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  1. Evaluation Summary:

    Embryonic behavior is a widespread phenomenon that remains poorly understood in any system. Ardiel et al. describe new quantitative methods for imaging late embryo behavior in C. elegans, which will be of great interest as a technical innovation. They identify a novel rhythmic behavior (which they call slow wave twitch) in very late embryogenesis that includes repeated periods of quiescence, and show that this behavior depends on a known pro-sleep neuron and neuropeptide. Although the biological function of the rhythmic sleep behavior is unclear, it has the potential to serve as a model for understanding the mechanisms and purposes of sleep in other model organisms.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)

  2. Reviewer #1 (Public Review):

    The authors combined light-sheet-based imaging with computational tools to track C. elegans motor behavior throughout the last ~5hrs of embryonic development. Using PCA and quantitative methods, the authors identified postures and movements along developmental time. Early on, embryonic movements are continuous and dominated by dorsoventral "flips". The embryos then enter a period of low activity followed by a phase where episodic sinusoidal waves are predominant. The authors later defined this episodic behavior as "slow wave twitch" (SWT). These phases are stereotyped across embryos, and the early flipping phase depends on neuronal synaptic transmission. Using a brightfield high-throughput method the authors implicated neuropeptides in SWT. Finally, they demonstrated that a somnogenic neuropeptide secreted from RIS neurons mediates the quiescent periods observed during SWT.

    At a high level, the authors developed a pipeline to capture behavior during late embryonic development to make the following conclusions: 1) Embryonic behaviors followed a stereotyped trajectory, with early flipping and a late-stage dominated by episodic sinusoidal crawling-like waves. 2) Synaptic transmission is necessary for late-stage episodic movements. 3) A peptidergic neuron known to promote a sleep-like state in the hatched animals promotes quiescent periods observed during SWT. Overall these conclusions are well supported by the presented data. This work focuses on the late stages of development when behaviors emerge, a heavily understudied period. The study provides some of the first insights into embryonic behaviors in C.elegans and lays the groundwork for further studies using this system. Therefore, this work should have a significant impact on the fields of neurodevelopment and neuroscience.

  3. Reviewer #2 (Public Review):

    While larval and adult C. elegans behavior has been extensively studied over the past few decades there has been little attention to the behavior of embryos during the second half of embryogenesis. One reason for the paucity of work is the inherent difficulty of monitoring the behavior of a worm spinning inside an eggshell. Previous careful observation and genetic studies have indicated that early movements represent spontaneous contractions of muscle and that later, more coordinated movement, depends on a developed and functioning nervous system. The authors developed a semi-automated tracking analysis system, including new software, that allows the capture of moving worm embryos. In their initial analysis, they use GFP-tagged skin cells to digitize worm postures and mathematically-simplify worm movements into four principles components, which they term eigen-embryos. The result is an effective methodology for mathematical description of late embryo behavior. The fact that it accurately captures the major features of late embryonic movement that are known make the convincing case that the methodology is sound and accurately reflects biological reality. In addition, there is some novelty in defining some nuances of embryonic behavior that were not previously described.

    The authors then develop a brightfield assay that allows high-throughput analysis of late embryonic movements. They demonstrate that this method can capture much of the detail that they described in the eigen-embryo analysis, but can be adapted to screen multiple embryos simultaneously. As such, this is an important tool that should allow the authors and other researchers in the field to conduct genetic screens into late embryonic behavior, something that has been difficult to date.

    Most interesting, from a biological perspective, is the authors' discovery of a period of rhythmic quiescence in the last two hours of embryogenesis. They identify a 20-40 mHz cycle of repeated (mostly forward it appears) movement punctuated by a period of quiescence, which they term slow wave twitch (SWT). They use genetic and optogenetic strategies to demonstrate that this depends on both synaptic activity of the nervous system and the production of FLP-11 neuropeptide by the pro-sleep neuron RIS. These observations tie the phenomenon to the well-studied mechanisms of worm sleep during larval development, suggesting that the embryo is "practicing" periods of sleep in the hours before hatching, that these periods of sleep are an important physiological component of the anabolic process of cuticle synthesis, and/or that the sleep bouts are important for consolidation of nervous system function. The possibility that this could serve as a model for understanding the biological functions of mammalian in utero sleep is exciting, even if this has nothing to do with understanding autism-spectrum disorders as the authors speculate.

  4. Reviewer #3 (Public Review):

    In this manuscript by Ardiel et al, the authors develop a novel automated approach to behavioral classification of C elegans embryos. They provide detailed validation of this system, and in exploiting it, identity a previously unknown period of behavioral quiescence in the late embryo that is likely dependent on synaptic transmission. Then shifting to a high throughput assay to focus on this specific period, they provide evidence for a sleep/quiescent like state. The highly technical approaches they develop can potentially be used by many labs, and the rich behavioral dataset can likewise serve as a foundation for numerous future studies. However, I have major concerns. Foremost is that at its core, there are very limited new biological conclusions to come out of this work, which will dampen impact of the techniques described. Other major issues:

    1. The period of quiescence/SWT is intriguing, though I believe the authors are premature in their conclusions. SWT shares molecular features of worm sleep, but the work does not go far enough to prove quiescence. Are the animals paralyzed? Does SWT have features of sleep homeostasis? I do not think the authors need to prove every feature exhaustively, but at a minimum, should demonstrate that it is a reversible state. Moreover, the authors convert midway through the work to calling this slow wave twitch (SWT). These are all words that are likely chosen specifically to evoke a sense of "sleeping" from readers, but the behavior does not really seem like twitching, and are these really slow waves?

    2. For the high throughput portion, the authors find some mutants that disrupt SWT. they should also test to see whether earlier embryonic behaviors are affected (as was tested with unc13), as this would very much alter the interpretation

    3. The Discussion really overreaches. There is a heavy focus on sleep and autism, despite no clear evidence that SWT is sleep. I certainly agree discussions can be speculative, but the tone here seems to make claims that are absolutely not supported by the data. I would suggest ending the manuscript with "Together, these similarities suggest that SWT may be akin to the developmentally timed sleep associated with each larval molt" which underscores to readers that the data really ends short of showing SWT is indeed sleep.

    4. The manuscript feels disjointed as a whole in some respects, as the authors put huge effort into the methodology of Figures 1-4, and then completely shift approaches. Perhaps they can reframe the work to better emphasize how MHHT led to an important biological discovery, and then better justify why moving to a new system was necessary. Also important - the manuscript portion describing Figs 1-4 is so technical that most readers will not be able to follow. Perhaps there are ways to better hand hold for a broad audience.

    5. Fig 6g attempts to show that the correlation between RIS calcium transients and motion is reduced in FLP-11 mutants. While this reduction is evident, it still seems like a very strong correlation, undercutting the idea that FLP-11 is required for SWT, as it is for sleep. This further calls into question whether SWT is the same at lethargus.