Congenital heart defects due to the aberrant development of the atrioventricular (AV) valves and septum are among the most common developmental abnormality in newborns and cause significant neonatal morbidity and mortality. A key point in cardiac morphogenesis occurs when cells within the endocardial cushions (ECCs), the precursors for the AV valvoseptal complex, delaminate and undergo an epithelial-to-mesenchymal transformation (EMT). The mesenchymal cells then proliferate and the cushion area elongates to form the AV valves and portions of the AV septae. The signals that initiate region-specific EMT during heart development are unknown. Cilia, known for their role in establishing left-right (LR) asymmetry, function to receive and integrate extracellular signals, including fluid flow, in a range of other organ systems. We hypothesize that cilia could also have a direct role in heart development outside of their role in LR development. Using immunohistochemistry, we demonstrated the presence of cilia on the myocardium, epicardium, and ECCs of wild-type mouse hearts at embryonic day (e) 9.5 and e12.5. To characterize the potential role of these cilia, we compared mice with mutations affecting ciliary biogenesis, motility, and mechanosensation. Using bright field microscopy and in situ hybridization, we analyzed the embryonic heart structure and the expression pattern of Gata4, an EMT transcription factor. We showed that compared to mice with immotile but structurally normal cilia, the mice without cilia had hypocellular ECCs, a thinned compact myocardium (CM), and an up-regulated expression of Gata4. These observations suggest that a subset of cilia called cardiac cilia have a role in cardiogenesis outside of their role in LR development and affect Gata4 expression. One possible function of cardiac cilia is as mechanosensors, integrating fluid flow and influencing cardiac morphogenesis including EMT and development of the CM.

Indiana University-Purdue University Indianapolis (IUPUI) / Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.

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