Marianne Bronner Reviewing Editor; California Institute of Technology, United States In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your work entitled "Hypocretin underlies the evolution of sleep loss in the Mexican cavefish" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Senior Editor. Our decision has been reached after extensive consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife in its present form.

While all three reviewers found the work to potentially of great interest, they also raised significant concerns. In particular, the reviewers would like to see addition of loss of function experiments to confirm the role of hypocretin. We would be open in the future to a new submission that addresses the concerns of the reviewers, such that every effort would be made to return the paper to the original reviewers. We hope you find the comments of the reviewers, appended below, helpful in revising the manuscript.

Reviewer #1:

The manuscript "Hypocretin underlies the evolution of sleep loss in the Mexican cavefish" by Jaggard et al. addresses the neurological basis of sleep loss in cavefish. The authors found that the number of hypocretin neurons is increased in cavefish correlating with the previously observed sleep loss in cavefish populations. The authors use pharmacological treatments to dissect the neurological basis of the sleep phenotype in cave and surface fish. In addition, the authors link the changes in hypocretin activity to physiological phenotypes in the cavefish suggesting not only a plastic behavioral response but also plasticity in the underlying neuronal workings of hypocretin. The study is interesting and carefully executed. I fully support publication in eLife. I have only two comments, one technical, one conceptual.

I have some slight concerns with the quantitative PCR data. For example, I was not able to find the primer sequences for rpl13α in the publicly available genome information. In the case for gapdh the primers are designed poorly as they span only a very small intron and would give a similarly sized product from genomic DNA which is a common contamination in cDNA preparations no matter how careful the cDNA is prepared. Given that the qPCR data is crucial for the message of the manuscript, I would like to see some better designed controls.

While the plasticity part is quite exciting, I have some trouble to fully comprehend its implications. I can see that starvation is triggering a reduction in wakefulness, presumably through hypocretin. I am not sure, however, how novel or important this is for this study and how this compares to other systems? Also, how can the authors distinguish between causality and it being simply a readout of sleep need? Same is true for the lateral line part, what is the mechanistic basis of the reduction in lateral line signaling leading to a reduction in hypocretin signaling and how does this correlate with the presumably developmental differences that the authors mention in the Discussion (first paragraph)? I feel the manuscript would profit from an extended explanation of this part.

Reviewer #2:

This is a very important manuscript that provides a new link between the wakefulness promoting neuropeptide hypocretin and previously discovered differences in sleep behavior in surface- and cave-dwelling Astyanax mexicanus. The major finding is that upregulation of hypocretin at both the transcript and protein levels is correlated with increased wakefulness in cavefish. Pharmacological data support a direct relationship between the neuropeptide function and the cavefish behavior. Because hypocretin neurons are found in the hypothalamus this discovery provides a possible explanation for why this part of the brain is modified during the evolution of cavefish. The novel information within this manuscript will markedly improve our general understanding of the conserved role of hypocretin in the evolution of sleep regulation.

The following comments are offered to improve the manuscript.

The results show that knocking down hypocretin with a pharmacological inhibitor increases sleep duration in cavefish. However, there is no effect on surface fish. Could an effect on surface fish be achieved by knocking up hypocretin, possibly by injecting transcripts, an expression construct containing the gene, or the protein?

To extend the above comment, there is no explanation given as to why pharmacological inhibition of hypocretin does not affect surface fish if this neuropeptide is indeed a general regulator of sleep behavior.

Although controls for the quantitative PCR analyses are mentioned in the Materials and methods, I cannot find a place where the control data is illustrated in the manuscript. Ideally, the control data for quantitative PCR should be presented for evaluation alongside the experimental results. I understand that this may be a part of the "normalization" process, but it is stated that normalization was relative to surface fish controls (subsection “Quantitative PCR (qPCR)”). Perhaps the "control" genes themselves change between surface fish and cavefish. More information is needed about this possibility.

