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. We describe here 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 8 somites. To investigate what regulates endocardial identity, we employed npas4l/cloche, etv2/etsrp and scl loss-of-function models. Endocardial expression is lost in cloche mutants, significantly reduced in etv2 mutants and only modestly effected upon scl loss-of-function. Bmp signalling was also examined: Overactivation of Bmp signalling increased endocardial expression, whilst 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.

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