eLife Assessment

This useful study presents the first detailed and comprehensive description of brain sulcus anatomy of a range of carnivoran species based on a robust manual labeling model allowing species comparisons. Although the database is recognized and the method for reconstructing cortical surfaces is convincing, the evidence supporting the conclusions is incomplete due to the lack of appropriate quantitative measurements and analyses. Considering additional specimens to assess intraspecies variations, as well as exploring the functional correlates of interspecies differences would increase the scope of the study. Setting an instructive foundation for comparative anatomy, this study will be of interest to neuroscientists and neuroimaging researchers interested in that field, as well as in brain morphology and sulcal patterns, their phylogeny, and ontogeny in relation to functional development and behaviour.

Reviewer #1 (Public review):

Summary:

The paper by Boch and colleagues, entitled Comparative Neuroimaging of the Carnivore Brain: Neocortical Sulcal Anatomy, compares and describes the cortical sulci of eighteen carnivore species, and sets a benchmark for future work on comparative brains.

Based on previous observations, electrophysiological, histological and neuroimaging studies and their own observations, the authors establish a correspondence between the cortical sulci and gyri of these species. The different folding patterns of all brain regions are detailed, put into perspective in relation to their phylogeny as well as their potential involvement in cortical area expansion and behavioral differences.

Strengths:

This is a pioneering article, very useful for comparative brain studies and conducted with great seriousness and based on many past studies. The article is well-written and very didactic. The different protocols for brain collection, perfusion, and scanning are very detailed. The images are self-explanatory and of high quality. The authors explain their choice of nomenclature and labels for sulci and gyri on all species, with many arguments. The opening on ecology and social behavior in the discussion is of great interest and helps to put into perspective the differences in folding found at the level of the different cortexes. In addition, the authors do not forget to put their results into the context of the laws of allometry. They explain, for example, that although the largest brains were the most folded and had the deepest folds in their dataset, they did not necessarily have unique sulci, unlike some of the smaller, smoother brains.

Weaknesses:

The article is aware of its limitations, not being able to take into account inter-individual variability within each species, inter-hemispheric asymmetries, or differences between males and females. However, this does not detract from their aim, which is to lay the foundations for a correspondence between the brains of carnivores so that navigation within the brains of these species can be simplified for future studies. This article does not include comparisons of morphometric data such as sulci depth, sulci wall surface, or thickness of the cortical ribbon around the sulci.

Reviewer #2 (Public review):

Summary:

The authors have completed MRI-based descriptions of the sulcal anatomy of 18 carnivoran species that vary greatly in behaviour and ecology. In this descriptive study, different sulcal patterns are identified in relation to phylogeny and, to some extent, behaviour. The authors argue that the reported differences across families reflect behaviour and electrophysiology, but these correlations are not supported by any analyses.

Strengths:

A major strength of this paper is using very similar imaging methods across all specimens. Often papers like this rely on highly variable methods so that consistency reduces some of the variability that can arise due to methodology.

The descriptive anatomy was accurate and precise. I could readily follow exactly where on the cortical surface the authors referring. This is not always the case for descriptive anatomy papers, so I appreciated the efforts the authors took to make the results understandable for a broader audience.

I also greatly appreciate the authors making the images open access through their website.

Weaknesses:

Although I enjoyed many aspects of this manuscript, it is lacking in any quantitative analyses that would provide more insights into what these variations in sulcal anatomy might mean. The authors do discuss inter-clade differences in relation to behaviour and older electrophysiology papers by Welker, Campos, Johnson, and others, but it would be more biologically relevant to try to calculate surface areas or volumes of cortical fields defined by some of these sulci. For example, something like the endocast surface area measurements used by Sakai and colleagues would allow the authors to test for differences among clades, in relation to brain/body size, or behaviour. Quantitative measurements would also aid significantly in supporting some of the potential correlations hinted at in the Discussion.

Although quantitative measurements would be helpful, there are also some significant concerns in relation to the specimens themselves. First, almost all of these are captive individuals. We know that environmental differences can alter neocortical development and humans and nonhuman animals and domestication affects neocortical volume and morphology. Whether captive breeding affects neocortical anatomy might not be known, but it can affect other brain regions and overall brain size and could affect sulcal patterns. Second, despite using similar imaging methods across specimens, fixation varied markedly across specimens. Fixation is unlikely to affect the ability to recognize deep sulci, but variations in shrinkage could nevertheless affect overall brain size and morphology, including the ability to recognize shallow sulci. Third, the sample size = 1 for every species examined. In humans and nonhuman animals, sulcal patterns can vary significantly among individuals. In domestic dogs, it can even vary greatly across breeds. It therefore remains unclear to what extent the pattern observed in one individual can be generalized for a species let alone an entire genus or family. The lack of accounting for inter-individual variability makes it difficult to make any firm conclusions regarding the functional relevance of sulcal patterns.

Author response:

eLife Assessment

This useful study presents the first detailed and comprehensive description of brain sulcus anatomy of a range of carnivoran species based on a robust manual labeling model allowing species comparisons. Although the database is recognized and the method for reconstructing cortical surfaces is convincing, the evidence supporting the conclusions is incomplete due to the lack of appropriate quantitative measurements and analyses. Considering additional specimens to assess intraspecies variations, as well as exploring the functional correlates of interspecies differences would increase the scope of the study. Setting an instructive foundation for comparative anatomy, this study will be of interest to neuroscientists and neuroimaging researchers interested in that field, as well as in brain morphology and sulcal patterns, their phylogeny, and ontogeny in relation to functional development and behaviour. 

We are pleased that our primary objective of creating a comprehensive framework to navigate carnivoran brains is considered as successfully achieved and that our work is expected to be of broad interest to various disciplines, as it provides the foundation for future investigations into carnivoran brain organization.

As we will set out below, a description of the major sulci is an appropriate measure for large-scale comparative anatomy — it is stable enough in the population of each species to not require a large N, provides a suitable variability across species, and can be related to other aspects of between-species diversity. We will include a number of additional species to increase the scope of the study, as suggested. Although a quantitative assessment of functional correlates is, in principle, beyond the scope of this first foundational paper, we will provide a first start of this as well. We emphasize, however, that this was a secondary outcome, emerging after first application of the framework.

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

The paper by Boch and colleagues, entitled Comparative Neuroimaging of the Carnivore Brain: Neocortical Sulcal Anatomy, compares and describes the cortical sulci of eighteen carnivore species, and sets a benchmark for future work on comparative brains. 

Based on previous observations, electrophysiological, histological and neuroimaging studies and their own observations, the authors establish a correspondence between the cortical sulci and gyri of these species. The different folding patterns of all brain regions are detailed, put into perspective in relation to their phylogeny as well as their potential involvement in cortical area expansion and behavioral differences. 

Strengths: 

This is a pioneering article, very useful for comparative brain studies and conducted with great seriousness and based on many past studies. The article is well-written and very didactic. The different protocols for brain collection, perfusion, and scanning are very detailed. The images are self-explanatory and of high quality. The authors explain their choice of nomenclature and labels for sulci and gyri on all species, with many arguments. The opening on ecology and social behavior in the discussion is of great interest and helps to put into perspective the differences in folding found at the level of the different cortexes. In addition, the authors do not forget to put their results into the context of the laws of allometry. They explain, for example, that although the largest brains were the most folded and had the deepest folds in their dataset, they did not necessarily have unique sulci, unlike some of the smaller, smoother brains. 

Weaknesses: 

The article is aware of its limitations, not being able to take into account inter-individual variability within each species, inter-hemispheric asymmetries, or differences between males and females. However, this does not detract from their aim, which is to lay the foundations for a correspondence between the brains of carnivores so that navigation within the brains of these species can be simplified for future studies. This article does not include comparisons of morphometric data such as sulci depth, sulci wall surface, or thickness of the cortical ribbon around the sulci. 

We thank the reviewer for their overwhelmingly positive evaluation of our work. As noted by the reviewer, our primary aim was to establish a framework for navigating carnivoran brains to lay the foundation for future research. We are pleased that this objective is deemed as successfully achieved.