Why are the critical results for gentamycin treatment printed so lightly in Figures 3B, D, such that even the orbits are difficult to distinguish? Are there no neuromasts at all that survive the treatment? More contrast needs to be included in the photographs for the reader to be convinced about the effects of the treatment on the levels of DASPEI stained neuromasts. This is particularly true for cavefish, in which superficial neuromasts in the head are more numerous than in surface fish.

The quantitative results shown in Figure 3F do not seem to correspond well to the representative photographs in Figure 3I, K. It seems like there are more hypocretin stained cells in Figure 3I than in K, not only a decrease in the level of staining in each cell, and this is not evident in the quantification in Figure 3F. The same is true for Figures 4B, E, and G. Perhaps the images need to be enlarged.

To continue with the comment immediately above, it appears that there are variable levels of hypocretin staining in cell clusters in different parts of the section. Is this real, and if so, does it correspond to particular centers within the hypothalamus?

In the Discussion, it states that there are "no differences in the (sic hypocretin) genomic sequence" between the two morphs. A correction is needed because only exon sequences could have been determined from the transcriptome data of Gross et al. (2013).

In the fourth paragraph of the Discussion, the authors point out that the wake-promoting role of HCRT neurons is dependent on norepinephrine signaling. However, they fail to point out that norepinephrine has been demonstrated to be increased in cavefish (see Bilandzija et al., 2013, PloS ONE 8 (11): e80823). This information is important here. It should be added and the Discussion revised accordingly.

Reviewer #3:

Jaggard et al. examines hypocretin neurons in two different populations of the Mexican cavefish, Astyanax mexicanus. They identified difference in hypocretin neuron cell numbers in the surface-dwelling vs cave-dwelling populations. Pharmacological inhibition of HCRT signaling increases sleep duration in cavefish. Further they perform manipulations that promote sleep, such as lateral line ablation and starvation, and show that hcrt expression is inhibited in cavefish and not in surface fish. Based on these results the authors conclude that alteration in HCRT signaling contribute to the evolution of sleep loss.

Given the conserved role of HCRT in sleep regulation in different species, the conclusion of this study seems logical and expected. However, their experimental results are insufficient to support this conclusion. The difference in hypocretin neuron cells numbers between these two species are clear, yet beyond this correlation the evidence that the study provides attempting to causally link HCRT and sleep loss is weak.

1) The authors draw their conclusion based on three experimental manipulations- pharmacological manipulation of Hcrt receptor 2, lateral line ablation using gentamicin, and starvation. The only "specific" manipulation for Hcrt system is the pharmacological manipulation using TCSOX229 (TCS). The authors cite Plaza-Zabala et al. 2012, which shows the application of this HCRTR2 in mice. A big issue in this study is that there is no control to show that the application actually affects hypocretin neurons in zebrafish. What is the half-life of TCS in water? How do we know that TCS is selective for HCRTR2 in zebrafish? The protein sequences of HCRTR2 in zebrafish and mouse are different and what is the evidence that TCS is a specific HCRTR2 antagonist in zebrafish? Even if this has been shown elsewhere, the authors need to demonstrate that it works in their lab in the context of this study as well. For example, the authors could show altered activity of cells expressing HCRTR upon TCS treatment using c-fos, p-ERK staining, etc. Also how is the dosage determined? What is the evidence that TCS actually penetrates and reaches the hypothalamic hypocretin neurons when supplied in bath water at this concentration? In mice TCS is administered by IP route in a volume of 5 mL/kg body weight. In this study the TCS doses were based on a pilot experiment testing the effects of this antagonist on the hyperactivity induced by hypocrein-2 in C57BL/6J mice. A similar experiment using hypocretin overexpression or knock out could be done to determine the effective concentration.

2) Pharmacological manipulations can have pleiotropic effects depending on the dosage even if the authors show that it targets HCRTR2. The possibility that it can target other proteins in addition to HCRT will be difficult to rule out. Therefore the authors need to show independent lines of evidence that HCRT is involved in sleep loss using genetic manipulation using morpholinos against HCRT/HCRTR and overexpressing HCRT/HCRTR, which should show the opposite phenotype from morpholino treatment.

