Establishment and transcriptome analysis of single blastomerederived cell lines from zebrafish

Maintaining chromosome euploidy in zebrafish embryonic cells is challenging because of the degradation of genomic integrity during cell passaging.In this study,we report the derivation of zebrafish cell lines from single blastomeres.These cell lines have a stable chromosome status attributed to BMP4 and exhibit continuous proliferation in vitro.Twenty zebrafish cell lines are successfully established from single blastomeres.Single-cell transcriptome sequencing analysis confirms the fidelity of gene expression profiles throughout long-term culturing of at least 45 passages.The long-term cultured cells are specialized into epithelial cells,exhibiting similar expression patterns validated by integrative transcriptomic analysis.Overall,this work provides a protocol for establishing zebrafish cell lines from single blastomeres,which can serve as valuable tools for in vitro investigations of epithelial cell dynamics in terms of lifeedeath balance and cell fate determination during normal homeostasis.

Draft Genome of White-blotched River Stingray Provides Novel Clues for Niche Adaptation and Skeleton Formation

The white-blotched river stingray(Potamotrygon leopoldi)is a cartilaginous fish native to the Xingu River,a tributary of the Amazon River system.As a rare freshwater-dwelling cartilaginous fish in the Potamotrygonidae family in which no member has the genome sequencing information available,P.leopoldi provides the evolutionary details in fish phylogeny,niche adaptation,and skeleton formation.In this study,we present its draft genome of 4.11 Gb comprising 16,227 contigs and 13,238 scaffolds,with contig N50 of 3937 kb and scaffold N50 of 5675 kb in size.Our analysis shows that P.leopoldi is a slow-evolving fish that diverged from elephant sharks about 96 million years ago.Moreover,two gene families related to the immune system(immunoglobulin heavy constant delta genes and T-cell receptor alpha/delta variable genes)exhibit expansion in P.leopoldi only.We also identified the Hox gene clusters in P.leopoldi and discovered that seven Hox genes shared by five representative fish species are missing in P.leopoldi.The RNA sequencing data from P.leopoldi and other three fish species demonstrate that fishes have a more diversified tissue expression spectrum when compared to mammals.Our functional studies suggest that lack of the gc gene encoding vitamin D-binding protein in cartilaginous fishes(both P.leopoldi and Callorhinchus milii)could partly explain the absence of hard bone in their endoskeleton.Overall,this genome resource provides new insights into the niche adaptation,body plan,and skeleton formation of P.leopoldi,as well as the genome evolution in cartilaginous fishes.

Derivation of zebrafish heart-related haploid cells

The first long-term cultured haploid embryonic stem cell(ESC)line was established from medaka fish,which represented a breakthrough in vertebrates(Yi et al.,2009).Subsequently,haploid ESC lines were established from mammals and used for genetic analysis and complex genetic manipulation(Elling et al.,2011;Yang et al.,2012).The diploidization of haploid cells is an interesting biological phenomenon and has been widely studied,but the mechanism of diploidization has not yet been fully resolved(Sun et al.,2020).The zebrafish is an excellent vertebrate animal model used in biological studies(Huang et al.,2022).Researchers have isolated many cell lines from zebrafish,such as the zebrafish fibroblast line ZF4(Driever and Rangini,1993).However,there have been no reports regarding stable haploid cell lines of zebrafish,and whether diploidization occurs in zebrafish is unclear.