As the reviewer points out, we do not quantify within-species intraindividual differences. This is a conscious choice; we aimed to emphasize breadth of species over individuals, as is standard in large-scale comparative anatomy (cf. Heuer et al., 2023, eLife; Suarez et al., 2022, eLife). Following the logic of phylogenetic relationships, the presence of a particular sulcus in related species is also a measure of reliability. We felt safe in this choice, as previous work in both primates and carnivorans has shown that differences across major sulci across individuals are a matter of degree rather than a case of presence or absence (Connolly, 1950, External morphology of the primate brain, C.C. Thomas; Hecht et al., 2019 J Neurosci; Kawamuro 1971 Acta Anat., Kawamuro & Naito, 1977, Acta Anat.). In our revised manuscript, we aim to include some additional individuals of selected species as supplementary material, further illustrating this point.

We feel that measures such as sulci depth, sulci wall surface, or thickness of the cortical ribbon are measures that vary more across individuals and we have therefore not included them in the study. In addition, these are measures that are not generally used as between-species comparative measures, whereas sulcal patterning is (cf. Amiez et al., 2019, Nat Comms; Connolly, 1950; Miller et al., 2021, Brain Behav Evol; Radinsky 1975, J Mammal; Radinsky 1969, Ann N Y Acad Sci; Welker & Campos 1963 J. Comp Neurol).

Reviewer #2 (Public review): 

Summary: 

The authors have completed MRI-based descriptions of the sulcal anatomy of 18 carnivoran species that vary greatly in behaviour and ecology. In this descriptive study, different sulcal patterns are identified in relation to phylogeny and, to some extent, behaviour. The authors argue that the reported differences across families reflect behaviour and electrophysiology, but these correlations are not supported by any analyses. 

Strengths: 

A major strength of this paper is using very similar imaging methods across all specimens. Often papers like this rely on highly variable methods so that consistency reduces some of the variability that can arise due to methodology. 

The descriptive anatomy was accurate and precise. I could readily follow exactly where on the cortical surface the authors referring. This is not always the case for descriptive anatomy papers, so I appreciated the efforts the authors took to make the results understandable for a broader audience. 

I also greatly appreciate the authors making the images open access through their website. 

Weaknesses: 

Although I enjoyed many aspects of this manuscript, it is lacking in any quantitative analyses that would provide more insights into what these variations in sulcal anatomy might mean. The authors do discuss inter-clade differences in relation to behaviour and older electrophysiology papers by Welker, Campos, Johnson, and others, but it would be more biologically relevant to try to calculate surface areas or volumes of cortical fields defined by some of these sulci. For example, something like the endocast surface area measurements used by Sakai and colleagues would allow the authors to test for differences among clades, in relation to brain/body size, or behaviour. Quantitative measurements would also aid significantly in supporting some of the potential correlations hinted at in the Discussion. 

Although quantitative measurements would be helpful, there are also some significant concerns in relation to the specimens themselves. First, almost all of these are captive individuals. We know that environmental differences can alter neocortical development and humans and nonhuman animals and domestication affects neocortical volume and morphology. Whether captive breeding affects neocortical anatomy might not be known, but it can affect other brain regions and overall brain size and could affect sulcal patterns. Second, despite using similar imaging methods across specimens, fixation varied markedly across specimens. Fixation is unlikely to affect the ability to recognize deep sulci, but variations in shrinkage could nevertheless affect overall brain size and morphology, including the ability to recognize shallow sulci. Third, the sample size = 1 for every species examined. In humans and nonhuman animals, sulcal patterns can vary significantly among individuals. In domestic dogs, it can even vary greatly across breeds. It, therefore, remains unclear to what extent the pattern observed in one individual can be generalized for a species, let alone an entire genus or family. The lack of accounting for inter-individual variability makes it difficult to make any firm conclusions regarding the functional relevance of sulcal patterns. 

We thank the reviewer for their assessment of our work. The primary aim of this study was to establish a framework for navigating carnivoran brains by providing a comprehensive overview of all major neocortical sulci across eighteen different species. Given the inconsistent nomenclature in the literature and the lack of standardized criteria (“recipes”) for identifying the major sulci, we specifically focused on homogenizing the terminology and creating recipes for their identification. Moreover, we also generated digital surfaces of all brains and will also add sulcal masks to further facilitate future research building on our framework. We are pleased to hear that we succeeded in our primary objective.

We respectfully disagree with the reviewer on two accounts, where we believe the reviewer is not judging the scope of the current work.

The first is with respect to individual differences. To the best of our knowledge, differences between captive and wild animals, or indeed between individuals, do not affect the presence or absence of any major sulci. No differences in sulcal patterns were detected between captive and (semi-)wild macaques (cf. Sallet et al., 2011, Science; Testard et al., 2022, Sci Adv), different dog breeds (Hecht et al., 2019 J Neurosci) or foxes selectively bred to simulate domestication, compared to controls (Hecht et al., 2021 J. Neurosci). Indeed, we do not find major differences between wolf-like canid species, suggesting that a difference between individuals of the same species is even more unlikely. Nevertheless, we agree with the reviewer that building up a database like ours will benefit from providing as much information about the samples as possible to enable these issues to be tested. We, therefore, will update our table to include if the animals were from captive or wild populations. Moreover, we aim, where possible, to include both wild and captive animals of the same species if they are available in our revision.

The second is in the quantification of structure/function relationships. We believe the sulci atlases themselves are the main deliverables of this project. We felt it prudent to include some qualitative descriptions of the relationship between sulci as we observed them and behaviours as known from the literature as an illustration of the possibilities that this foundational work opens us. This approach also allowed us to confirm previous findings based on observations from a less diverse range of carnivoran species and families (Radinsky 1968 J Comp Neurol; Radinsky 1969, Ann N Y Acad Sci; Welker & Campos 1963 J Comp Neurol; Welker & Seidenstein, 1959 J Comp Neurol). However, a full statistical framework for analysis is beyond the scope of this paper. Our group has previously worked on methods to quantitatively compare brain organization across species — indeed, we have developed a full framework for doing so (Mars et al., 2021, Annu Rev Neurosci), based on the idea that brains that differ in size and morphology should be compared based on anatomical features in a common feature space. Previously, we have used white matter anatomy (Mars et al., 2018, eLife) and spatial transcriptomics (Beauchamp et al., 2021, eLife). The present work presents the foundation for this approach to be expanded to sulcal anatomy, but the full development of this approach will be the topic of future communications.

Nevertheless, we aim to include a first step quantitative analysis of the relationship between the presence and absence of particular sulci and the two behaviours of interest in our manuscript.

We also would like to emphasize that we strongly believe that looking at measures of brain organization at a more detailed level than brain size or relative brain size is informative. Indeed, studies looking at correlations between brain size and particular behavioural variables, although very prominent in the literature, have found it very difficult to distinguish between competing behavioural hypotheses (Healy, 2021, Adaptation and the brain, OUP). In contrast, connectivity has a much more direct relationship to behavioural differences across species (Bryant et al., 2024, bioRxiv), as does sulcal anatomy (Amiez et al., 2019, Nat Comms; Miller et al., 2021, Brain Behav Evol). Moreover, such measures are less sensitive to the effects of fixation since that will affect brain size but not the presence or absence of a sulcus.

Following the reviewer’s recommendations, we will endeavour to include an even broader range of species in the revised version.

eLife Assessment

This important paper by Lechler and colleagues describes the transcriptomic signature and fate of intermediate cells (ICs), a transient and poorly defined embryonic cell type in the skin. The paper convincingly shows through lineage tracing that ICs are granular and not spinous cell precursors, and through ectopic expression in vivo, that cell contractility, a mechanical feature of ICs, lies upstream of differentiation.

