A scalable and tunable platform for functional interrogation of peptide hormones in fish
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eLife assessment
Moises and Harel generate an important set of novel molecular tools in African turquoise killifish, an innovative model to study aging biology. The new solid tools described in this paper can boost this buddying model system for broad biotechnological applications. The authors showcase the efficacy of their tools in the context of peptide hormones involved in growth and gonad development. The killifish community will greatly benefit from these novel tools and the relevance of the developed methods will likely go beyond the killifish community.
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Abstract
Pituitary hormones play a central role in shaping vertebrate life history events, including growth, reproduction, metabolism, and aging. The regulation of these traits often requires precise control of hormone levels across diverse timescales. However, fine tuning circulating hormones in-vivo has traditionally been experimentally challenging. Here, using the naturally short-lived turquoise killifish ( N. furzeri ), we describe a high-throughput platform that combines loss- and gain-of-function of peptide hormones. Mutation of three primary pituitary hormones, growth hormone ( gh1 ), follicle stimulating hormone ( fshb ), and thyroid stimulating hormone ( tshb ), alters somatic growth and reproduction. Thus, suggesting that while the killifish undergoes extremely rapid growth and maturity, it still relies on vertebrate-conserved genetic networks. As the next stage, we developed a gain-of-function vector system in which a hormone is tagged using a self-cleavable fluorescent reporter, and ectopically expressed in-vivo through intramuscular electroporation. Following a single electroporation, phenotypes, such as reproduction, are stably rescued for several months. Notably, we demonstrate the versatility of this approach by using multiplexing, dose-dependent, and doxycycline-inducible systems to achieve tunable and reversible expression. In summary, this method is relatively high-throughput, and facilitates large-scale interrogation of life-history strategies in fish. Ultimately, this approach could be adapted for modifying aquaculture species and exploring pro-longevity interventions.
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eLife assessment
Moises and Harel generate an important set of novel molecular tools in African turquoise killifish, an innovative model to study aging biology. The new solid tools described in this paper can boost this buddying model system for broad biotechnological applications. The authors showcase the efficacy of their tools in the context of peptide hormones involved in growth and gonad development. The killifish community will greatly benefit from these novel tools and the relevance of the developed methods will likely go beyond the killifish community.
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Reviewer #1 (Public Review):
Moises and Harel develop an impressive set of novel molecular tools in African turquoise killifish, which include hormone tagging by a self-cleavable fluorescent reporter, intramuscular electroporation for ectopic transgene expression and a doxycycline-inducible system. All these tools are per se fundamental technological innovations in killifish. The authors apply their advanced techniques to modulate growth and gonad development in killifish, showing that the methods work and that it is possible to modulate these fundamental developmental milestones through the use of their molecular tools.
Strengths:
The tools developed are effective, convincing and will likely be adopted as a reference for future work, beyond the field of peptide hormones. I congratulate the authors for their ingenuity and …
Reviewer #1 (Public Review):
Moises and Harel develop an impressive set of novel molecular tools in African turquoise killifish, which include hormone tagging by a self-cleavable fluorescent reporter, intramuscular electroporation for ectopic transgene expression and a doxycycline-inducible system. All these tools are per se fundamental technological innovations in killifish. The authors apply their advanced techniques to modulate growth and gonad development in killifish, showing that the methods work and that it is possible to modulate these fundamental developmental milestones through the use of their molecular tools.
Strengths:
The tools developed are effective, convincing and will likely be adopted as a reference for future work, beyond the field of peptide hormones. I congratulate the authors for their ingenuity and resourcefulness. The figures are clear and of high standard.
Weaknesses:
The manuscript does not obviously follow a question-driven flow and the authors do not make a compelling case about the necessity of developing such platform.
The manuscript should be framed as a tool/resource, showcasing the interventions with gh and fshb to support the tool.The manuscript is not thoroughly edited and the authors should check and review extensively for improvements to the use of English. Overall, I find a disconnect between the way in which the manuscript is written and the quality of the figures. While the figures have a very high quality standard, the Abstract, Introduction and Discussion are not doing justice to the work done.
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Reviewer #2 (Public Review):
In this article, Moses and Harel present genetic knock-out and partial rescue of the phenotypes of neuropeptides gh1 and fshb, and tshb in a short-lived vertebrate African turquoise killifish Nothobranchius furzeri. Neuropeptides are among the key regulators of growth, reproduction, and metabolism. Understanding their mechanisms of action has important implications for vertebrate physiology.
The authors first characterize the loss of function phenotypes of gh1, fshb, and tshb in killifish, followed by attempts to rescue the loss of function phenotypes through ectopic expression of two of the neuropeptides. The primary strength and innovation of this work are partially rescuing the phenotypes by muscular injection of plasmids followed by electroporation, including a doxycycline-inducible system for tunable …
Reviewer #2 (Public Review):
In this article, Moses and Harel present genetic knock-out and partial rescue of the phenotypes of neuropeptides gh1 and fshb, and tshb in a short-lived vertebrate African turquoise killifish Nothobranchius furzeri. Neuropeptides are among the key regulators of growth, reproduction, and metabolism. Understanding their mechanisms of action has important implications for vertebrate physiology.
The authors first characterize the loss of function phenotypes of gh1, fshb, and tshb in killifish, followed by attempts to rescue the loss of function phenotypes through ectopic expression of two of the neuropeptides. The primary strength and innovation of this work are partially rescuing the phenotypes by muscular injection of plasmids followed by electroporation, including a doxycycline-inducible system for tunable expression control. The techniques for tunable expression control and rescue of knock-out phenotypes have not been established for killifish and will be useful to expand the technical repertoire of this emerging model organism. Once established, these techniques can be extended to other categories of genes to rapidly evaluate their function and the impact of their loss or gain of function on killifish and other fish models.
However, the phenotypes discussed need further characterization, many technical details are unclear, and it seems that appropriate controls are missing for some of the experiments. The rescued phenotypes also need more validation.
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Reviewer #3 (Public Review):
This manuscript describes the development of CRISPR knockouts for gh, fsh and tsh in the fast-aging Nothobranchius furzeri grz strain. CRISPR knockouts have been published before, and the strength of the paper is that here, the authors include a novel, easy and fast way of rescuing the loss of function in the entire body by electroporation in muscle. This offers flexibility in timing and dosage, and leads to intriguing results regarding the role of these hormones in growth and fertility. Finally they also add a conditional doxycycline-dependent overexpression model that would allow even more control over the modalities of the rescue. The phenotypes of the knockouts were not the key message of the paper and remained at times only superficially described. The doxycycline-dependent overexpression was only …
Reviewer #3 (Public Review):
This manuscript describes the development of CRISPR knockouts for gh, fsh and tsh in the fast-aging Nothobranchius furzeri grz strain. CRISPR knockouts have been published before, and the strength of the paper is that here, the authors include a novel, easy and fast way of rescuing the loss of function in the entire body by electroporation in muscle. This offers flexibility in timing and dosage, and leads to intriguing results regarding the role of these hormones in growth and fertility. Finally they also add a conditional doxycycline-dependent overexpression model that would allow even more control over the modalities of the rescue. The phenotypes of the knockouts were not the key message of the paper and remained at times only superficially described. The doxycycline-dependent overexpression was only minimally validated, and here it is not yet clear how robust this system is in terms of overexpression levels, timing, and reversibility.
Overall this study brings a new set of tools in the killifish toolbox that can have much wider applications and will be appreciated also in other teleost models.
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