message of Figure 2C regarding transfer within and across participants? Why are the stats presentation different between the Cognitive model and the EIDT framework plots? In Figure 3, it's unclear what these dots and clusters represent and how they support the authors' claim that the same individual forms clusters. And isn't this experiment have 98 subjects after exclusion, this plot has way less than 98 dots as far as I can tell. Furthermore, I find Figure 5 particularly confusing, as the underlying claim it is meant to illustrate is unclear. Clearer figures and more informative captions are needed to guide the reader effectively.

We agree that several figures and analyses in the original manuscript were unclear, and we have thoroughly revised our figures and their captions to improve clarity.

The confusing analysis in the old Figures 2C and 5 (Original/Others comparison) have been completely removed. The unclear visualization of the latent space for the test pool (old Figure 3 showing representations only from test participants) has also been removed to avoid confusion. For visualization of the overall latent space, we now use models trained on all data (Figures S3 and S7) and have clarified this in the captions. In place of these removed analyses, we have introduced a new, more intuitive "cross-individual" analysis (presented in Figures 4, 11, S5, S8, and S9). As explained in the new, more detailed captions, this analysis directly plots prediction performance as a function of the distance in latent space, providing a much clearer demonstration of how the latent representation relates to predictive accuracy.

I also find the writing somewhat difficult to follow. The subheadings are confusing, and it's often unclear which specific claim the authors are addressing. The presentation of results feels disorganized, making it hard to trace the evidence supporting each claim. Also, the excessive use of acronyms (e.g., SX, SY, CG, EA, ES, DA, DS) makes the text harder to parse. I recommend restructuring the results section to be clearer and significantly reducing the use of unnecessary acronyms.

Thank you for this feedback. We have made significant revisions to improve the clarity and organization of the manuscript. We have renamed confusing acronyms: "Situation SX" is now "Within- Condition Prediction," and "Situation SY" is now "Cross-Condition Transfer." We also added a table to clarify the MNIST condition acronyms (EA/ES/DA/DS) in Figure 7.

The Results section has been substantially restructured with clearer subheadings.

Regarding the decline in age-standardized incidence rates, we expect that as diagnostic tools improve and early detection advances, more cases will be identified, which may lead to an increase in this indicator. I think it might be better to relate this factor to the improvement of preventive strategies.

Results currently under-discussed. - State outliers in Table 1 not discussed; New York largest declines; New Mexico's significant male prevalence increase; briefly discuss why these states merit targeted evaluation. - Non-fatal burden (YLDs) is absent from the discussion.

This study does not quantify risk-factor contributions. I think it might be better to shorten this paragraph to 1–2 key examples and remove non-essential details.

eLife Assessment

This study presents compelling new data that combine two FTD-tau mutations, P301L/S320F (PL-SF), that reliably induce spontaneous full-length tau aggregation across multiple cellular systems. The findings are important for the field of neurodegenerative disease. The strength of evidence is solid; however, several conclusions would benefit from more validation.

Reviewer #1 (Public review):

Summary:

This study presents compelling new data that combine two FTD-tau mutations P301L/S320F (PL-SF), that reliably induce spontaneous full-length tau aggregation across multiple cellular systems. However, several conclusions would benefit from more validation. Key findings rely on quantification of overexposed immunoblot, and in some experiments, the tau bands shift in molecular weight that are not explained (and in some instances vary between experiments). The effect seems to be driven by the S320F mutation, with the P301L mutation enhancing the effect observed with S320F alone. Although the observation that Hsp70, but not the related Hsc70, enhances aggregation is intriguing, the mechanistic basis for these differences remains unclear despite both Hsp70 and Hsc70 binding to tau. Additional experiments clarifying which PL-SF tau species Hsp70 engages, how this interaction alters tau conformational landscapes, and whether other chaperones or cofactors contribute to this effect would help solidify the conclusions and build a mechanistic picture. Overexpression of Hsp70 in the context of PL tau did not increase tau aggregation, which raises questions about whether the observed effects are specific to the SF mutation. Hsp70 functions in the context of a larger network of chaperones and has been proposed to cooperate with other proteins/machinery to disassemble tau amyloids, perhaps to produce more seeds. This would be consistent with the presented observations. For example, co-IP experiments using Hsp70 as bait combined with proteomics could really help build a more complete picture of what tau species Hsp70 binds and what other factors cooperate to yield the observed increases in aggregation. As it stands, the Hsp70 component of the paper is not fully developed, and additional experiments to address these questions would strengthen this manuscript beyond simply presenting a new tool to study spontaneous tau aggregation.

Strengths:

(1) The PL-SF FL tau mutant aggregates spontaneously in different cellular systems and shows hallmarks of tau pathology linked to disease.