Reviewer #1 (Public review):

Summary:

The authors address a fundamental question for cell and tissue biology using the skin epidermis as a paradigm and ask how stratifying self-renewing epithelia induce differentiation and upward migration in basal dividing progenitor cells to generate suprabasal barrier-forming cells that are essential for a functional barrier formed by such an epithelium. The authors show for the first time that an increase in intracellular actomyosin contractility, a hallmark of barrier-forming keratinocytes, is sufficient to trigger terminal differentiation. Hence the data provide in vivo evidence of the more general interdependency of cell mechanics and differentiation. The data appear to be of high quality and the evidences are strengthened through a combination of different genetic mouse models, RNA sequencing, and immunofluorescence analysis.

To generate and maintain the multilayered, barrier-forming epidermis, keratinocytes of the basal stem cell layer differentiate and move suprabasally accompanied by stepwise changes not only in gene expression but also in cell morphology, mechanics, and cell position. Whether any of these changes is instructive for differentiation itself and whether consecutive changes in differentiation are required remains unclear. Also, there are few comprehensive data sets on the exact changes in gene expression between different states of keratinocyte differentiation. In this study, through genetic fluorescence labeling of cell states at different developmental time points the authors were able to analyze gene expression of basal stem cells and suprabasal differentiated cells at two different stages of maturation: E14 (embryonic day 14) when the epidermis comprises mostly two functional compartments (basal stem cells and suprabasal so-called intermediate cells) and E16 when the epidermis comprise three (living) compartments where the spinous layer separates basal stem cells from the barrier-forming granular layer, as is the case in adult epidermis. Using RNA bulk sequencing, the authors developed useful new markers for suprabasal stages of differentiation like MafB and Cox1. The transcription factor MafB was then shown to inhibit suprabasal proliferation in a MafB transgenic model.

The data indicate that early in development at E14 the suprabasal intermediate cells resemble in terms of RNA expression, the barrier-forming granular layer at E16, suggesting that keratinocytes can undergo either stepwise (E16) or more direct (E14) terminal differentiation.

Previous studies by several groups found an increased actomyosin contractility in the barrier-forming granular layer and showed that this increase in tension is important for epidermal barrier formation and function. However, it was not clear whether contractility itself serves as an instructive signal for differentiation. To address this question, the authors use a previously published model to induce premature hypercontractility in the spinous layer by using spastin overexpression (K10-Spastin) to disrupt microtubules (MT) thereby indirectly inducing actomyosin contractility. A second model activates myosin contractility more directly through overexpression of a constitutively active RhoA GEF (K10-Arhgef11CA). Both models induce late differentiation of suprabasal keratinocytes regardless of the suprabasal position in either spinous or granular layer indicating that increased contractility is key to induce late differentiation of granular cells. A potential weakness of the K10-spastin model is the disruption of MT as the primary effect which secondarily causes hypercontractility. However, their previous publications provided some evidence that the effect on differentiation is driven by the increase in contractility (Ning et al. cell stem cell 2021). Moreover, the data are confirmed by the second model directly activating myosin through RhoA. These previous publications already indicated a role for contractility in differentiation but were focused on early differentiation. The data in this manuscript focus on the regulation of late differentiation in barrier-forming cells. These important data help to unravel the interdependencies of cell position, mechanical state, and differentiation in the epidermis, suggesting that an increase in cellular contractility in most apical positions within the epidermis can induce terminal differentiation. Importantly the authors show that despite contractility-induced nuclear localization of the mechanoresponsive transcription factor YAP in the barrier-forming granular layer, YAP nuclear localization is not sufficient to drive premature differentiation when forced to the nucleus in the spinous layer.

Overall, this is a well-written manuscript and a comprehensive dataset. Only the RNA sequencing result should be presented more transparently providing the full lists of regulated genes instead of presenting just the GO analysis and selected target genes so that this analysis can serve as a useful repository. The authors themselves have profited from and used published datasets of gene expression of the granular cells. Moreover, some of the previous data should be better discussed though. The authors state that forced suprabasal contractility in their mouse models induces the expression of some genes of the epidermal differentiation complex (EDC). However, in their previous publication, the authors showed that major classical EDC genes are actually not regulated like filaggrin and loricrin (Muroyama and Lechler eLife 2017). This should be discussed better and necessitates including the full list of regulated genes to show what exactly is regulated.

Reviewer #2 (Public review):

Summary:

The manuscript from Prado-Mantilla and co-workers addresses mechanisms of embryonic epidermis development, focusing on the intermediate layer cells, a transient population of suprabasal cells that contributes to the expansion of the epidermis through proliferation. Using bulk-RNA they show that these cells are transcriptionally distinct from the suprabasal spinous cells and identify specific marker genes for these populations. They then use transgenesis to demonstrate that one of these selected spinous layer-specific markers, the transcription factor MafB is capable of suppressing proliferation in the intermediate layers, providing a potential explanation for the shift of suprabasal cells into a non-proliferative state during development. Further, lineage tracing experiments show that the intermediate cells become granular cells without a spinous layer intermediate. Finally, the authors show that the intermediate layer cells express higher levels of contractility-related genes than spinous layers and overexpression of cytoskeletal regulators accelerates the differentiation of spinous layer cells into granular cells.

Overall the manuscript presents a number of interesting observations on the developmental stage-specific identities of suprabasal cells and their differentiation trajectories and points to a potential role of contractility in promoting differentiation of suprabasal cells into granular cells. The precise mechanisms by which MafB suppresses proliferation, how the intermediate cells bypass the spinous layer stage to differentiate into granular cells, and how contractility feeds into these mechanisms remain open. Interestingly, while the mechanosensitive transcription factor YAP appears deferentially active in the two states, it is shown to be downstream rather than upstream of the observed differences in mechanics.

Strengths:

The authors use a nice combination of RNA sequencing, imaging, lineage tracing, and transgenesis to address the suprabasal to granular layer transition. The imaging is convincing and the biological effects appear robust. The manuscript is clearly written and logical to follow.

Weaknesses:

While the data overall supports the authors' claims, there are a few minor weaknesses that pertain to the aspect of the role of contractility, The choice of spastin overexpression to modulate contractility is not ideal as spastin has multiple roles in regulating microtubule dynamics and membrane transport which could also be potential mechanisms explaining some of the phenotypes. Use of Arghap11 overexpression mitigates this effect to some extent but overall it would have been more convincing to manipulate myosin activity directly. It would also be important to show that these manipulations increase the levels of F-actin and myosin II as shown for the intermediate layer. It would also be logical to address if further increasing contractility in the intermediate layer would enhance the differentiation of these cells.

The gene expression analyses are relatively superficial and rely heavily on GO term analyses which are of course informative but do not give the reader a good sense of what kind of genes and transcriptional programs are regulated. It would be useful to show volcano plots or heatmaps of actual gene expression changes as well as to perform additional analyses of for example gene set enrichment and/or transcription factor enrichment analyses to better describe the transcriptional programs

Claims of changes in cell division/proliferation changes are made exclusively by quantifying EdU incorporation. It would be useful to more directly look at mitosis. At minimum Y-axis labels should be changed from "% Dividing cells" to % EdU+ cells to more accurately represent findings

Despite these minor weaknesses the manuscript is overall of high quality, sheds new light on the fundamental mechanisms of epidermal stratification during embryogenesis, and will likely be of interest to the skin research community.

Reviewer #3 (Public review):

Summary:

This is an interesting paper by Lechler and colleagues describing the transcriptomic signature and fate of intermediate cells (ICs), a transient and poorly defined embryonic cell type in the skin. ICs are the first suprabasal cells in the stratifying skin and unlike later-developing suprabasal cells, ICs continue to divide. Using bulk RNA seq to compare ICs to spinous and granular transcriptomes, the authors find that IC-specific gene signatures include hallmarks of granular cells, such as genes involved in lipid metabolism and skin barrier function that are not expressed in spinous cells. ICs were assumed to differentiate into spinous cells, but lineage tracing convincingly shows ICs differentiate directly into granular cells without passing through a spinous intermediate. Rather, basal cells give rise to the first spinous cells. They further show that transcripts associated with contractility are also shared signatures of ICs and granular cells, and overexpression of two contractility inducers (Spastin and ArhGEF-CA) can induce granular and repress spinous gene expression. This contractility-induced granular gene expression does not appear to be mediated by the mechanosensitive transcription factor, Yap. The paper also identifies new markers that distinguish IC and spinous layers and shows the spinous signature gene, MafB, is sufficient to repress proliferation when prematurely expressed in ICs.

