Genome editing reveals reproductive and developmental dependencies on specific types of vitellogenin in zebrafish (Danio rerio)

Abstract

ABSTRACTOviparous 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.