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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. [ABSTRACT FROM AUTHOR]

Sonic hedgehog signaling regulates processes of embryonic development across multiple tissues, yet factors regulating context-specific Shh signaling remain poorly understood. Exome sequencing of families with polymicrogyria (disordered cortical folding) revealed multiple individuals with biallelic deleterious variants in TMEM161B , which encodes a multi-pass transmembrane protein of unknown function. Tmem161b null mice demonstrated holoprosencephaly, craniofacial midline defects, eye defects, and spinal cord patterning changes consistent with impaired Shh signaling, but were without limb defects, suggesting a CNS-specific role of Tmem161b. Tmem161b depletion impaired the response to Smoothened activation in vitro and disrupted cortical histogenesis in vivo in both mouse and ferret models, including leading to abnormal gyration in the ferret model. Tmem161b localizes non-exclusively to the primary cilium, and scanning electron microscopy revealed shortened, dysmorphic, and ballooned ventricular zone cilia in the Tmem161b null mouse, suggesting that the Shh-related phenotypes may reflect ciliary dysfunction. Our data identify TMEM161B as a regulator of cerebral cortical gyration, as involved in primary ciliary structure, as a regulator of Shh signaling, and further implicate Shh signaling in human gyral development.