Strengths:

Overall this is a well-executed study, and the data are clearly presented and the findings convincing. It provides an important contribution to the skin field by characterizing the features and fate of ICs, a much-understudied cell type, at high levels of spatial and transcriptomic detail. The conclusions challenge the assumption that ICs are spinous precursors through compelling lineage tracing data. The demonstration that differentiation can be induced by cell contractility is an intriguing finding and adds a growing list of examples where cell mechanics influence gene expression and differentiation.

Weaknesses:

A weakness of the study is an over-reliance on overexpression and sufficiency experiments to test the contributions of MafB, Yap, and contractility in differentiation. The inclusion of loss-of-function approaches would enable one to determine if, for example, contractility is required for the transition of ICs to granular fate, and whether MafB is required for spinous fate. Second, whether the induction of contractility-associated genes is accompanied by measurable changes in the physical properties or mechanics of the IC and granular layers is not directly shown. The inclusion of physical measurements would bolster the conclusion that mechanics lies upstream of differentiation.

Finally, whether the expression of granular-associated genes in ICs provides them with some sort of barrier function in the embryo is not addressed, so the role of ICs in epidermal development remains unclear. Although not essential to support the conclusions of this study, insights into the function of this transient cell layer would strengthen the overall impact.

Author response:

Public Reviews: 

Reviewer #1 (Public review): 

Summary: 

The authors address a fundamental question for cell and tissue biology using the skin epidermis as a paradigm and ask how stratifying self-renewing epithelia induce diCerentiation and upward migration in basal dividing progenitor cells to generate suprabasal barrier-forming cells that are essential for a functional barrier formed by such an epithelium. The authors show for the first time that an increase in intracellular actomyosin contractility, a hallmark of barrier-forming keratinocytes, is suCicient to trigger terminal diCerentiation. Hence the data provide in vivo evidence of the more general interdependency of cell mechanics and diCerentiation. The data appear to be of high quality and the evidences are strengthened through a combination of diCerent genetic mouse models, RNA sequencing, and immunofluorescence analysis. 

To generate and maintain the multilayered, barrier-forming epidermis, keratinocytes of the basal stem cell layer diCerentiate and move suprabasally accompanied by stepwise changes not only in gene expression but also in cell morphology, mechanics, and cell position. Whether any of these changes is instructive for diCerentiation itself and whether consecutive changes in diCerentiation are required remains unclear. Also, there are few comprehensive data sets on the exact changes in gene expression between diCerent states of keratinocyte diCerentiation. In this study, through genetic fluorescence labeling of cell states at diCerent developmental time points the authors were able to analyze gene expression of basal stem cells and suprabasal diCerentiated cells at two diCerent stages of maturation: E14 (embryonic day 14) when the epidermis comprises mostly two functional compartments (basal stem cells and suprabasal so-called intermediate cells) and E16 when the epidermis comprise three (living) compartments where the spinous layer separates basal stem cells from the barrier-forming granular layer, as is the case in adult epidermis. Using RNA bulk sequencing, the authors developed useful new markers for suprabasal stages of diCerentiation like MafB and Cox1. The transcription factor MafB was then shown to inhibit suprabasal proliferation in a MafB transgenic model. 

The data indicate that early in development at E14 the suprabasal intermediate cells resemble in terms of RNA expression, the barrier-forming granular layer at E16, suggesting that keratinocytes can undergo either stepwise (E16) or more direct (E14) terminal diCerentiation. 

Previous studies by several groups found an increased actomyosin contractility in the barrier-forming granular layer and showed that this increase in tension is important for epidermal barrier formation and function. However, it was not clear whether contractility itself serves as an instructive signal for diCerentiation. To address this question, the authors use a previously published model to induce premature hypercontractility in the spinous layer by using spastin overexpression (K10-Spastin) to disrupt microtubules (MT) thereby indirectly inducing actomyosin contractility. A second model activates myosin contractility more directly through overexpression of a constitutively active RhoA GEF (K10Arhgef11CA). Both models induce late diCerentiation of suprabasal keratinocytes regardless of the suprabasal position in either spinous or granular layer indicating that increased contractility is key to induce late diCerentiation of granular cells. A potential weakness of the K10-spastin model is the disruption of MT as the primary eCect which secondarily causes hypercontractility. However, their previous publications provided some evidence that the eCect on diCerentiation is driven by the increase in contractility (Ning et al. cell stem cell 2021). Moreover, the data are confirmed by the second model directly activating myosin through RhoA. These previous publications already indicated a role for contractility in diCerentiation but were focused on early diCerentiation. The data in this manuscript focus on the regulation of late diCerentiation in barrier-forming cells. These important data help to unravel the interdependencies of cell position, mechanical state, and diCerentiation in the epidermis, suggesting that an increase in cellular contractility in most apical positions within the epidermis can induce terminal diCerentiation. Importantly the authors show that despite contractility-induced nuclear localization of the mechanoresponsive transcription factor YAP in the barrier-forming granular layer, YAP nuclear localization is not suCicient to drive premature diCerentiation when forced to the nucleus in the spinous layer. 

Overall, this is a well-written manuscript and a comprehensive dataset. Only the RNA sequencing result should be presented more transparently providing the full lists of regulated genes instead of presenting just the GO analysis and selected target genes so that this analysis can serve as a useful repository. The authors themselves have profited from and used published datasets of gene expression of the granular cells. Moreover, some of the previous data should be better discussed though. The authors state that forced suprabasal contractility in their mouse models induces the expression of some genes of the epidermal diCerentiation complex (EDC). However, in their previous publication, the authors showed that major classical EDC genes are actually not regulated like filaggrin and loricrin (Muroyama and Lechler eLife 2017). This should be discussed better and necessitates including the full list of regulated genes to show what exactly is regulated. 

We thank all the reviewers for their suggestions and comments.

Thank you especially for the reminder to include gene lists. We had an excel document with all this data but neglected to upload it with the initial manuscript decision. This includes all the gene signatures for the diCerent cell compartments across development. We will also include a page that lists all EDC genes and whether they were up-regulated in intermediate cells and cells in which contractility was induced. Further, we note that all the RNA-Seq datasets are available for use on GEO. 

In our previous publication, we indeed included images showing a lack of change in loricrin and filaggrin in the embryos where spastin was expressed in the diCerentiated epidermis. Consistent with this, there is no change in Lor mRNA levels by RNA-Seq, (it is one of the rare EDC genes that is unchanged). In contrast, Flg mRNA was up in the RNASeq, though we didn’t see a dramatic change in protein levels. We have not further pursued whether this reflects translational regulation. That said, our data clearly show that other genes associated with granular fate were increased in the contractile skin.  

Reviewer #2 (Public review): 

Summary: 

The manuscript from Prado-Mantilla and co-workers addresses mechanisms of embryonic epidermis development, focusing on the intermediate layer cells, a transient population of suprabasal cells that contributes to the expansion of the epidermis through proliferation. Using bulk-RNA they show that these cells are transcriptionally distinct from the suprabasal spinous cells and identify specific marker genes for these populations. They then use transgenesis to demonstrate that one of these selected spinous layer-specific markers, the transcription factor MafB is capable of suppressing proliferation in the intermediate layers, providing a potential explanation for the shift of suprabasal cells into a non-proliferative state during development. Further, lineage tracing experiments show that the intermediate cells become granular cells without a spinous layer intermediate. Finally, the authors show that the intermediate layer cells express higher levels of contractilityrelated genes than spinous layers and overexpression of cytoskeletal regulators accelerates the diCerentiation of spinous layer cells into granular cells. 

Overall the manuscript presents a number of interesting observations on the developmental stage-specific identities of suprabasal cells and their diCerentiation trajectories and points to a potential role of contractility in promoting diCerentiation of suprabasal cells into granular cells. The precise mechanisms by which MafB suppresses proliferation, how the intermediate cells bypass the spinous layer stage to diCerentiate into granular cells, and how contractility feeds into these mechanisms remain open. Interestingly, while the mechanosensitive transcription factor YAP appears deferentially active in the two states, it is shown to be downstream rather than upstream of the observed diCerences in mechanics. 

