FSHβ links photoperiodic signalling to seasonal reproduction in Japanese quail

Reviewer #1 (Public Review):

The authors investigated the molecular correlates in potential neural centers in the Japanese quail brain associated with photoperiod-induced life-history states. The authors simulated photoperiod to attain winter and summer-like physiology and samples of neural tissues at spring, and autumn life-history states, daily rhythms in transcripts in solstices and equinox, and lastly studies FSHb transcripts in the pituitary. The experiments are based on a series of changes in photoperiod and gave some interesting results. The experiment did not have a control for no change in photoperiod so it seems possible that endogenous rhythms could be another aspect of seasonal rhythms that lack in this study. The short-day group does not explain the endogenous seasonal response.

The manuscript would benefit from further clarity in synthesizing different sections. Additionally, there are some instances of unclear language and numerous typos throughout the manuscript. A thorough revision is recommended, including addressing sentence structure for improved clarity, reframing sentences where necessary, correcting typos, conducting a grammar check, and enhancing overall writing clarity.

Data analysis needs more clarity particularly how transcriptome data explains different physiological measures across seasonal life-history states. It seems the discussion is built around a few genes that have been studied in other published literature on quail seasonal response. Extending results on the promotor of DEGs and building discussion is an extrapolating discussion on limited evidence and seems redundant.

Last, I wondered if it would be possible to add an ecological context for the frequent change in the photoperiod schedule and not take account of the endogenous annual response. Adding discussion on ecological relevance would make more sense.

Reviewer #2 (Public Review):

This study is carefully designed and well executed, including a comprehensive suite of endpoint measures and large sample sizes that give confidence in the results. I have a few general comments and suggestions that the authors might find helpful.

1. I found it difficult to fully grasp the experimental design, including the length of light treatment in the three different experiments (which appears to extend from 2 weeks up to 8 weeks). A graphical description of the experimental design along a timeline would be very helpful to the reader. I suggest adding the respective sample sizes to such a graphic, because this information is currently also difficult to keep track of.

2. The authors use a lot of terminology that is second nature to a chronobiologist but may be difficult for the general reader to keep track of. For example, what is the difference between "photoinducibility" and "photosensitivity"? Similarly, "vernal" and "autumnal" should be briefly explained at the outset, or maybe simply say "spring equinox" and "fall equinox."

3. What was the rationale for using only male birds in this study? The authors may want to include a brief discussion on whether the expected results for females might be similar to or different from what they found in males, and why.

4. The authors used the Bonferroni correction method to account for multiple hypothesis testing of measures of testes mass, body mass, fat score, vimentin immunoreactivity and qPCR analyses in Study 1. I don't think Bonferroni is ever appropriate for biological data: these methods assume that all variables are independent of each other, an assumption that is almost never warranted in biology. In fact, the data show clear relationships between these endpoint measures. Alternatively, one might use Benjamini-Hochberg's FDR correction or various methods for calculating the corrected alpha level.

5. The graphical interpretations of the results shown in Figure 1n and Figure 3e, along with the hypothesized working model shown in Figure S5, might best be combined into a single figure that becomes part of the Discussion. As is, I do not think these interpretative graphics (which are well done and super helpful!) are appropriate for the Results section.

Reviewer #3 (Public Review):

It is well known that as seasonal day length increases, molecular cascades in the brain are triggered to ready an individual for reproduction. Some of these changes, however, can begin to occur before the day length threshold is reached, suggesting that short days similarly have the capacity to alter aspects of phenotype. This study seeks to understand the mechanisms by which short days can accomplish this task, which is an interesting and important question in the field of organismal biology and endocrinology.

The set of studies that this manuscript presents is comprehensive and well-controlled. Many of the effects are also strong and thus offer tantalizing hints about the endo-molecular basis by which short days might stimulate major changes in body condition. Another strength is that the authors put together a compelling model for how different facets of an animal's reproductive state come "on line" as day length increases and spring approaches. In this way, I think the authors broadly fulfill their aims.

I do, however, also think that there are a few weaknesses that the authors should consider, or that readers should consider when evaluating this manuscript. First, some of the molecular genetic analyses should be interpreted with greater caution. By bioinformatically showing that certain DNA motifs exist within a gene promoter (e.g., FSHbeta), one is not generating robust evidence that corresponding transcription factors actually regulate the expression of the gene in question. In fact, some may argue that this line of evidence only offers weak support for such a conclusion. I appreciate that actually running the laboratory experiments necessary to generate strong support for these types of conclusions is not trivial, and doing so may even be impossible. I would therefore suggest a clear admission of these limitations in the paper.

Second, I have another issue with the interpretation of data presented in Figure 3. The data show that FSHbeta increases in expression in the 8Lext group, suggesting that endogenous drivers likely act to increase the expression of this gene despite no change in day length. However, more robust effects are reported for FSHbeta expression in the 10v and 12v groups, even compared to the 8Lext group. Doesn't this suggest that both endogenous mechanisms and changes in day length work together to ramp up FSHbeta? The rest of the paper seemed to emphasize endogenous mechanisms and gloss over the fact that such mechanisms likely work additively with other factors. I felt like there was more nuance to these findings than the authors were getting into.

Third, studies 1 - 3 are well controlled; however, I'm left wondering how much of an effect the transitions in day length might have on the underlying molecular processes that mediate changes in body condition. While the changes in day length are themselves ecologically relevant, the transitions between day length states are not. How do we know, for example, that more gradual changes in day length that occur over long timespans do not produce different effects at the levels of the brain and body? This seemed especially relevant for study 3, where animals experience a rather sudden change in day length. I recognize that these experimental methods are well described in the literature, and they have been used by endocrinologists for a long time; nonetheless, I think questions remain.