AgRP1 modulates breeding season-dependent feeding behavior in female medaka

Reviewer #1 (Public review):

Summary:

The authors use the teleost medaka as an animal model to study the effect of seasonal changes in day-length on feeding behaviour and oocyte production. They report a careful analysis of how day-length affects female medakas and a thorough molecular genetic analysis of genes potentially involved in this process. They show a detailed analysis of two genes and include a mutant analysis of one gene to support their conclusions

Strengths:

The authors pick their animal model well and exploit the possibilities to examine in this laboratory model the effect of a key environmental influence, namely the seasonal changes of day-length. The phenotypic changes are carefully analysed and well-controlled. The mutational analysis of the agrp1 by a ko-mutant provides important evidence to support the conclusions. Thus this report exceeds previous findings on the function of agrp1 and npyb as regulators of food-intake and shows how in medaka these genes are involved in regulating the organismal response to an environmental change. It thus furthers our understanding of how animals react to key exogenous stimuli for adaptation.

Weaknesses:

The authors are too modest when it comes to underscoring the importance of their findings. Previous animal models used to study the effect of these neuropeptides on feeding behaviour have either lost or were most likely never sensitive to seasonal changes of day length. Considering the key importance of this parameter on many aspects of plant and animal life it could be better emphasised that a suitable animal model is at hand that permits this.
The molecular characterization of the agrp1 ko-mutant that the authors have generated lacks some details that would help to appreciate the validity of the mutant phenotype. Additional data would help in this respect.

Reviewer #2 (Public review):

Summary:

The authors investigated the mechanisms behind breeding season-dependent feeding behavior using medaka, a well-known photoperiodic species, as a model. Through a combination of molecular, cellular, and behavioral analyses, including tests with mutants, they concluded that AgRP1 plays a central role in feeding behavior, mediated by ovarian estrogenic signals.

Strengths:

This study offers valuable insights into the neuroendocrine mechanisms that govern breeding season-dependent feeding behavior in medaka. The multidisciplinary approach, which includes molecular and physiological analyses, enhances the scientific contribution of the research.

Weaknesses:

While medaka is an appropriate model for studying seasonal breeding, the results presented are insufficient to fully support the authors' conclusions.

Specifically, methods and data analyses are incomplete in justifying the primary claims:
- the procedure for the food intake assay is unclear;
- the sample size is very small;
- the statistical analysis is not always adequate.

Additionally, the discussion fails to consider the possible role of other hormones that may be involved in the feeding mechanism.

Reviewer #3 (Public review):

Summary:

Understanding the mechanisms whereby animals restrict the timing of their reproduction according to day length is a critical challenge given that many of the most relevant species for agriculture are strongly photoperiodic. However, the principal animal models capable of detailed genetic analysis do not respond to photoperiod so this has inevitably limited progress in this field. The fish model medaka occupies a uniquely powerful position since its reproduction is strictly restricted to long days and it also offers a wide range of genetic tools for exploring, in depth, various molecular and cellular control mechanisms.

For these reasons, this manuscript by Tagui and colleagues is particularly valuable. It uses the medaka to explore links bridging photoperiod, feeding behaviour, and reproduction. The authors demonstrate that in female, but not male medaka, photoperiod-induced reproduction is associated with an increase in feeding, presumably explained by the high metabolic cost of producing eggs on a daily basis during the reproductive period. Using RNAseq analysis of the brain, they reveal that the expression of the neuropeptides agrp and npy that have been previously implicated in the regulation of feeding behaviour in mice are upregulated in the medaka brain during exposure to long photoperiod conditions. Unlike the situation in mice, these two neuropeptides are not co-expressed in medaka neurons, and food deprivation in medaka led to increases in agrp but also a decrease in npy expression. Furthermore, the situation in fish may be more complicated than in mice due to the presence of multiple gene paralogs for each neuropeptide. Exposure to long-day conditions increases agrp1 expression in medaka as the result of increases in the number of neurons expressing this neuropeptide, while the increase in npyb levels results from increased levels of expression in the same population of cells. Using ovariectomized medaka and in situ hybridization assays, the authors reveal that the regulation of agrp1 involves estrogen acting via the estrogen receptor esr2a. Finally, a loss of agrp1 function mutant is generated where the female mutants fail to show the characteristic increase in feeding associated with long-day enhanced reproduction as well as yielding reduced numbers of eggs during spawning.

Strengths:

This manuscript provides important foundational work for future investigations aiming to elucidate the coordination of photoperiod sensing, feeding activity, and reproduction function. The authors have used a combination of approaches with a genetic model that is particularly well suited to studying photoperiodic-dependent physiology and behaviour. The data are clear and the results are convincing and support the main conclusions drawn. The findings are relevant not only for understanding photopriodic responses but also provide more general insight into links between reproduction and feeding behaviour control.

Weaknesses:

Some experimental models used in this study, namely ovariectomized female fish and juvenile fish have not been analysed in terms of their feeding behaviour and so do not give a complete view of the position of this feeding regulatory mechanism in the context of reproduction status. Furthermore, the scope of the discussion section should be expanded to speculate on the functional significance of linking feeding behaviour control with reproductive function.

Author response:

Reviewer #1 (Public review):

Summary:

The authors use the teleost medaka as an animal model to study the effect of seasonal changes in day-length on feeding behaviour and oocyte production. They report a careful analysis of how day-length affects female medakas and a thorough molecular genetic analysis of genes potentially involved in this process. They show a detailed analysis of two genes and include a mutant analysis of one gene to support their conclusions

Strengths:

The authors pick their animal model well and exploit the possibilities to examine in this laboratory model the effect of a key environmental influence, namely the seasonal changes of day-length. The phenotypic changes are carefully analysed and well-controlled. The mutational analysis of the agrp1 by a ko-mutant provides important evidence to support the conclusions. Thus this report exceeds previous findings on the function of agrp1 and npyb as regulators of food-intake and shows how in medaka these genes are involved in regulating the organismal response to an environmental change. It thus furthers our understanding of how animals react to key exogenous stimuli for adaptation.

