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 a gh1-T2A-GFP plasmid 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.