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

Abstract

12Oviparous vertebrates produce multiple forms of vitellogenin (Vtg), the major source of yolk 13 nutrients, but little is known about their individual contributions to reproduction and development. This14 study employed a CRISPR/Cas9 genome editing to assess essentiality and functionality of zebrafish 15 (Danio rerio) type-I and -III Vtgs. The multiple CRISPR approach employed to knock out (KO) all genes 16 encoding type-I vtgs (vtg1, 4, 5, 6, and 7) simultaneously (vtg1-KO), and the type-III vtg (vtg3) 17 individually (vtg3-KO). Results of PCR genotyping and sequencing, qPCR, LC-MS/MS and Western 18blotting showed that only vtg6 and vtg7 escaped Cas9 editing. In fish whose remaining type-I vtgs were 19 incapacitated (vtg1-KO), and in vtg3-KO fish, significant increases in Vtg7 transcript and protein levels 20 occurred in liver and eggs, a heretofore-unknown mechanism of genetic compensation to regulate Vtg 21 homeostasis. Fecundity was more than doubled in vtg1-KO females, and fertility was ~halved in vtg3-KO 22 females. Substantial mortality was evident in vtg3-KO eggs/embryos after only 8 h of incubation and in 23 vtg1-KO embryos after 5 d. Hatching rate and timing were markedly impaired in vtg mutant embryos and 24 pericardial and yolk sac/abdominal edema and spinal lordosis were evident in the larvae, with feeding and 25 motor activities also being absent in vtg1-KO larvae. By late larval stages, vtg mutations were either 26 2 completely lethal (vtg1-KO) or nearly so (vtg3-KO). These novel findings offer the first experimental 27 evidence that different types of vertebrate Vtg are essential and have disparate requisite functions at 28 different times during both reproduction and development. 29 30

Molecular mechanisms involved in Atlantic halibut (Hippoglossus hippoglossus) egg quality: impairments at transcription and protein folding levels induce inefficient protein and energy homeostasis during early development

“…All these pathways seem to be interconnected (34)(35)(36)(37)(38) [Chen et al, 2010, Youle et al, 2012, Rugarli and Langer, 2012, Labbadia et al, 2013, Tublin et al, 2019 and previously reported to be linked to perturbations in mitochondrial maintenance, localization, and activity along with aberrant protein folding (37,39) [Williams andPaulson, 2008, Labbadia et al, 2013], all of whom signatures were observed in our study, leading to subsequent impairments in normal development (38) [Tublin et al, 2019]. The cardiac muscle contraction pathway was previously linked to a cardiac and yolk sac edema phenotype observed in zebrafish eggs lacking certain vtgs in their genomes (23,24) (Yilmaz et al, 2019(Yilmaz et al, , 2021. Further research targeting morphological observations on development of offspring originating from different quality halibut egg batches are needed.…”

“…Our recent research comparing the global proteomes of different quality eggs from zebrafish revealed a number of proteins as potential markers, but more importantly, several molecular mechanisms and related physiological processes to be associated with egg quality in this species (22) (Yilmaz et al, 2017). In a most up to date study, the consecutive changes in global proteome of 1-cell-stage egg after invalidation of certain types of Vtgs (vtg1, 4 and 5; vtg1-KO and vtg3; vtg3-KO) were investigated using CRISPR/Cas9 genome editing technology (23,24) (Yilmaz et al, 2019, Yilmaz et al, 2021. The collective results of these studies delivered a clear portrait of the impaired molecular mechanisms that impacts egg and offspring developmental competence with striking similarities between vtg-KO and poor quality egg proteome profiles in zebrafish.…”

Cloning, partial sequencing and 17β‐estradiol modulation of hepatic vitellogenin gene of the Neotropical catfish Rhamdia quelen

“…This basal level of vtg expression in males was already observed in some other fish species, as C. auratus (Silva de and Scatophagus argus (Cui et al, 2017). To the best of our knowledge, there is no discussion about possible mechanisms associated to this observation, although some studies have reported that some Vtg can present others functions but embryos nourishment (Finn et al, 2009;Finn & Kristoffersen, 2007;Reading et al, 2009;Yilmaz et al, 2019). Since we do not evaluate the plasma Vtg concentrations, we cannot directly associate vtg mRNA levels with Vtg protein levels.…”

Vitellogenin gene family in vertebrates: evolution and functions

“…Recently Yilmaz et al (2018b) reported the first experimental evidence of selective knockout of multiple vtg forms in zebrafish. Their findings have revealed not only a role of Vtg in development of embryo and larvae but also new regulatory effects on fecundity and fertility.…”