Hedgehog signaling is required for differentiation of endocardial progenitors in zebrafish

Abstract

Endocardial cells form the inner endothelial layer of the heart tube, surrounded by the myocardium. Signaling pathways that regulate endocardial cell specification and differentiation are largely unknown and the origin of endocardial progenitors is still being debated. To study pathways that regulate endocardial differentiation in a zebrafish model system, we isolated zebrafish NFATc1 homolog which is expressed in endocardial but not vascular endothelial cells. We further demonstrate that Hedgehog (Hh) but not VegfA or Notch signaling is required for early endocardial morphogenesis. Pharmacological inhibition of Hh signaling with cyclopamine treatment resulted in nearly complete loss of the endocardial marker expression. Simultaneous knockdown of the two zebrafish sonic hedgehog homologs, shh and twhh or Hh co-receptor smoothened (smo) resulted in similar defects in endocardial morphogenesis. Inhibition of Hh signaling resulted in the loss of fibronectin (fn1) expression in the presumptive endocardial progenitors as early as the 10-somite stage which suggests that Hh signaling is required for the earliest stages of endocardial specification. We further show that the endoderm plays a critical role in migration but not specification or differentiation of the endocardial progenitors while notochord-derived Hh is a likely source for the specification and differentiation signal. Mosaic analysis using cell transplantation shows that Smo function is required cell-autonomously within endocardial progenitor cells. Our results argue that Hh provides a critical signal to induce the specification and differentiation of endocardial progenitors.

Endoderm convergence controls subduction of the myocardial precursors during heart-tube formation

“…To test further whether the migration of ALPM cells relies on endoderm convergence, we injected a MO targeting sox32, a transcription factor required for endoderm development (Wong et al, 2012;Yelon et al, 1999). In such morphants, which lack endoderm, the Kaede-expressing myocardial precursors failed to migrate towards the midline, remaining in the same position from 12-18 s (supplementary material Fig.…”

“…The previously validated MOs targeting the following genes were injected into embryos at 1-cell stage: gna13a and gna13b (2-3 ng each) (Lin et al, 2005), s1pr2/mil (15 ng) (Kawahara et al, 2009) and sox32 (4 ng) (Wong et al, 2012).…”

Myocardium and BMP signaling are required for endocardial differentiation

“…Markers associated with endocardial differentiation, such as nfatc1, notch1b and klf2a, are expressed throughout the endocardium at 24-48 hpf stages in wild-type embryos and are subsequently enriched in the atrioventricular canal (AVC) (Westin and Lardelli, 1997;Oates et al, 2001;Walsh and Stainier, 2001;Wong et al, 2012). Endocardial expression of these markers was severely reduced or completely absent in hand2 −/− embryos at the analyzed stages of 22 hpf, 30 hpf (nfatc1) and 53 hpf (nfatc1, notch1b and klf2a) (Fig.…”

“…We have previously demonstrated that hedgehog (Hh) signaling is required for endocardial differentiation in zebrafish embryos (Wong et al, 2012). In the absence of Hh signaling, endocardial progenitors fail to migrate to the midline and do not initiate endocardial nfatc1 expression, while endothelial differentiation of blood vessels is not affected.…”

“…Subsequently, the cardiac disk is transformed into a cardiac cone that elongates to form a tube and initiates heart beat. Intriguingly, endocardial nfatc1 expression is only initiated at 21-22 hpf, shortly after myocardial cells come into close contact with endocardial progenitors (Wong et al, 2012). Therefore, we hypothesized that the myocardium may provide a signal critical for endocardial differentiation.…”