A scalable platform for functional interrogation of peptide hormones in fish

Abstract

Fish display a remarkable diversity of life-history traits, including body size, age at maturity, and longevity. Although pituitary hormones are conserved mediators of life-history transitions, regulatory networks are less understood in fish. However, the relatively long life-cycles and germline-dependent maturation of classical fish models are less compatible with rapid exploration of adult physiology, particularly in females. Here, we describe a high-throughput platform that combines, for the first time, loss- and gain-of-function of peptide hormones in a naturally short-lived fish. As a proof-of-principle, we first manipulate growth by mutating growth hormone (gh1) in the turquoise killifish (N. furzeri). Next, to rescue growth defects, we designed a vector in which hormones are tagged by a self-cleavable fluorescent reporter, and are ectopically expressed using intramuscular electroporation. A single injection of agh1-T2A-GFPplasmid was sufficient to produce a stable expression of tag-free hormone and rescue growth phenotypes. This, in contrast to current practice for which multiple injections of recombinant hormones are required. We demonstrate the versatility of our platform by rescuing female sterility, which is induced by manipulating the follicle stimulating hormone (fshb). As killifish maturation is germline-independent, both sexes can be explored in genetic models with germline defects. Finally, we describe a doxycycline-inducible system for tunable expression control. Together, this platform significantly advances the state-of-the-art by allowing high-throughput functional dissection of distinct life-history strategies in fish. This method could be multiplexed to facilitate various applications, including optimizing commercially valuable traits in aquaculture, or screening pro-longevity hormonal interventions in aging.

A scalable and tunable platform for functional interrogation of peptide hormones in fish

“…Tissues samples were processed as described previously ( Harel et al, 2015 ; Astre et al, 2022b ; Harel and Brunet, 2015 ; Astre et al, 2021 ; Harel et al, 2022 ; Nathan et al, 2008 ; Theis et al, 2010 ; Harel and Tzahor, 2012 ; Harel et al, 2012 ; Benayoun et al, 2019 ; Rozenberg et al, 2023b ; Astre et al, 2022a ; Harel et al, 2016 ; Rozenberg et al, 2023a ; Van Keymeulen et al, 2009 ; Moses and Harel, 2023 ; Valenzano et al, 2015 ; Harel et al, 2009 ; Gruenbaum-Cohen et al, 2012 ). Briefly, fish were euthanized with 500 mg/l tricaine (MS222, #A5040, Sigma).…”

A genetic toolbox for the turquoise killifish identifies sporadic age-related cancer

“…Crossing Cre-injected adult Killibow fish with WT fish produced offspring that were uniformly colored in a variety of hues (Figure 2c), indicating efficient labeling of the killifish germline. Finally, we utilized our recently-developed in-vivo electroporation system 43 to induce Cre-mediated recombination at will (Figure 2d, left). Specifically, we electroporated a Cre-GFP plasmid following intramuscular injection.…”

“…The copyright holder for this preprint this version posted May 1, 2023. ; https://doi.org/10.1101/2023.05.01.538839 doi: bioRxiv preprint cell-line that expresses RFP in a robust and uniform manner (Figure 4b). We then modified our recently developed doxycycline-inducible system in killifish 43 to induce Cre recombination at will. To this end we tested two plasmids that use the tamoxifen-inducible Cre system 47,48 , namely the pCAG-ERT2-Cre-ERT2 plasmid (Addgene #13777), and the Tol2-ubi-ERT2-Cre-ERT2 plasmid (a kind gift from Wang Wei).…”

The killifish germline regulates longevity and somatic repair in a sex-specific manner

“…Thus, providing a unique opportunity for rapid exploration of vertebrate physiology. The turquoise killifish has vertebrate-conserved organs and systems, including the HPG reproductive axis, and a recently developed state-of-the-art genome engineering platform 30–38 . However, the regulatory mechanisms of reproduction and gonadal development in killifish are largely unknown.…”