Strengths: 

The authors use a nice combination of RNA sequencing, imaging, lineage tracing, and transgenesis to address the suprabasal to granular layer transition. The imaging is convincing and the biological eCects appear robust. The manuscript is clearly written and logical to follow. 

Weaknesses: 

While the data overall supports the authors' claims, there are a few minor weaknesses that pertain to the aspect of the role of contractility, The choice of spastin overexpression to modulate contractility is not ideal as spastin has multiple roles in regulating microtubule dynamics and membrane transport which could also be potential mechanisms explaining some of the phenotypes. Use of Arghap11 overexpression mitigates this eCect to some extent but overall it would have been more convincing to manipulate myosin activity directly. It would also be important to show that these manipulations increase the levels of F-actin and myosin II as shown for the intermediate layer. It would also be logical to address if further increasing contractility in the intermediate layer would enhance the diCerentiation of these cells. 

We agree with the reviewer that the development of additional tools to precisely control myosin activity will be of great use to the field. That said, our series of publications has clearly demonstrated that ablating microtubules results in increased contractility and that this phenocopies the eCects of Arhgef11 induced contractility (Ning et al, Cell Stem Cell 2021). Further, we showed that these phenotypes were rescued by myosin inhibition with blebbistatin. Our prior publications also showed a clear increase in junctional acto-myosin through expression of either spastin or Arhgef11, as well as increased staining for the tension sensitive epitope of alpha-catenin (alpha-18) (also in Ning et al, 2021).  We are not aware of tools that allow direct manipulation of myosin activity that currently exist in mouse models.  

The gene expression analyses are relatively superficial and rely heavily on GO term analyses which are of course informative but do not give the reader a good sense of what kind of genes and transcriptional programs are regulated. It would be useful to show volcano plots or heatmaps of actual gene expression changes as well as to perform additional analyses of for example gene set enrichment and/or transcription factor enrichment analyses to better describe the transcriptional programs 

We will include an excel document that lists all the gene signatures. Additionally, all of our data are deposited in GEO for others to perform their own analyses.  

Claims of changes in cell division/proliferation changes are made exclusively by quantifying EdU incorporation. It would be useful to more directly look at mitosis. At minimum Y-axis labels should be changed from "% Dividing cells" to % EdU+ cells to more accurately represent findings 

We will change the axis label to precisely match our analysis.  

Despite these minor weaknesses the manuscript is overall of high quality, sheds new light on the fundamental mechanisms of epidermal stratification during embryogenesis, and will likely be of interest to the skin research community. 

Reviewer #3 (Public review): 

Summary: 

This is an interesting paper by Lechler and colleagues describing the transcriptomic signature and fate of intermediate cells (ICs), a transient and poorly defined embryonic cell type in the skin. ICs are the first suprabasal cells in the stratifying skin and unlike laterdeveloping suprabasal cells, ICs continue to divide. Using bulk RNA seq to compare ICs to spinous and granular transcriptomes, the authors find that IC-specific gene signatures include hallmarks of granular cells, such as genes involved in lipid metabolism and skin barrier function that are not expressed in spinous cells. ICs were assumed to diCerentiate into spinous cells, but lineage tracing convincingly shows ICs diCerentiate directly into granular cells without passing through a spinous intermediate. Rather, basal cells give rise to the first spinous cells. They further show that transcripts associated with contractility are also shared signatures of ICs and granular cells, and overexpression of two contractility inducers (Spastin and ArhGEF-CA) can induce granular and repress spinous gene expression. This contractility-induced granular gene expression does not appear to be mediated by the mechanosensitive transcription factor, Yap. The paper also identifies new markers that distinguish IC and spinous layers and shows the spinous signature gene, MafB, is suCicient to repress proliferation when prematurely expressed in ICs. 

Strengths: 

Overall this is a well-executed study, and the data are clearly presented and the findings convincing. It provides an important contribution to the skin field by characterizing the features and fate of ICs, a much-understudied cell type, at high levels of spatial and transcriptomic detail. The conclusions challenge the assumption that ICs are spinous precursors through compelling lineage tracing data. The demonstration that diCerentiation can be induced by cell contractility is an intriguing finding and adds a growing list of examples where cell mechanics influence gene expression and diCerentiation. 

Weaknesses: 

A weakness of the study is an over-reliance on overexpression and suCiciency experiments to test the contributions of MafB, Yap, and contractility in diCerentiation. The inclusion of loss-of-function approaches would enable one to determine if, for example, contractility is required for the transition of ICs to granular fate, and whether MafB is required for spinous fate. Second, whether the induction of contractility-associated genes is accompanied by measurable changes in the physical properties or mechanics of the IC and granular layers is not directly shown. The inclusion of physical measurements would bolster the conclusion that mechanics lies upstream of diCerentiation. 

We agree that loss of function studies would be useful. For MafB, these have been performed in cultured human keratinocytes, where loss of MafB and its ortholog cMaf results in a phenotype consistent with loss of spinous diCerentiation (Lopes-Pajares, Dev Cell 2015). Due to the complex genetics involved, generating these double mutant mice is beyond the scope of this study. Loss of function studies of myosin are also complicated by genetic redundancy of the non-muscle type II myosin genes, as well as the role for these myosins in actin cross linking in addition to contractility. In addition, we have found that these myosins are quite stable in the embryonic intestine, with loss of protein delayed by several days from the induction of recombination. Therefore, elimination of myosins by embryonic day e14.5 with our current drivers is not likely possible. Thus, generation of inducible inhibitors of contractility is a valuable future goal. 

A number of recent papers have used AFM of skin sections to probe tissue rigidity. We have not attempted these studies and are unclear about the spatial resolution and whether, in the very thin epidermis at these stages we could spatially resolve diCerences. That said, we previously assessed the macro-contractility of tissues in which myosin activity was induced and demonstrated that there was a significant increase in this over a tissue-wide scale (Ning et al, Cell Stem Cell, 2021).  

Finally, whether the expression of granular-associated genes in ICs provides them with some sort of barrier function in the embryo is not addressed, so the role of ICs in epidermal development remains unclear. Although not essential to support the conclusions of this study, insights into the function of this transient cell layer would strengthen the overall impact.  

By traditional dye penetration assays, there is no epidermal barrier at the time that intermediate cells exist. One interpretation of the data is that cells are beginning to express mRNAs (and in some cases, proteins) so that they are able to rapidly generate a barrier as they become granular cells. We have attempted experiments to ablate intermediate cells with DTA expression - this resulted in ineCicient and delayed cell death and thus did not yield strong conclusions. Our findings that transcriptional regulators of granular diCerentiation (such as Grhl3 and Hopx) are also present in intermediate cells, should allow future analysis of the eCects of their ablation on the earliest stages of granular diCerentiation from intermediate cells.

Getting Started with the Tin Whistle by [[whistletutor]]

Starter flutes: - Gary Humphrey - Feadóg Original Irish Whistle - Terry McGee - Windward

Beginner Tunes:<br /> - Down by the Sally Gardens - Raglan Road

Merkle Linking is all that you need

tru names for immutable and mutable things

Yin = Mutable Yang = Immutable

mutable imutable distinction merkel tree

Stanford Seminar - IPFS and the Permanent Web Juan Benet

this is really cool concept that shows how much connection and relationships between humans and nature were important for them.

I agree with this because there are so many feelings and experinces that has happend there will be so many mixed thoughts and will not be able to think straight.

They were always in the common pot no matter what they are the reason Natives have changed.

It seems as if the colonists were included in the common pot, but chose to only participate in the parts that benefitted themselves. I find it interesting that this ceremony emphasized the human responsibility to rebalance the network, which is something that colonists largely ignored after greatly tipping the scales in their favor.