Weaknesses:

The authors are too modest when it comes to underscoring the importance of their findings. Previous animal models used to study the effect of these neuropeptides on feeding behaviour have either lost or were most likely never sensitive to seasonal changes of day length. Considering the key importance of this parameter on many aspects of plant and animal life it could be better emphasised that a suitable animal model is at hand that permits this. The molecular characterization of the agrp1 ko-mutant that the authors have generated lacks some details that would help to appreciate the validity of the mutant phenotype. Additional data would help in this respect.

We would like to thank Reviewer #1 for the really constructive advice. In the revised manuscript, we will try to provide more information on the molecular characterization of the agrp1 KO-mutant and to emphasize the importance of our present animal model that permits the analysis of neuropeptide effects on feeding behavior in response to seasonal changes of day length.

Reviewer #2 (Public review):

Summary:

The authors investigated the mechanisms behind breeding season-dependent feeding behavior using medaka, a well-known photoperiodic species, as a model. Through a combination of molecular, cellular, and behavioral analyses, including tests with mutants, they concluded that AgRP1 plays a central role in feeding behavior, mediated by ovarian estrogenic signals.

Strengths:

This study offers valuable insights into the neuroendocrine mechanisms that govern breeding season-dependent feeding behavior in medaka. The multidisciplinary approach, which includes molecular and physiological analyses, enhances the scientific contribution of the research.

Weaknesses:

While medaka is an appropriate model for studying seasonal breeding, the results presented are insufficient to fully support the authors' conclusions.

Specifically, methods and data analyses are incomplete in justifying the primary claims:
- the procedure for the food intake assay is unclear;

- the sample size is very small;

- the statistical analysis is not always adequate.

Additionally, the discussion fails to consider the possible role of other hormones that may be involved in the feeding mechanism.

We would like to thank Reviewer #2 for the helpful comments. As the reviewer suggested, we will try to edit the paragraph describing the procedure for the food intake assay to make it much easier for the readers to understand in the revised manuscript. In Figure 1-Supplementary figure 2, RNAseq was performed to search for the candidate neuropeptides, and that’s why the sample size was the minimum. On the other hand, each group in the other experiments consist of n ≥ 5 samples, which is usually accepted to be an adequate sample size in various studies (cf. Kanda et al., Gen Comp Endocrinol., 2011, Spicer et al., Biol Reprod., 2017). As for the statistical analyses, we will revise our manuscript so that the readers may be convinced with the validity of our statistical analyses.

Reviewer #3 (Public review):

Summary:

Understanding the mechanisms whereby animals restrict the timing of their reproduction according to day length is a critical challenge given that many of the most relevant species for agriculture are strongly photoperiodic. However, the principal animal models capable of detailed genetic analysis do not respond to photoperiod so this has inevitably limited progress in this field. The fish model medaka occupies a uniquely powerful position since its reproduction is strictly restricted to long days and it also offers a wide range of genetic tools for exploring, in depth, various molecular and cellular control mechanisms.

For these reasons, this manuscript by Tagui and colleagues is particularly valuable. It uses the medaka to explore links bridging photoperiod, feeding behaviour, and reproduction. The authors demonstrate that in female, but not male medaka, photoperiod-induced reproduction is associated with an increase in feeding, presumably explained by the high metabolic cost of producing eggs on a daily basis during the reproductive period. Using RNAseq analysis of the brain, they reveal that the expression of the neuropeptides agrp and npy that have been previously implicated in the regulation of feeding behaviour in mice are upregulated in the medaka brain during exposure to long photoperiod conditions. Unlike the situation in mice, these two neuropeptides are not co-expressed in medaka neurons, and food deprivation in medaka led to increases in agrp but also a decrease in npy expression. Furthermore, the situation in fish may be more complicated than in mice due to the presence of multiple gene paralogs for each neuropeptide. Exposure to long-day conditions increases agrp1 expression in medaka as the result of increases in the number of neurons expressing this neuropeptide, while the increase in npyb levels results from increased levels of expression in the same population of cells. Using ovariectomized medaka and in situ hybridization assays, the authors reveal that the regulation of agrp1 involves estrogen acting via the estrogen receptor esr2a. Finally, a loss of agrp1 function mutant is generated where the female mutants fail to show the characteristic increase in feeding associated with long-day enhanced reproduction as well as yielding reduced numbers of eggs during spawning.

Strengths:

This manuscript provides important foundational work for future investigations aiming to elucidate the coordination of photoperiod sensing, feeding activity, and reproduction function. The authors have used a combination of approaches with a genetic model that is particularly well suited to studying photoperiodic-dependent physiology and behaviour. The data are clear and the results are convincing and support the main conclusions drawn. The findings are relevant not only for understanding photopriodic responses but also provide more general insight into links between reproduction and feeding behaviour control.

Weaknesses:

Some experimental models used in this study, namely ovariectomized female fish and juvenile fish have not been analysed in terms of their feeding behaviour and so do not give a complete view of the position of this feeding regulatory mechanism in the context of reproduction status. Furthermore, the scope of the discussion section should be expanded to speculate on the functional significance of linking feeding behaviour control with reproductive function.

We would like to thank Reviewer #3 for the insightful advice. We will try to revise several pertinent sentences describing the ovariectomized female fish and juvenile fish so that our present experimental results will give more complete view of their feeding regulatory mechanism in the context of reproduction status. We will also try to expand and revise the discussion section to incorporate the valuable suggestion of Reviewer #3.