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

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 utilized clustered regularly interspaced short palindromic repeats/CRISPR‐associated protein 9 (CRISPR/Cas9) genome editing to assess essentiality and functionality of zebrafish (Danio rerio) type‐I and type‐III Vtgs. A multiple CRISPR approach was employed to knockout (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 polymerase chain reaction (PCR) genotyping and sequencing, quantitative PCR, liquid chromatography‐tandem mass spectrometry, and Western blot analysis 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, revealing a heretofore‐unknown mechanism of genetic compensation regulating Vtg homeostasis. Egg numbers per spawn were elevated more than 2‐fold in vtg1‐KO females, and egg fertility was approximately halved in vtg3‐KO females. Substantial mortality was evident in vtg3‐KO eggs/embryos after only 8 hr of incubation and in vtg1‐KO embryos after 5 days. Hatching rate and timing were markedly impaired in embryos from vtg mutant mothers 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.

Knock out of Specific Maternal Vitellogenins in Zebrafish (Danio Rerio) Evokes Vital Changes in Egg Proteomic Profiles that resemble the Phenotype of Poor Quality Eggs

“…Highly signi cant overrepresentation of these pathways altogether in vtg-KO zebra sh eggs are likely connected to the aberrant development and function of cardiovascular system, which leads to pericardial edema and related in ammatory responses culminating in death. The enrichment of vtg-KO eggs by proteins related to the cardiac muscle contraction KEGG pathway revealed by network enrichment analysis ( Table 1 and Table 2) and the previously reported zebra sh phenotypes related to vtg-KO, in which pericardial and yolk sac edema were apparent [33] support the idea that Vtgs do somehow contribute to heart development and function in zebra sh. Along with the overrepresentation of proteins related to cell cycle, division, growth and fate related activities in vtg-KO sh (e.g.…”

“…Furthermore, results of our recent study of zebra sh examining the effects of simultaneous knock-out of multiple type I vtgs, versus the effects of KO of vtg3 only, revealed substantial mortality of vtg3-KO eggs/embryos after only 8 h post-fertilization, whereas signi cant mortality of vtg1-KO embryos did not occur until ~ 5 days later. These observations and similar disparities seen during late larval stages provided the rst experimental evidence that different types of Vtg are essential and have disparate requisite functions at different times of development in zebra sh, a well-established biomedical model for research on reproduction and developmental biology [33].…”

“…The potential ER and mitochondrial dysfunctions discussed above may culminate in abnormal aspects of FFA biosynthesis and phospholipid metabolism, as is observed in vtg3-KO eggs. However, despite the interrelation of these molecular changes, surviving vtg3-KO offspring did not exhibit obvious problems with mobility and, similarly to Wt offspring, they were swimming and feeding actively, which was not observed for surviving vtg1-KO offspring [33]. The speci c contribution of Vtg3 to energy and lipid metabolism mechanisms and/or apoptosis remain unelucidated and need to be further investigated.…”

“…Furthermore, any failure of mitochondrial quality control could disable removal of damaged mitochondria, facilitating apoptosis during high levels of cellular stress. Based on the resulting substantial proteomic changes, it seems certain that the absence of knocked out Vtgs induces severe cellular stress on the way toward its induction of the observed developmental disorders [33]. However, the underlying mechanisms by which Vtg3 forestalls these disorders are unclear.…”

CEH-60/PBX regulates vitellogenesis and cuticle permeability through intestinal interaction with UNC-62/MEIS in Caenorhabditis elegans

“…Vitellogenesis is the process of maternal yolk formation. In many organisms, including insect and vertebrate representatives [1,2], it is deemed essential for embryonic development, although the necessity of abundant yolk synthesis for development has been questioned in some species, including C . elegans [3,4].…”