| Cardiovascular diseases (CVD) are the leading cause of death in developing and developed countries. Congenital heart disease is one type of CVD derived from abnormal heart development during embryogenesis, and affects up to 5% of newborns. Understanding of the mechanisms involved in establishing the early heart and regulating its morphogenesis are crucial.The earliest cardiac progenitor comes from lateral plate mesoderm and consists of two separate progenitor cell lineages, first heart field (FHF) and second heart field (SHF). The FHF uniquely contributes to the left ventricular (LV), while the SHF contributes to outflow tract (OFT), right ventricle (RV), sinus venosus (SV), and left and right atria (LA and RA, respectively). Signals including fibroblast growth factors (Fgfs), bone morphogenetic proteins (Bmps), and Wnt proteins are critical for inducing the expression of the cardiogenic transcription factors which controls early cardiac development. However, how the signaling pathways and cardiac transcriptional regulators coordinate with eath other to regulate cardiogenesis is still unclear.Hand2, as well as other cardiac transcription factors such as Isll, Mef2c, Fgf8/10 and FoxH, is required for second heart field development. Germline mutation of Hand2 results in embryonic lethality with failure of heart looping, so we specifically deleted Hand2 using Mef2c-cre mice to study the role of this gene in early cardiac development, particularly in SHF development. Loss of Hand2 in SHF results in right ventricle hypoplasia and outflow tract defects, and ultimately causes embryonic lethality at around E13.5, indicating the essential role of Hand2 in this region. Immunostaing assay revealed decreased cell proliferation, increased apoptosis and autophagy, but no changes in differentiation is observed in Hand2 mutants. In addition, ablation of Hand2 leads to downregulation of ERK activity. Fgf8 and Fgf10, which directly regulate ERK activity, are also downregulated in Hand.2 KO embryos. We further investigated that Fgf10 is a direct downstream target of Hand2 in SHF development. However, enhanced ERK activity by deletion of Pten could partially reverse the phenotypes of Hand2 single mutant both at the molecular level and in the mouse model. Genetic ablation of Hand2 and Pten can also extend the life span of Hand.2 mutant mice.In early embryonic development, the activation of the MAPK/ERK and the PI3K/AKT pathway, directly induced by FGF signaling, are essential for cell survival, proliferation and differentiation. Previous studies indicated the importance of Pten as a tumour suppressor, which negatively regulates PI3K/AKT and MAPK/ERK signaling pathway. However, a role for Pten in vertebrate development has not been elucidated. To investigate whether the disruption of these two major pathways by deletion of Pten can influence embryonic development, particularly in early heart morphogenesis, we focus our study on the function of Pten in SHF development. Histological sections analysis of both embryonic and adult mice reveals that deletion of Pten in the SHF results not only in cardiac hypertrophy, ventricular septal hyperplasia, and outflow tract defects, but also cardiac valve and trabecular defects. Meanwhile, the ablation of Pten leads to excessive proliferation and premature differentiation of SHF progenitor cells. Further investigation reveals that Pten negatively regulates SHF cell proliferation through AKT-FOXO mediated upregulation of BMP-SMAD signaling pathway. In addition, we find that Pten could upregulate NOTCH signaling to promote ventricular trabeculation development. BAF complexes also participate, with an Akt-dependent manner, in regulating RV development of Pten KO mice, suggesting that Pten is involved in epigenetic regulation during SHF development.Taken together, our results demonstrate that Hand2 and Pten play important roles in regulating SHF progenitor cells proliferation and cardiac morphogenesis. |
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