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Programmed cell death is prominent in developing nervous systems across evolution, but its function remains obscure. Recent work suggests that it might impact behavior, but an examination of its effects on behavior and underlying neuronal circuits in intact organisms has not been determined. In this manuscript we report that programmed cell death sculpts the developing nervous system and shapes innate behavior. Using synaptic labeling, in vivo calcium imaging, targeted rescue of programmed cell death, and automated high-resolution analysis of cell death mutants, we find that loss of programmed cell death alters animal behavior. These findings reveal that neuronal cell death during development provides a reservoir of fates and circuit connections that could be accessed on evolutionary time scales to modify innate behavioral programs. Our manuscript thus answers one of the major outstanding questions in developmental neuroscience—why programmed cell death is so prevalent—by identifying consequences for brain function at the subcellular, cellular, circuit, and behavioral level. This study will be of interest to those interested in evolution of the nervous system and behavior, developmental biology, and neural circuit development.
We thank the reviewers for their careful attention to the manuscript. Both reviewers were enthusiastic about the work. Here we address their suggestions. As noted below, we have already addressed most of their points, and we discuss in detail the remaining point—whether it is possible to perform experiments for a more specific targeting of the undead RIM cell death event to provide additional evidence for its role in altering reversal behavior.
2. Description of the planned revisions
*Reviewer 1: “1. The argument that that differences in reversal behavior are likely attributable to the difference in RIM neuron numbers in the ced-3 rescue studies is very plausible. Nonethless, there remains the possibility that for some reason in animals with 4 RIMs there may be a more global effect on the fate of cells slated to die, unrelated to the number of RIMs. I think there are two ways to test this. (1) quantify the behavior in 2- vs. 4- RIM neurons in animals also containing a marker for other undead neurons, and see if there is any correlation between 4 RIMs and survival of unrelated neurons (but preferably reasonably closely related by lineage- in case that's the issue). (2) Since the authors are able to distinguish the undead cells, can they perform laser ablations on these cells and assess whether behavior is restored to normal values?” *
- *We agree that this point is already very plausible. We also appreciate the reviewer’s suggestions on how to extend this conclusion.
Regarding suggestion (1): Unfortunately there is not a reliable marker for undead neurons (although a current project in the lab is indeed to develop one). However, we note that the undead RIM sister cells adopt a RIM neuron fate in 96% of ced-3 mutants, while with other undead cells investigated neuron fate adoption ranged from 59% (ASEL) to 77% (ASER). This suggests that the undead RIM fate adoption is not strongly correlated with the fates of other undead cells.
Regarding suggestion (2): We attempted to perform laser ablation of undead RIM neurons in ced-3 mutants, but we could not overcome the technical hurdles (despite our lab’s expertise in laser axotomy). We found that we could not reliably remove both undead RIMs without damaging the wildtype RIM that is in close proximity, especially in the quantities of animals necessary for behavioral experiments.
As an alternative, we plan to perform more targeted experiments to manipulate cell death in the undead RIM to address the points raised by both reviewers. Our goal is to generate two strains. In one, programmed cell death is prevented specifically in the RIM neurons in wild type animals. We hope to achieve this by either transgenic expression of a gain-of-function mutation of ced-9, or else by RIM-specific RNAi against egl-1, ced-3 or ced-4. To do this we will use the RIM promoter tdc-1, which is confined to RIM and RIC. The second strain will allow cell death to occur only in RIM (and RIC) in animals that otherwise have no cell death. Here, we will drive wild-type ced-3 or ced-4 under the tdc-1 promoter in the corresponding mutant background.
We note 2 caveats for both of these approaches: 1) RIC also has an undead sister; 2) Most probably, the tdc-1 promoter will not be active in time to block cell death. Caveat #2 is actually the reason why we did not do these experiments initially (instead we used the most specific promoter we could find that is expressed early in the RIM lineage, before RIM is born).
However, we agree that if successful these experiments would complement the existing experiments, and we will build all these strains.