(2) PL-SF 4delta mutant reverses the spontaneous aggregation phenotype, consistent with these residues being critical for tau aggregation.

(3) PL-SF 4delta also loses the ability to recruit Hsp70/Hsc70, consistent with these residues also being critical for chaperone recruitment.

(4) The PL-SF tau mutant establishes a new system to study spontaneous tau assembly and to begin to compare it to seeded tau aggregation processes.

Weaknesses:

(1) Mechanistic insight into how Hsp70 but not Hsc70 increase PL-SF FL tau aggregation/pathology is missing. This is despite both chaperones binding to PL-SF FL tau. What species of tau does Hsp70 bind, and what cofactors are important in this process?

(2) The study relies heavily on densitometry of bands to draw conclusions; in several instances, the blots are overexposed to accurately quantify the signal.

Reviewer #2 (Public review):

Summary:

This study developed a novel tauopathy model combining two mutations, P301L and S320F, termed the PL-SF model. This model shows rapid tau protein aggregation.

Strengths:

The authors demonstrated pathogenicity through solid in vivo and in vitro experiments. Simultaneously, they used this model to investigate the role of the heat shock protein Hsp70 in tau protein aggregation, finding that Hsp70 promotes rather than inhibits tau pathology, which differs from previous findings.

Weaknesses:

(1) Although the PL-SF model can accelerate tau aggregation, it is crucial to determine whether this aligns with the temporal progression and spatial distribution of tau pathology in the brains of patients with tauopathies.

(2) The authors did not elucidate the specific molecular mechanism by which Hsp70 promotes tau aggregation.

(3) Some figures in this study show large error bars in the quantitative data (some statistical analysis figures, MEA recordings, etc.), indicating significant inter-sample variability. It is recommended to label individual data points in all quantitative figures and clearly indicate them in figure legends.

Author response:

Reviewer #1

(1) Mechanistic insight into how Hsp70 but not Hsc70 increase PL-SF FL tau aggregation/pathology is missing. This is despite both chaperones binding to PL-SF FL tau. What species of tau does Hsp70 bind, and what cofactors are important in this process?

We agree that explaining why Hsp70, but not Hsc70, promotes tau aggregation would strengthen the study. Although both chaperones bind tau, they diverge slightly in 1) protein sequence, 2) biochemical activity, and 3) co-chaperone engagement.

Sequence: Hsp70 has an extra cysteine residue (Cys306) that is highly reactive to oxidation and a glycine residue that is critical for cysteine oxidation (Gly557). Both residues are specific to Hsp70 (not present in Hsc70) and may alter Hsp70 conformation or client handling (Hong et al., 2022).

Biochemical activity: Prior studies indicate that Hsp70’s ATPase domain (NBD) is critical for tau interactions (Jinwal et al., 2009; Fontaine et al., 2015; Young et al., 2016) and can be disrupted with point mutations including K71E and E175S for ATPase and A406G/V438G for substrate binding (Fontaine et al., 2015).

Co-chaperone engagement: Hsp70 recruits the co-chaperone and E3 ubiquitin ligase CHIP/Stub1 more strongly than Hsc70, suggesting co-chaperone engagement could lead to differences in tau processing (Jinwal et al., 2013).

To directly test how the two closely related chaperones could differentially impact tau, we plan to perform the following experiments:

(a) We will mutate residues responsible for cysteine reactivity in Hsp70 including the cysteine itself (Cys306) and the critical glycine that facilitates cysteine reactivity (Gly557). These residues will be deleted from Hsp70 or alternatively inserted into Hsc70 to determine whether cysteine reactivity is the reason for Hsp70’s ability to drive tau aggregation.

(b) We will generate Hsp70 mutants lacking ATPase- or substrate-binding mutants to determine which Hsp70 domains are responsible for driving tau aggregation.

(c) We will perform seeding assays in stable tau-expressing cell lines to determine whether Hsp70/Hsc70 overexpression or depletion alters seeded tau aggregation.

(d) We will perform confocal microscopy to determine the extent of co-localization of Hsp70 or Hsc70 with phospho-tau, oligomeric tau, or Thioflavin-S (ThioS) to identify which tau species are engaged by Hsp70/Hsc70.

(e) We will perform immunoprecipitation pull-downs followed by mass spectrometry to globally identify any relevant Hsp70/Hsc70 interacting factors that might account for the differences in tau aggregation.

(2) The study relies heavily on densitometry of bands to draw conclusions; in several instances, the blots are overexposed to accurately quantify the signal.

