Genomic disruption of type II vitellogenin leads to vitellin membrane deficiencies and significant mortalities at early stages of embryonic development in zebrafish (Danio rerio)

Type II vitellogenin (Vtg2), the second most abundant type of vitellogenin in zebrafish eggs, is a major source of nutrients for early embryonic development. The main objective of this study was to determine the specific functions and essentiality of vtg2 in zebrafish early development using CRISPR/Cas9 genome editing tool. A 2811 bp deletion on gDNA was detected in vtg2 -mutant zebrafish via PCR genotyping and sequencing. Introduced mutation caused vitelline membrane deficiencies and significant mortalities of mutant offspring. Further effects on female fecundity, egg fertilization rate and vtg2 gene expression and Vtgs abundance in liver were observed in F2, while effects on embryo hatching, survival rates, proteomic profiles and abundances were observed in F3 generations of the vtg2 -mutant line. No change in vtg2 transcript has been detected, however, Vtg2 abundance in F2 female liver was 5x, and in 1 hpf F3 vtg2 -mutant embryos was 3.8x less than Wt ( p < 0.05). All Vtgs, except Vtg7, declined in abundance in 1 hpf F3 vtg2 -mutant embryos ( p < 0.05). Fecundity was unaffected while fertilization rate was more than halved in F2 vtg2 -mutant females ( p < 0.05). Hatching rate was significantly higher in F3 vtg2 -mutant embryos in comparison to Wt embryos. Survival rate declined drastically to 29 % and 18 % at 24 hpf and 20 dpf, respectively, in F3 vtg2 -mutant embryos. Pericardial, yolk sac/abdominal edema and spinal lordosis were evident at later stages in the surviving F3 vtg 2 - mutant larvae. Overrepresentation and high expression of histones, zona pellucida proteins, lectins, and protein degradation related proteins in F3 vtg2 -mutant embryos provide evidence to impaired mechanisms involved in vitellin membrane formation. Findings of this study imply a potential function of Vtg2 in acquisition of vitellin membrane integrity, among other reproductive functions, and therefore, its essentiality in early zebrafish embryo development. AUTHOR SUMMARY Vitellogenins (Vtgs) are major yolk nutrient precursors supporting early vertebrate development. Most species have multiple forms of Vtg, but little is known about their individual roles in reproduction and it is uncertain which forms are essential for successful development or at what stage(s) of development they are required. This study employed a CRISPR/Cas9 gene knock out (KO) to assess essentiality and functionality of Vtg2 in zebrafish, in continuation to a previously published work on type I and type III vtgs KO. The findings of this study, in combination with the previous findings, present a new model of Vtg functionality. Accordingly, Vtg2 contribute to regulation of fecundity and fertilization in female reproduction while make essential contributions to embryonic morphogenesis, hatching and embryonic and larval kinetics and survival. In addition, Vtg2 is critically important to proper formation of the vitellin membrane, and thus, to embryogenesis and later development. Our novel findings provide, for the first time, empirical evidence that the Vtg2 are essential, having critical requisite functions during oogenesis and embryonic and larval development. The overall results substantiate the concept that each type of Vtg is specialized to play unique roles in reproduction and development. ### Competing Interest Statement The authors have declared no competing interest.