1. Genome editing reveals reproductive and developmental dependencies on specific types of 2 vitellogenin in zebrafish (Danio rerio)
- Author
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Yilmaz, Ozlem, Patinote, Amélie, Nguyen, Thuy Thao Vi, Com, Emmanuelle, Pineau, Charles, Bobe, Julien, ProdInra, Migration, Proteomic Profiling and Knock-out Analysis of Key Components of the Zebrafish Egg: Discovering Vitellogenin Contributions to Fish Egg Quality - FISHEGG - - EC:FP7:PEOPLE2015-03-01 - 2017-02-28 - 626272 - VALID, Laboratoire de Physiologie et Génomique des Poissons (LPGP), Institut National de la Recherche Agronomique (INRA)-Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique ), Norwegian Institute of Marine Research, Institut National de la Santé et de la Recherche Médicale (INSERM), This study was supported by the Region Bretagne in France (SAD-2013)-FishEgg (8210), Project #13009218), and Maternal Legacy (ANR-13-BSV7-0015)., European Project: 626272,EC:FP7:PEOPLE,FP7-PEOPLE-2013-IEF,FISHEGG(2015), and Structure Fédérative de Recherche en Biologie et Santé de Rennes ( Biosit : Biologie - Santé - Innovation Technologique )-Institut National de la Recherche Agronomique (INRA)
- Subjects
vitellogenine ,[SDV]Life Sciences [q-bio] ,édition de gènes ,CRISPR/Cas9 ,knock out ,vitellogenins ,zebrafish ,poisson zèbre ,[INFO] Computer Science [cs] ,[SDV] Life Sciences [q-bio] ,reproduction ,fertilité ,poisson ,danio rerio ,cyprinidae ,fécondite ,[INFO]Computer Science [cs] - Abstract
Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk nutrients, but little is known about their individual contributions to reproduction and development. This study employed a CRISPR/Cas9 genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type-I and -III Vtgs. The multiple CRISPR approach employed to knock out (KO) all genes encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) individually (vtg3-KO). Results of PCR genotyping and sequencing, qPCR, LC-MS/MS and Western blotting showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels occurred in liver and eggs, a heretofore-unknown mechanism of genetic compensation to regulate Vtg homeostasis. Fecundity was more than doubled in vtg1-KO females, and fertility was ~halved in vtg3-KO females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 h of incubation and in vtg1-KO embryos after 5 d. Hatching rate and timing were markedly impaired in vtg mutant embryos and pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at different times during both reproduction and development.
- Published
- 2018