All immunoblots were acquired as 16-bit TIFFs with exposure settings chosen to prevent pixel saturation, and quantification was performed on raw, unsaturated images. Brightness and contrast adjustments were applied only for visualization and did not alter pixel values used for analysis. All quantified bands fell within the linear range of the detector, with one exception in Figure 7B, which we removed from quantification. We will add both low- and high-exposure versions of immunoblots to the revised figures to demonstrate signal linearity and dynamic range.

Reviewer #2

(1) Although the PL-SF model can accelerate tau aggregation, it is crucial to determine whether this aligns with the temporal progression and spatial distribution of tau pathology in the brains of patients with tauopathies.

No single tauopathy model fully recapitulates the temporal and spatial progression of human tauopathies. The PL-SF system is not intended to model the disease course. Rather, it is an excellent model for mechanistic studies of mature tau aggregation, which is otherwise challenging to study. We note that prior studies showed that PL-SF tau expression in transgenic mice (Xia et al., 2022 and Smith et al., 2025) and rhesus monkeys (Beckman et al., 2021) led to prion-like tau seeding and aggregation in hippocampal and cortical regions. Indeed, the spatial and temporal tau aggregation patterns aligned with features of human tauopathies. So far, these findings all support PL-SF as a valid accelerated model of tauopathy than can be used to interrogate pathogenic mechanisms that impact tau processing, degradation, and/or aggregation.

(2) The authors did not elucidate the specific molecular mechanism by which Hsp70 promotes tau aggregation.

We agree that a deeper understanding of the molecular mechanism is needed. The revision experiments outlined above (Reviewer #1, point #1) will define how Hsp70 promotes tau aggregation by testing sequence contributions, dissecting ATPase and substrate-binding domain requirements, and mapping Hsp70/Hsc70 interactors to directly address this mechanistic question.

(3) Some figures in this study show large error bars in the quantitative data (some statistical analysis figures, MEA recordings, etc.), indicating significant inter-sample variability. It is recommended to label individual data points in all quantitative figures and clearly indicate them in figure legends.

We acknowledge the inter-sample variability in some of the quantitative datasets. This level of variability can occur in primary neuronal cultures (e.g., MEA recordings) that are sensitive to growth and surface adhesion conditions, leading to many technical considerations. To improve transparency and interpretation, we will revise all quantitative figures to display individual data points overlaid on summary statistics and will update figure legends to clearly indicate sample sizes and statistical tests used.

References

Hong Z, Gong W, Yang J, Li S, Liu Z, Perrett S, Zhang H. Exploration of the cysteine reactivity of human inducible Hsp70 and cognate Hsc70. J Biol Chem. 2023 Jan;299(1):102723. doi: 10.1016/j.jbc.2022.102723. Epub 2022 Nov 19. PMID: 36410435; PMCID: PMC9800336.

Jinwal UK, Miyata Y, Koren J 3rd, Jones JR, Trotter JH, Chang L, O'Leary J, Morgan D, Lee DC, Shults CL, Rousaki A, Weeber EJ, Zuiderweg ER, Gestwicki JE, Dickey CA. Chemical manipulation of hsp70 ATPase activity regulates tau stability. J Neurosci. 2009 Sep 30;29(39):12079-88. doi: 10.1523/JNEUROSCI.3345-09.2009. PMID: 19793966; PMCID: PMC2775811.

Fontaine SN, Rauch JN, Nordhues BA, Assimon VA, Stothert AR, Jinwal UK, Sabbagh JJ, Chang L, Stevens SM Jr, Zuiderweg ER, Gestwicki JE, Dickey CA. Isoform-selective Genetic Inhibition of Constitutive Cytosolic Hsp70 Activity Promotes Client Tau Degradation Using an Altered Co-chaperone Complement. J Biol Chem. 2015 May 22;290(21):13115-27. doi: 10.1074/jbc.M115.637595. Epub 2015 Apr 11. PMID: 25864199; PMCID: PMC4505567

Young ZT, Rauch JN, Assimon VA, Jinwal UK, Ahn M, Li X, Dunyak BM, Ahmad A, Carlson G, Srinivasan SR, Zuiderweg ERP, Dickey CA, Gestwicki JE. Stabilizing the Hsp70‑Tau Complex Promotes Turnover in Models of Tauopathy. Cell Chem Biol. 2016 Aug 4;23(8):992–1001. doi:10.1016/j.chembiol.2016.04.014.

Jinwal UK, Akoury E, Abisambra JF, O'Leary JC 3rd, Thompson AD, Blair LJ, Jin Y, Bacon J, Nordhues BA, Cockman M, Zhang J, Li P, Zhang B, Borysov S, Uversky VN, Biernat J, Mandelkow E, Gestwicki JE, Zweckstetter M, Dickey CA. Imbalance of Hsp70 family variants fosters tau accumulation. FASEB J. 2013 Apr;27(4):1450-9. doi: 10.1096/fj.12-220889. Epub 2012 Dec 27. PMID: 23271055; PMCID: PMC3606536.