3) Presumably many other cell types other than HCRT could show differences between surface and cave-dwelling types apart from HCRT. Modulation of several neuropeptide/neurotransmitter systems could affect sleep dramatically. For example, differences in CRH (corticotropin-releasing- hormone) or histaminergic, or NPY system could alter sleep. The authors should examine differences in other major neuropeptide systems that are involved in sleep regulation between surface and cave fish.

4) I am confused about the some of the supplementary figure information. For example, what is the difference between Figure 2A, B vs. Figure 2—figure supplement 1A and B. Are they both treated with TCS as the figures themselves indicate?

[Editors’ note: what now follows is the decision letter after the authors submitted for further consideration.]

Congratulations, we are pleased to inform you that your article, "Hypocretin underlies the evolution of sleep loss in the Mexican cavefish", has been accepted for publication in eLife.

The authors investigate the role of a hypocretin in controlling sleep in the blind Mexican cavefish. The results suggest that evolutionary changes in hypocretin regulation may have been a driving factor in the evolution of sleep loss. On further discussion the reviewers agreed that the morpholino controls recommended by reviewer #2 (below) are not essential for the publication of this work.

Reviewer #1:

In the revised manuscript "Hypocretin underlies the evolution of sleep loss in the Mexican cavefish" by Jaggard et al. the authors have addressed all my previous comments. The manuscript is now substantially improved and provides several novel technical advances in the cavefish field. The use of Gal4 UAS in mosaic transgenics is elegant and very convincing. The morpholino results by its own would technically require some additional controls (e.g. RT-PCR of splice morpholino or RNA rescue), however, as they only serve as additional evidence, their use strengthen the other results significantly, especially as the authors have established their behavioral assays now in larval fish which will be useful for future studies.

There are still some oversight mistakes but I assume these can be taken care through editorial revision (e.g. Results, fifth, sixth, seventh and ninth paragraphs; Discussion second and third paragraphs).

Reviewer #2:

This manuscript is the resubmission of a previous manuscript submitted to eLife, that was not accepted for publication. The major criticism was that not enough evidence, above pharmacological studies, was included to strongly support the possibility that hypocretin is evolved in cavefish sleep loss, and that it was unlikely that the required information could be produced within the time frame required by the journal. The present manuscript answers some (but not all) of the previous objections, and is considerably improved over the previous submission.

An improvement is the development of a morpholino-based procedure for knockdown of the hypocretin gene, which resulted in an increase in sleep duration in cavefish, without affecting sleep in surface fish, as predicted by the authors’ hypothesis.

Morpholinos have been in use for some time in cavefish, most recently by Bilandzija (2013) for oca2 knockdown. In the latter study, it was simple to determine the morpholino effects because visible pigmentation was reduced or eliminated. In other morpholino studies, however, controls in addition to scrambled morpholinos were done to be certain the that morpholinos were working on the expected targets. Such controls are not reported here, and would be seem to be necessary for publication in eLife.

A translation blocking morpholino is used in these experiments, so the appropriate control would be to compare hypocretin protein levels using the available antibody, either in Western blots (preferred for more exact quantification) or in immuno-imaging experiments of the brain. These controls could be done rather quickly.

Reviewer #3:

In this manuscript, Jaggard et al. investigate HCRT and its role in controlling sleep in Astyanix mexicanus, the blind Mexican cavefish. Through pharmacological and genetic manipulations, the authors show that an increase in HCRT is associated with sleep loss in the Pachón cavefish. Comparing the results of the cave population to the surface population, along with previous zebrafish and mouse data, the authors conclude that evolutionary changes in HCRT function and regulation may have been a driving factor in the evolution of sleep loss in the Pachón cavefish.

This is a very interesting paper. The authors addressed all of the reviewer's concerns and the data presented will, no doubt, further understanding of HCRT and its evolutionary role in sleep regulation as well as provide fuel for further experiments addressing the evolution of sleep.

The data presented is very thorough and the authors' interpretation of the role of HCRT in sleep regulation of the Pachón cavefish population is very convincing.