Annotation #1-The effect of the COVOD-19 pandemic on infant development and maternal mental health the first 2 years. Investigations how exogenous variation in exposure to the Covid 19 on first year of life caused perceived stress. The pandemic has wreaked devastation on a global scale. In addition to causing millions of deaths. While the pandemic substantially impacted all New Yorkers lives mothers and infants may have been particularly affected. Masking requirements may have interfered with infants abilities to learn and communicate, correlational work has suggested that mothers reported decline in mental health during teb pandemic during both perinatal and postpartum periods. The long-term effects has impacted children as well as future generations, the brain damage inflicted by Covid in infants is a matter for concern it affected development in the most crucial stages and it also caused anxiety and stress on the motherf and the baby the long term effect would be mental illness for future generations.

Annotation #2 According the National Library of Medicine-Pandemic-related disruptions may have led to changes in parent-child interactions in way that could plausibly support or hinder language and socioemotional skill development. Furthermore, accumulating evidence has indicated declines in maternal mental health due to pandemic related stressors. The effect of the pandemic on maternal mental health is an important public health concern in its own right and also has potential implications for infant development. We hypothesized that greater duration of pandemic exposure over the first year of life would be associated with lower infant language skills, more infant socioemotional problems, and greater maternal anxiety, depressive, and perceived stress symptoms when infants were 12-24 months.

Like 前人種樹，後人乘涼

It seems as though Thomas believes that there is a positive impact that can be taken away from divulging in games, and that has to mainly do with the soul. He tends to weave in different ideas in religious texts and words of people who contain lots of wisdom to form this idea that the soul of an individual needs to find delight in these kinds of games. He is essentially saying that words such as playful and humorous were invented so that individuals could discover different ways to create such strong or positive emotions that come from different activities. If playing some kind of medieval game creates "humor" or inspires "joy" in someone's heart, then it was a game that was meant to exist and be played by all.

This is like the old adage 'all work and no play.' People need to be able to reset their minds and games are apart of that.

It is a good idea but would would take a lot of time.

The have been and are fabricated

This is the "check the links" advice Wineburg talks about.

This also seems complex and time consuming IT requires deep, close reading.

But that will only catch bad, sloppy AI disinfo sites. Soon they will be better..

This is CRAAP all over again.

It is definitely interesting to explain how it even seemed like the horses themselves were quivering in excitement. It seems like the infectious energy that comes from these kinds of events affects everyone and everything! The time and energy that goes into planning and executing these medieval games has great effects, as individuals who wish to compete in future competitions spend lots of money investing in animals or objects that will benefit them in the end. Whether the horses are to lead chariots of have races of their own in "the starting gates," (Isidore of Seville, Etymologies 367) there are numerous different ways that they can be used as pieces of entertainment for these people, and it seems as though they feed into the energy (probably an effect from being bred to be stronger and faster).

I really like this section, as it highlights the different kinds of people that were around during this time, and puts emphasis on their work lives. It brings to light how different people are from each other, which definitely can reflect their views on games and pastimes. Whether the opinion was that medieval games provided an outlet of energy and a way to relax or that these games were useless and did not teach any life lessons, it is relevant to connect these opinions to those who are giving them (Milliman 586). For example, an individual who sells goods all day might not want their young children to indulge in playing games, as it can be considered a waste of time, however, a different individual who sells different goods might think it is a good way to keep their children occupied whilst they are working.

I really like this section because it not only highlights a casual version of gaming vs. a more intense version, but the way they play with each other is exactly how people play now.

This line seems to me that some young men used their games as a way of practicing for their future lives, such as impending knighthood.

When I am trying to learn or memorize new information, I often find myself taking handwritten notes. The physical act of taking notes helps you lock the information into memory better. In my first semester of college, I had a professor who emphasized handwritten notes and explained a study he found that proved handwriting notes is more effective than typing them. Since then, I have primarily found myself handwriting notes, which has proved to be my most effective study method.

born a dead man pretty much

what effect is this gonna have on him now?

interesting...

decieivng

all skeletons are pretty much the same color....what

dude should've trusted his gut feelings

I don't like the term "score" in here. it feels like a wrong word choice, I don't know

focusing in on American here and what this is saying about being American

still wondering about the underscores

eyes finally opening

um

point of contact

why????

"above" hm...power dynamic

coming off as more than just a servant...

hm okay then

like.... a date?

hm interesting way to put this

dun dun dun!! foreshadowing has taken effect

the dynamic between the two is still so weird based on how they talk to one another

woah dude just kept rolling with the punches

so the openings are useless?

there is such an odd dynamic here of the servant sort of "parenting" their master

this is just sad

so..why must the race be mentioned...again?

super interesting imagery here

something being "alike" between the two is uncommon in these pieces of works we've read

interesting way of doing things...

so much tension here

toxic masculinity; perceives any assistance as a threat to his authority

something unexpected happening; like something small and weak (a child) controlling/leading something more powerful and dangerous (a bull)

Probably 6pm, though I'd consider that more evening than afternoon (maybe I'm just being nitpicky lol)

What logic is this? All of our skeletons look the same

Where Benito was!

Not surprised that this is coming from a crusade

Again - what is this saying about being American?

Here it says women get paid less and that men have it different just cause they are men.

Here Hicks describes a computation: The EMIDEC read characters from a tape and printed out statements according to them

Here Hicks describes a social norm: Women get paid less in the work place and receive different job titles/grades than men do

It is very important to examine how many other sources have cited or mentioned this specific source to discover its credibility. Since there are 19 mentions of this source in other documents, it is likely that other academics have read and used this source, helping to show that this source has valuable and credible information. It would be good to look at the sources mentioning this source to determine exactly how credible it is because if more well-known and respected sources mention this source for the same reason you wish to mention or cite it, it is likely a great source for you to use.

One of the authors of this source Terrance Ruth is a former school teacher and PHD graduate. His extensive education gives and number of degrees aligns with the point of view and opinion of this source. Due to the fact that he has received higher than average education, it would be logical to assume this affects his opinion and makes him more likely to promote higher education.

i can agree with this and how education and going to school will open you up to be more verbal as immigrants who come from different countries. It helps you learn the language to be able to communicate and learn culture. It's actually really fascinating to see how interested people can be with culture when they're open to hearing about others and not just with one mindset that they're better than others.

Schooling does open your eyes to reality. It does give you a different perception as to how choosing what you like as a career will give you a passion to want to push for more. It shows you the reality of how expensive everything is and how things just keep getting worse. Its not something we cant already see without going to school but it does show different outcomes to what jobs will help you get put of that dark hole. Today i see how the generations don't want to have kids because of how expensive things are if we could barely afford ourselves.

I would honestly say yes. Boys and girls experience the migration journey to their full capacity only because number 1 out parents did not have the resources we had growing up so when it comes down to us needing help they don't know how to help us. We learn how to grow up faster and develop ourselves faster because there is no one to speak up for us especially when it comes to language. I remember being in elementary and learning how to translate things to my parents from english to spanish in order for them to understand. So yes the journey is different.

This new generation and the following generations will have a different outcome in life based on how everything has been. Its been hard and successful seeing the new generation of children who come from immigrant parents have gone to school and will be ablle to check off the box of bachelors or higher for their future of jobs and their future kids based on questions. Me myself who comes from immigrant parents and soon to be first generation to graduate, I would always check the box or fill in that my family had a high school degree or not even fully completed high school. Times are changing and we see the hard work and effort put in to want to make a change even if it does not include the success of education.

The significance of the social force to face reality has been a scary factor for immigrants. They're faced with opportunities to see if education or work is the way to go. I know immigrants look for the opportunity to work because of how much they escape the reality of what they're fleeing. The "American Dream" is pushed to believe that the outcome is education but once you live in a society where education is a luxury because of the life they lived.

Dissecting the title of the Long View of immigrants to me long view means the out come or hard reality of a situation and here the immigrants. The education system for immigrants seems to fail depending on the society and area you live in. Sometimes the accommodation is not always what it seems and immigrants look for a outcome of success.

According to the Brennan Center of Justice's mission page, it is an advocate of nonpartisan laws. This means that they try to remain unbiased in the policies they push and try to do things that are better for everyone as a whole, rather than one group. This proves that anything they publish likely is also non partisan and has little bias, ensuring author credibility.

