Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2023.07.24.550261

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

YILMAZ, O.·Com, E.·Pineau, C.·Bobe, J.
View original source ↗Export metadata ↓

Abstract

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 vtg2-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 SUMMARYVitellogenins (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.

Explore related subjects

molecular biologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

YILMAZ, O., Com, E., Pineau, C., Bobe, J.. 2023-07-24. Genomic disruption of type II vitellogenin leads to vitellin membrane deficiencies andsignificant mortalities at early stages of embryonic development in zebrafish (Danio rerio). https://doi.org/10.1101/2023.07.24.550261

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Hidden Biodiversity in Wildlife Trade Networks: DNA Barcoding Reveals Fish and Crocodilian Species in Commercialized Swim Bladders

International wildlife trade represents one of the major drivers of biodiversity exploitation worldwide. However, the true taxonomic diversity embedded within commercial wildlife products often remains unknown because processing removes diagnostic morphological characteristics, preventing reliable species identification. Consequently, biodiversity assessments based solely on product labels may substantially underestimate the diversity of species involved in trade networks. To investigate hidden biodiversity within wildlife trade products, we applied DNA barcoding based on the mitochondrial cytochrome c oxidase subunit I (COI) gene to 77 products commercialized as fish swim bladders and seized at Guarulhos International Airport, Brazil. Molecular analyses successfully identified all samples and revealed the presence of four species: Plagioscion auratus (n = 38), Cynoscion acoupa (n = 7), Melanosuchus niger (n = 17), and Caiman crocodilus (n = 15). Fish species accounted for 71.4% of all samples, whereas crocodilians represented 28.6%, demonstrating that products marketed under a single commercial category may conceal substantial taxonomic diversity. Notably, the occurrence of two Amazonian crocodilian species within a trade chain traditionally associated with fish products reveals a previously undocumented component of the international wildlife trade. Our findings demonstrate that DNA barcoding is an effective tool for uncovering hidden biodiversity within processed wildlife products and provide evidence that wildlife trade networks may involve a broader spectrum of species than suggested by commercial labels. These results highlight the importance of molecular surveillance for biodiversity monitoring, wildlife trade regulation, and conservation planning.

molecular biology↗
bioRxiv2026

Plasmid architecture determines the stability of inverted terminal repeats in adeno-associated virus vectors

Recombinant vectors derived from adeno-associated viruses (rAAVs) are a mainstay of human gene therapy. rAAVs are produced from plasmids containing transgene cassettes flanked by inverted terminal repeats (ITRs), which form structured DNA elements that stabilize the ends of the single-stranded viral genome and are the only viral sequences required in cis for genome packaging. For decades, it has been recognized that propagation of ITR-containing plasmids can result in deletions and other mutations, prompting the use of specialized bacterial strains, modified growth conditions, and truncated or altered ITRs. Despite these practices, ITR instability remains a persistent source of plasmid heterogeneity. To identify determinants of ITR stability, we evaluated ITR integrity in one of the original cloned AAV2 genome isolates, a reconstructed AAV2 plasmid, and a synthetic rAAV vector containing full-length native AAV2 ITRs. We established a quantitative bioinformatic workflow for analyzing ITR-containing plasmids and virus preparations from raw Oxford Nanopore sequencing data. These experiments showed that ITRs were highly stable during short-term culture, whereas prolonged culture revealed strong positional effects, with preferential loss or mutation of the ITR nearest the plasmid origin of replication. Consistent with this model, a survey of 7,041 sequence-verifiable AAV plasmids from the Addgene repository identified a widely disseminated 11-bp ITR deletion in 4,773 plasmids; among analyzable two-ITR plasmids, this deletion was located in the origin-proximal ITR in 95.3% of cases. Guided by these findings, we constructed a novel rAAV entry vector with stable full-length native AAV2 ITRs that enabled efficient packaging of a 4,750-bp all-in-one CRISPR-Cas9 cassette. Finally, we developed a cell-based strategy to compare the effects of ITR mutations on rAAV genome integration, providing preliminary evidence that ITR sequence variation can influence integration outcomes. Together, these findings show that ITR instability is a preventable, position-dependent property of plasmid architecture and identify ITR integrity as an important variable in rAAV vector design and quality control.

molecular biology↗
bioRxiv2026

Single-point mutation alters odorant receptor sensitivity associated with host plant specialization in Spodoptera moths

Host specialization in herbivorous insects is often associated with divergence in chemosensory abilities. Here, we investigated the possible contribution of odorant receptors (ORs) in host plant restriction in the lily moth Spodoptera picta, a species specialized on Amaryllidaceae. Manual annotation of S. picta ORs in its genome revealed a repertoire similar in size and composition to those of its polyphagous sister species, S. littoralis and S. litura, suggesting that specialization did not involve major gene loss or expansion in the lily moth. To assess functional divergence beyond gene number, we applied a large scaled structure-based virtual screening approach to the entire OR repertoires of these three Spodoptera species, generating ligand-binding profiles for 120,591 volatile compounds. Among 69 1:1:1 OR orthologs, 24 exhibited divergent predicted binding spectra. We pinpointed OR29 that we also found to be highly expressed in both male and female antennae of S. picta through a RNAseq approach. Functional assays demonstrated that S. picta OR29 acquired heightened sensitivity to limonene enantiomers, volatiles emitted by host Amaryllidaceae inflorescences. Site-directed mutagenesis revealed that a single amino acid substitution within the predicted binding region underlies this shift in sensitivity. These results show that host specialization in S. picta has not been accompanied by significant OR repertoire remodeling, but rather by subtle molecular changes that fine-tune receptor sensitivity to host-derived volatiles.

molecular biology↗