During heart development and regeneration, coronary vascularization is tightly coupled with cardiac growth. Although inhibiting vascularization causes defects in the innate regenerative response of zebrafish to heart injury, angiogenic signals are not known to be sufficient for triggering regeneration events. Here, by using a transgenic reporter strain, we found that regulatory sequences of the angiogenic factor are active in epicardial cells of uninjured animals, as well as in epicardial and endocardial tissue adjacent to regenerating muscle upon injury. Additionally, we find that induced cardiac overexpression of in zebrafish results in overt hyperplastic thickening of the myocardial wall, accompanied by indicators of angiogenesis, epithelial-to-mesenchymal transition, and cardiomyocyte regeneration programs. Unexpectedly, overexpression in the context of cardiac injury enabled ectopic cardiomyogenesis but inhibited regeneration at the site of the injury. Our findings identify Vegfa as one of a select few known factors sufficient to activate adult cardiomyogenesis, while also illustrating how instructive factors for heart regeneration require spatiotemporal control for efficacy.

Objectives Smad4 is a central mediator of transforming growth factor-β/bone morphogenetic protein signaling that controls numerous developmental processes as well as homeostasis in the adult. The present studies sought to understand the function of Smad4 expressed in vascular smooth muscle cells (VSMC) in vascular development and the underlying mechanisms. Methods and results Breeding of Smad4flox/flox mice with SM22α-Cre mice resulted in no viable offspring with SM22α-Cre;Smad4flox/flox genotype in a total of 165 newborns. Subsequent characterization of 301 embryos between embryonic day 9.5 (E9.5) and E14.5 demonstrated that mice with SM22α-Cre;Smad4flox/flox genotype died between E12.5 and E14.5, due to decreased cell proliferation and increased apoptosis in the embryonic heart and arteries. Additionally, deletion of Smad4 more specifically in SMC with the inducible SMMHC-Cre mice, in which decreased cell proliferation was observed only in the artery but not the heart, also caused lethality of the knockout embryos at E12.5 and E14.5. The Smad4 deficient VSMC lacked smooth muscle α-actin filaments, decreased expression of SMC-specific gene markers, and markedly reduced cell proliferation, migration and attachment. Using specific pharmacological inhibitors and small-interfering RNAs, we demonstrated that inhibition of TGF-β signaling and its regulatory Smad 2/3 decreased VSMC proliferation, migration and expression of SMC-specific gene markers, while inhibition of BMP signaling only affected VSMC migration. Conclusions SMC-specific deletion of Smad4 results in vascular defects that lead to embryonic lethality in mice, which may be attributed to decreased VSMC differentiation, proliferation, migration, as well as cell attachment and spreading. The TGF-β signaling pathway contributes to VSMC differentiation and function; while the BMP signaling pathway regulates VSMC migration. These studies provide important insight into the role of Smad4 and its upstream Smads in regulating smooth muscle cell function and vascular development of mice.

We show for the first time that TGFβ signalling is directly regulated by the miRNA mechanism during myocardial wall morphogenesis. Increased TGFβ activity plays a major role in the cardiac defects caused by myocardial deletion of Dicer1. Thus, miRNA-mediated regulation of TGFβ signalling is indispensable for normal cardiogenesis.

MYCN is a highly conserved transcription factor with multifaceted roles in development and disease. Mutations in MYCN are associated with Feingold syndrome, a developmental disorder characterized in part by congenital heart defects. Mouse models have helped elucidate MYCN functions; however its cardiac-specific roles during development remain unclear. We employed a Cre/loxp strategy to uncover the specific activities of MYCN in the developing mouse myocardium. Myocardial deletion of Mycn resulted in a thin-myocardial wall defect with dramatically reduced trabeculation. The mutant heart defects strongly resemble the phenotype caused by disruption of BMP10 and Neuregulin-1 (NRG1) signaling pathways, two central mediators of myocardial wall development. Our further examination showed that expression of MYCN is regulated by both BMP and NRG1 signaling. The thin-wall defect in mutant hearts is caused by a reduction in both cell proliferation and cell size. MYCN promotes cardiomyocyte proliferation through regulating expression of cell cycle regulators (including CCND1, CCND2, and ID2) and promotes cardiomyocyte growth through regulating expression of p70S6K. In addition, expression of multiple sarcomere proteins is altered in Mycn myocardial-inactivation embryos, indicating its essential role for proper cardiomyocyte differentiation. In summary, Mycn acts downstream of BMP and NRG1 cardiogenic signaling pathways to promote normal myocardial wall morphogenesis.