I believe laws like this are such a touchy subject. Many individuals have different opinions on this topic. In order to have a full opinion on this, I believe it is important to do other research to completely understand. I think pay-to-vote limits many voter's capabilities. Paying to vote discriminates against individuals who do not have the financial means to do so. This is very exclusive. This part of the article really stuck out to me. This article can be verified through the links provided below that showed where this information was coming from.

Although this source was created in 2019, it was updated and recent information from 2020 up until 2022 was added. Readers can now have more present information, and are up to date on a new crucial resource (the NAACP Legal defense fund and ACLU lawyer help for potential voters). This shows the importance of updating research and keeping sources relevant to present-day when it is applicable.

This confuses me sometimes while reading articles - media as in mass media vs. communication tools such as Instagram or smartphone. The contextual use of the word can be helpful, but the meanings seem to even overlap at times.

This is very true. Media technology is always evolving in the digital world. It is getting more complicated and difficult to see all of it to understand it. Even a type of philosophy (Postphenomenology) is needed to understand it.

I didn't know this famous quote, but now it makes sense. - How information is delivered (like a YouTube vs. a newspaper) can be another message that influences how we understand the message. So it is important to choose what kind of media to use when delivering information.

Media ecology sees how different media impact our lives. Just like ecosystems in biology, the different media are connected and influenced by each other and change how we act, think, and communicate.

More than 10 hours a day! This makes me think that we should set a screen time on our phones, not only for children. I agree that it is time to think about how we can consume media in a healthy way.

#mindless media concept!

I resonate a lot with this quote. I try and make intentional choices to be present in my everyday life and surroundings. I hate being at a restaurant where everyone is on their phones. My friends and I often put our phones in a pile in the middle of the table to "unplug" and be present in the moment. We frequently find the best times are when no technology or media is involved!

This makes a lot of sense. Most forms of social media are permanent- people could have screenshots, old videos could resurface, or people could always associate you with a specific thing. However, I would not consider the radio to be a permanent platform. With this, it is important to analyze where you are posting, how you are posting, and how you are engaging with that medium.

This sentence helps build my understanding of the term "media literacy" as more than just what we engage in, it includes how we engage with it.

The idea of "located within a specific time" related to media technologies caught my eye. I can relate to this in that I have unofficial "designated" times when I can scroll on social media like Instagram and TikTok, almost like a brain break. For example, before I go to bed, I associate this with "scroll time."

#mindlessmedia trend once again!

This is what I was talking about prior. My district is moving in the direction of detoxing students from their cell phones, I would imagine eventually moving to no phones at all during the school day.

This is becoming a concern in my district. Administration is sharing a book to parents that touches on how this generation is riddled with anxiety and depression due to the constant access of their phones.

We have become so reliant on these technologies that they are essentially a piece of us now. Nobody can go anywhere without their phone

We have a constant access to communication

Everything you do and everywhere you go nowadays is saturated by media. It's everywhere

Like all new, innovative, revolutionary technologies, the prevailing opinion is "this is here to stay. How can we harness it instead of fight against it?" Media is a part of our lives now, so understanding and utilizing it is a goal we must work to achieve at some point.

As we have seen with literacy in earlier classes, the words we choose must be carefully crafted for our intended meaning to be drawn out. We must consider not only the words themselves, but the medium they are being presented, as well as the possible interpretations from perspectives different from ours.

I feel this is too important and likely a reason why it may be difficult to study human behavior in media technologies. We are much more than simply humans interacting on a website. What may be concluded easily through a more straightforward experiment cannot be done so through something like this. The intricacies of the human themselves can add a lot to their actions, not just the technology itself.

I have heard of this. It may also go by a more general name "dopamine detox." The goal is to reset your dopamine receptors through an either brief or extended recess from digital media, and I believe it does have merit in certain contexts, especially if you notice your concentration levels are significantly warped.

I think education does not necessarily have to equate to endorsement. I still firmly believe much of the internet is just not suited for children or even teenagers. That said, screen time is an inevitability in our world, so dedicating time to thoughtful, intentional time in media technologies is important, especially if they can be done in environments where learning and reflection can occur.

We have a professional skills course in our school where students spend dedicated class periods learning to write professional emails, and this comment reminded me of it. Actually practicing the skill is important!

I would argue that this allows us to communicate whatever the mood strikes as well. Even ignoring anonymity entirely, the strengthened confidence we attain when our words are thrown into an information superhighway and not another human being right in front of us most certainly impacts what we have to say, whether for better or for worse.

There is what we theorize will occur in media technologies, and then there is what will actually happen. Not that there is no place for skill-building or theoretical analysis, but to say the internet has surprised us again and again would be an understatement of great magnitude. Being immersed in and learning from practical experiences in media technologies is important.

The Blitzkrieg were german jets that also conquered france in 1940

I wonder if America would never have joined the war if Japan never attacked Pearl Harbor.

Hitler was the one who broke their alliance with the soviet union and it later came back to stab him in the back.

This makes me wonder if the attack on Pearl Harbor led to the mistreatment and hatred towards Japanese Americans.

Dropping a strong alliance was probably a bad decision.

This framework allows for a way to critically examine online privacy through the EU’s GDPR framework using the CIDER method, encouraging an inclusive perspective on user assumptions and biases. By engaging in each step of the method, people can develop a deeper understanding of privacy law implications and the diverse needs of digital users.

This passage talks about and highlights the risks and challenges of data security on social media platforms, where even big companies fail to protect users information. It emphasizes the importance of individual responsibility, such as enabling two-factor authentication, to help mitigate risks of potential data breaches and hacking attacks.

I feel like this might happen more naturally in Special Education because we spend a lot of time understanding the evaluation process, communicating with parents, managing student behavior and so on. Yay, sped!

I have only ever had one mentor actually co-teach with me, and I remember it so clearly. I also use the exact method he used in that moment very often. It is such a powerful technique. I am looking forward to doing this with my mentee. I am also quite nervous about it!

He kind of summarizes the events leading up to the declaration of war and the other countries doings. This is something that would not have to be done with congress because they would've been well informed of the tensions going on in the east.

His tone here is not respectful like the other speech. By painting the Japanese as gangsters, he can convey the message to the people that they should deserve the respect that other people should have.

Here, he is talking about accepting the challenge and not having to live in fear, implying they will begin a war with Japan. The other speech focuses on the events of that day, the loss of life, and the horrible betrayal carried out.

Here his tone is not respectful like the December 8th, 1941 speech. He expresses more anger and sadness for the lost lives. He refers to them as resourceful gangsters showing all loss of respect for them.

Roosvelt promotes unity in the American people, in order to keep standing strong.

Compared to the address to congress, the opening to the Fireside chat seems to be different in tone, as it uses fervont and informal vocabulary to the more grim and formal tone of the address.

Again, his terminology when addressing congress is formal, with the idea to send a serious message that could change the US

His manner of speech here emphasizes the importance of what he his addressing

Both passages end with some sort of mention of God, and how he will be with them.

Roosvelt uses repetition to show the various actions Japan has done that has affected the US, it is blatant call to action.

Thia shows the severity of the situations that the Japanese had started.

Also known as finger or hump coral. They have fused branches with round tips found in similar places at A. cervicornis including the Bahamas. It is seen as "least concern".

The sentence underscores the dual role of GI in public health and environmental management, emphasizing the need to consider human-induced landscape changes when evaluating health impacts related to climate change. Investing in GI not only promotes public health by reducing disease risk but also provides ecological benefits, such as enhanced biodiversity and improved air quality, showcasing the multifaceted benefits of such initiatives

The socio-economic aspects of zoonotic disease transmission, illustrating how cultural practices and livelihoods can contribute to increased human contact with potential disease reservoirs. The bushmeat trade has been linked to several zoonotic outbreaks, including Ebola, promoting discussion on sustainable alternatives that balance economic needs and health safety.