Reviewer 2: “Mosaic rescue of RIM via stochastic loss of a rescue array helped demonstrate the contribution RIMu have to the locomotor phenotype. As the authors emphasise these animals have many other undead cells (outside of the reverse network). A conditional rescue of only the RIMu would greatly improve the strength of the claims made. Would a conditional RIM egl-1 knockdown (via RNAi) be possible to selectively inhibit apoptosis in those neurons. This experiment should be considered OPTIONAL. It may be that such specific promoters do not allow for egl-1 RNAi to function at the right time to rescue death.”
- *We appreciate the reviewer’s suggestion. As stated above, we are working to perform an expanded version of these exact experiments, as well as their converse. However, as the reviewer notes, it is very possible that the timing of expression will prevent these approaches from working (Caveat #2 above).
Reviewer 2: There is a slight issue with interpretation of the data with the mosaic GLR-1::tagRFP Fig 2M which reveals the postsynaptic compartment of one RIM even though there are two present. There seems to be no obvious apposition between pre/post and they somewhat seem to be floating in space. Why is this the case? One would have imagined that the structures in Fig 2L would be tiled composites of both AIB & RIM pre and postsynaptic elements coalescing. Can the authors provide an alternative explanation for this phenotype. Nevertheless, the data on Fig 2L seems solid.. that is animals with extra undead RIM cells have additional cell-type specific synaptic terminals
We have selected a different micrograph that is more representative of the RIM post-synapses in ced-3 mutants. In this animal, the array labeling the post-synapses in RIM has been lost from one of the two RIM neurons, making it easier to discern that the post-synapses are apposed to the AIM pre-synaptic marker (Fig 1M).
Reviewer 2: Clarity should be improved around the use of 'expected number' in figure 1. The description of the metric 'The 'expected number' is defined as the number of neurons of the type present in wild-type animals, plus the number of lineage-proximate undead cells.' suggests that expected (blue) regions of pie charts represents lineages with expected sum total of wt and extra undead cells. However, in reference to panel H 'The wild-type animal has two RIM neurons, and the ced-3(n717) animal has two additional RIMlike cells and is counted as contributing to the orange "more than expected" sector in panel (A)' it is said that the animals with 2 WT accompanied by each undead sister contributes to more than expected (orange) region. These appear inconsistent. Can you qualify?
We thank the reviewer for this point and have added a schematic to clarify the quantification of undead cell fates (Fig. 1).
Reviewer 2: Specific observations shown in supplemental data SI-L despite being cited in the text is not explained or formally referenced. The details of these panels should either be briefly explained/their inclusion qualified in the text or simply remove from the figure
We have added reference to these figures in the main text “Undead cells are even capable of producing complex morphology, such as the highly branched dendrites of the PVD neurons (Figure S1I-L).” (p. 3)
Reviewer 2: The dual image photomicrographs could be in green/ magenta or red/cyan to make colourblind friendly.
We have updated micrograph colors to be colorblind friendly (Fig 1K-M, S1L).
Reviewer 2: Do the authors have data with the pRIMtagRFP egl-nucGFP. If they do it would be useful to show it.
We have added a micrograph of egl-1::GFP and RIM labeled using NeuroPAL (Fig. S2A).
Reviewer 1: 2. The authors speculate, if I understand correctly, that the mechanism by which reversal frequencies are decreased in 4 RIM animals may be that the reversal state is stabilized, resulting in longer reversals and consequently fewer reversal events. This is a nice model that is testable. The authors could, for example, examine the connections of RIM neurons to the AVA neuron, a main command interneuron for reversal initiation, and assess whether there are indeed more such synapses. Furthermore, the authors can assess whether the frequency of AVA firing is decreased. Of course, there are other plausible mechanisms involving connectivity of other neurons onto AVA which could explain the phenomenon. The authors may wish to add a comment regarding this in the discussion.
We thank the reviewer for this suggestion. There are multiple postsynaptic receptors expressed in AVA for RIM neurotransmitters and the contribution of each to reversal behavior is still being debated, making it challenging to dissect the contribution of each of these to the effects on reversal behavior mediated by the undead RIM. Given this, we believe that addressing this point experimentally is beyond the scope of this paper. We have added a sentence in the discussion commenting on this as a future direction for this work “The mechanism of the downstream circuit mediating the effects of the undead RIM could be determined through quantification of AVA postsynaptic receptors and examining reversal behavior of cell death mutants with knockouts of AVA receptors.”