Xia, Y., Prokop, S., Bell, B.M. et al. Pathogenic tau recruits wild-type tau into brain inclusions and induces gut degeneration in transgenic SPAM mice. Commun Biol 5, 446 (2022). https://doi.org/10.1038/s42003-022-03373-1.

Smith ED, Paterno G, Bell BM, Gorion KM, Prokop S, Giasson BI. Tau from SPAM Transgenic Mice Exhibit Potent Strain-Specific Prion-Like Seeding Properties Characteristic of Human Neurodegenerative Diseases. Neuromolecular Med. 2025 May 30;27(1):44. doi: 10.1007/s12017-025-08850-4. PMID: 40447946; PMCID: PMC12125038.

Beckman D, Chakrabarty P, Ott S, Dao A, Zhou E, Janssen WG, Donis-Cox K, Muller S, Kordower JH, Morrison JH. A novel tau-based rhesus monkey model of Alzheimer's pathogenesis. Alzheimers Dement. 2021 Jun;17(6):933-945. doi: 10.1002/alz.12318. Epub 2021 Mar 18. PMID: 33734581; PMCID: PMC8252011.

eLife Assessment

This study investigates the role of developmental oligodendrocytes in synchronising spontaneous activity in neuronal circuits and influencing cerebellar-dependent behaviour. The authors use advanced viral targeting techniques to deplete oligodendrocytes in a cell-specific manner, paired with in vivo calcium imaging of Purkinje cells, to establish a relationship between oligodendrocyte-mediated neuronal synchrony and complex brain function. The authors present compelling evidence of oligodendrocyte-regulated neuronal synchrony. Overall, this manuscript holds promise as an important contribution to neurodevelopment research.

Reviewer #1 (Public review):

Summary:

This study presents convincing findings that oligodendrocytes play a regulatory role in spontaneous neural activity synchronization during early postnatal development, with implications for adult brain function. Utilizing targeted genetic approaches, the authors demonstrate how oligodendrocyte depletion impacts Purkinje cell activity and behaviors dependent on cerebellar function. Delayed myelination during critical developmental windows is linked to persistent alterations in neural circuit function, underscoring the lasting impact of oligodendrocyte activity.

Strengths:

(1) The research leverages the anatomically distinct olivocerebellar circuit, a well-characterized system with known developmental timelines and inputs, strengthening the link between oligodendrocyte function and neural synchronization.

(2) Functional assessments, supported by behavioral tests, validate the findings of in vivo calcium imaging, enhancing the study's credibility.

(3) Extending the study to assess long-term effects of early life myelination disruptions adds depth to the implications for both circuit function and behavior.

Weaknesses:

(1) The study would benefit from a closer analysis of myelination during the periods when synchrony is recorded. Direct correlations between myelination and synchronized activity would substantiate the mechanistic link and clarify if observed behavioral deficits stem from altered myelination timing.

(2) Although the study focuses on Purkinje cells in the cerebellum, neural synchrony typically involves cross-regional interactions. Expanding the discussion on how localized Purkinje synchrony affects broader behaviors-such as anxiety, motor function, and sociality - would enhance the findings' functional significance.

(3) The authors discuss the possibility of oligodendrocyte-mediated synapse elimination as a possible mechanism behind their findings, drawing from relevant recent literature on oligodendrocyte precursor cells. However, there are no data presented supporting these assumptions. The authors should explain why they think the mechanism behind their observation extends beyond the contribution of myelination or remove this point from the discussion entirely.

Comment for resubmission: Although the argument on synaptic elimination has been removed, it has been replaced with similarly unclear speculation about roles for oligodendrocytes outside of conventional myelination or metabolic support, again without clear evidence. The authors measured MBP area but have not performed detailed analysis of oligodendrocyte biology to support the claims made in the discussion. Please consider removing this section or rephrasing it to align with the data presented.

(4) It would be valuable to investigate secondary effects of oligodendrocyte depletion on other glial cells, particularly astrocytes or microglia, which could influence long-term behavioral outcomes. Identifying whether the lasting effects stem from developmental oligodendrocyte function alone or also involve myelination could deepen the study's insights.

(5) The authors should explore the use of different methods to disturb myelin production for a longer time, in order to further determine if the observed effects are transient or if they could have longer-lasting effects.

(6) Throughout the paper, there are concerns about statistical analyses, particularly on the use of the Mann-Whitney test or using fields of view as biological replicates.