This establishes the core focus of infectious disease ecology, highlighting its dynamic nature and the interplay among various biological and environmental factors that contribute to disease transmission. This field has gained increased attention due to the rise of emerging infectious diseases, emphasizing the importance of interdisciplinary research in predicting and controlling outbreaks

Attending fully and decreasing our blocks to understanding are so important. You can really feel when these things are happening, or when they are not happening. So powerful to think about. One of the things that is happening for me is the need to have really strong boundaries with some of the members of my team, because they have a tendency to undermine and exhaust me. Its such an interesting dance to work with being open and attending, and also needing to not get manipulated by others' negativity.

definition: the movement of animals in response to light. Positive phototaxic is towards the light and negative phototaxic is moving away from the light.

As of 2023, urbanization in New Zealand was at 86.98%. very similar to past years, meaning there has been no break in growth for the biological environment to keep up.

I am going to add some of these to my PLC slides for our meeting this week. I love this idea.

My mentee seems to visibly relax when I do this.

I feel like all of the teachers in my department are feeling this way. There isn't a single person who hasn't cried with me, or said they wanted to quit since August.

Animalia seems to be a piece of a collection that every culture all over the world would have their own version of. Most cultures create stories and art about animals in their region. For example, Mesoamericans would have created artifacts of animals that live in the jungles of central America like snakes, or jaguars. And i a European cabinet of curiosity from this time period, it may not have been uncommon to find a Mesoamerican artifact of the animalia kind. This could have been brought over during the trans-Atlantic trade between the Americas and Europe. Or it could have simply been acquired by an explorer or conqueror.

The picture of this "basiliskos" seems as though it is exotic. However, by the way that this piece is persevered, we can infer that at one point this animal was alive. But we can not be too sure as to what it was. We can only assume that it is maybe a serpent-like creature.

Work cited: “The Cabinet of Curiosities - Google Arts & Culture.” Google, Google, artsandculture.google.com/theme/the-cabinet-of-curiosities/4QKSkqTAGnJ2LQ?hl=en. Accessed 27 Oct. 2024.

Could he be reiterating the structure of the poem in these lines? Beauty - lust truth - god and sprituality truth and beauty - love and lust in old age in the final stanza

Associated with either: 1. attending to livestock OR 2. being spiritual(notice pastor in the word)

You can still love when you are alive even if it is not young and lustfull.

**What is the affect of bordering a poem filled with symbols of spirituality and religion with sexuality and lust? **

Attic - Athenian Marble men - statues of gods and important people in ancient times Forest branches and the trodden weed - vases and urns played a large part of story telling in ancient times. This could suggest that the story of ones life was entirely lived in the forest.

Notes on Nymphs in Greek mythology: The Meliai (Meliae) were nymphs of the ash-trees. They were born when Gaia (Gaea, the Earth) was impregnated by the blood of the castrated Ouranos (Uranus, the Sky). They were wed by the men of the Silver Age--in the time before the first woman was created--and from them mankind was descended.

Context for sacrificing of a Heifer in Deuteronomy from

The Book of Deuteronomy explains how a heifer is to be sacrificed in atonement for innocent blood whenever a human being is found lying dead in the countryside and nobody knows how they have died (21:1-9)

Could this suggest that she who died with the urn was left to die alone in the forest? Is that the true story that the reader of this story is after?

Even the cow is preserved as beautiful in her death: **What sacrifice did the urn die for? **

From informational Christian website .-,GREEN,into%20the%20life%20of%20God.)

Green is a sign of life in nature and as such it represents growth, life and hope. Green is the colour worn most often during liturgies in Ordinary Time. It symbolises the graces that draw people into the life of God. Most of the Church's year is Ordinary Time.

**What does placing these scene during "ordinary time" suggest? **

"nor ever bid the Spring adieu?" These lines offer that the character is in a constant state of revival; does Keats call upon Spring in reference to Easter. Perhaps with this scenery he suggests that even those who died are constantly born again, eve if they are stuck being thought of differently forever.

Here, Keats argues that there is nothing to truly be sad about, as she cannot ever really leave since she is preserved in her death.

*Does Keats begin this as a poem of lust to suggest hopeless and unrequited passion to the point of wishing death for the sake of preservation? Or does he start with that passion as a distraction before offering a sobering recount of grieving someone who has really died? *

(From Oxford English Dictionary) "In Tempe" The proper name of a charming valley in Thessaly, watered by the Peneus, between Mounts Olympus and Ossa; used (already by the Roman writers) as a general name for a beautiful valley; hence for any delightful rural spot.

dales - a valley

Arcady - poetic version of Arcadia/home of hermes and Pan/rustic historic village

Have laptop out for pictures

(Oxford English Dictionary) To enhance the value of

Once again begins and ends with sexual imagery.

Question: How does beginning the first stanza with "unravish'd" and ending with "wild ecstasy" set the tone for the poem? Furthermore, how does historic imagery of peaceful isolation further or weaken this tone?

begins the subtle themes of lust/sex/purity throughout the poem. note the difference between reading this as "unravished bride of quietness" vs. "bride of unravished quietness"

As a graduate of the CU Denver IPTE program, I remember this very well. The people who went through the same program as I did were definitely on the forefront of the most recent research based methods.

This is the most important quality in ANY teacher.

Perhaps the local governments would work with their Fish and Wildlife communities to make more surveying jobs to monitor these less "notable" locations to avoid ignoring a possible invasive outbreak.

Definition: rapid increase in numbers or the rapid reproduction of a cell, part, or organism.

This is interesting. My Student teacher shared that he is very worried about understanding the curriculum, but everything else he worried about was personal stuff outside of school, like his living situation. There seems to be a lot outside of school that we are also all carrying. I hope to learn about how to support that aspect as well.

I don't think I will need to CREATE any challenges as they are already to prevalent in my school.

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Who was the leader/emperor of Russia at this time?

Cite your sources!

What is a "scorched earth policy?" Has the United States ever used this kind of policy?

Cite your sources!

Describe the Aleut sto us. Where do they live? What is their traditional lifestyle like? How do they live today?

Describe the Inuit to us. Where do they live? What is their traditional lifestyle like? How do they live today?

Interesting how acid rain transportation travels long distances through wind and eventually break down as acid precipitation. Though it leads to cross-border environmental damage, it proves to not only be a local issue but a regional and international concern that requires environmental policies and regulations to mitigate its widespread ecological and economic effects.

This is my biggest frustration as a student because many apps advertise as "free" but most of the features require a premium subscription. As a college student, this is not ideal and the amount of required personal information does not seem safe.

Just shows the significant focal points of combating acid rain due the large-scale emissions of pollutants that cause our environmental issues.

The fact that the pH levels just continued to decrease but in a dramatic way. Especially how it effected the aluminum concentrations.

I like that even though immigrants have been looked down upon for a long time, there is a lot of research today that is trying to bring about change and make life easier for immigrant children so that they feel part of school. With the help of interventions that many studies can determine, new resources can be found and the voices of many are heard.

Jacob was very lucky because he was able to find the support that many students don't find at school. Jacob found school to be a second home and he feels supported and has the support of his teachers, and I imagine also from other classmates.

Having that support from family or people close to me was always a great help in moving forward. Flor did not have that support and it makes me very sad to know that there are possibly students at the UCI who are struggling without family support, or without that phrase “you can do it, I believe in you.” That phrase is worth a lot because emotionally and psychologically it has helped me believe in myself.

Coping mechanism for poor people and lower classes laborers

Think about this

object of sublime terror crowds

Could this be the Malay? Probably not

the fact that revolution and instability repeated in the history causing a public and private anxiety, and causing the repetition evoking further anxiety and uncontrollability like a prophecy? terror evoking loop?

Sublime in Burke's aesthetics was based on the terror that the object of observance evoked in the eyes of the observer.

the model emphasizes the idea that HR practices as designed by the organization often differ form those actually implemented by managers and how they are perceived by employees

This still happens in current life. people with disability are asked to follow the rules of society, or they cannot live well. Although people purposely treat them differently and carefully, this is another type of discrimination. We should not expect and ask what they need to learn or do. Disability convenience should be a common thing.