Endocardial identity is established during early somitogenesis by Bmp signalling acting upstream ofnpas4landetv2

Abstract

The endocardium plays important roles in the development and function of the vertebrate heart; however, few molecular markers of this tissue have been identified and little is known about what regulates its differentiation. Here, we describe the Gt(SAGFF27C); Tg(4xUAS:egfp) line as a marker of endocardial development in zebrafish. Transcriptomic comparison between endocardium and pan-endothelium confirms molecular distinction between these populations and time-course analysis suggests differentiation as early as eight somites. To investigate what regulates endocardial identity, we employed npas4l, etv2 and scl loss-of-function models. Endocardial expression is lost in npas4l mutants, significantly reduced in etv2 mutants and only modestly affected upon scl loss-of-function. Bmp signalling was also examined: overactivation of Bmp signalling increased endocardial expression, whereas Bmp inhibition decreased expression. Finally, epistasis experiments showed that overactivation of Bmp signalling was incapable of restoring endocardial expression in etv2 mutants. By contrast, overexpression of either npas4l or etv2 was sufficient to rescue endocardial expression upon Bmp inhibition. Together, these results describe the differentiation of the endocardium, distinct from vasculature, and place npas4l and etv2 downstream of Bmp signalling in regulating its differentiation.

Single-cell transcriptomic analysis of vascular endothelial cells in zebrafish embryos

“…Endocardial cells are known to share a large transcriptional similarity with other vascular endothelial cells 33 , 60 – 63 , but they also have their unique identity known by expression of marker genes such as nfatc1 and gata5 64 , 65 . Recent studies have described endocardial transcriptome at 15 and 20-somite stages in zebrafish embryos 48 , 66 . However, endocardial transcriptome at 24 hpf stage has not been previously reported.…”

“…There was a significant overlap of marker genes, such as fn1a , gata5 , id2b, wnt11r , bmp16 and bambib between the two endocardial clusters (Table S7 ). A recent study reported the zebrafish endocardial transcriptome at the 15-somite stage 66 . There was an extensive overlap between our endocardial dataset and the one reported by Capon et al (Table S7 ), further validating endocardial identity of the nfatc1 + gata5 + cell cluster.…”

“…We have previously reported a transcriptional signature of zebrafish endocardial progenitors identified during scRNA-seq analysis of etv2 reporter embryos at the 20-somite stage (19 hpf) 48 . In addition, a recent study performed RNA-seq using an endocardial specific reporter line to describe the endocardial transcriptome at the 15-somite stage 66 . Here we performed subclustering based on the established markers gata5 and nfatc1 to define the endocardial transcriptome at 24 hpf.…”

Endocardium gives rise to blood cells in zebrafish embryos

“…Previous studies have demonstrated that endocardial-specific identity may be established as early as the 8-somite stage. 54 Therefore it is likely that endocardial-derived hematopoiesis initiates prior to the 24-hpf stage, and double-positive kdrl:GFP - and spi1b -positive cells observed at the 16-somite stage may mark early endocardial-derived myeloid cells.…”