Recent studies support the existence of a common progenitor for the cardiac and endothelial cell lineages, but the underlying transcriptional networks responsible for specification of these cell fates remain unclear. Here we demonstrated that Ets-related protein 71 (Etsrp71), a newly discovered ETS family transcription factor, was a novel downstream target of the homeodomain protein, Nkx2-5. Using genetic mouse models and molecular biological techniques, we demonstrated that Nkx2-5 binds to an evolutionarily conserved Nkx2-5 response element in the Etsrp71 promoter and induces the Etsrp71 gene expression in vitro and in vivo. Etsrp71 was transiently expressed in the endocardium/endothelium of the developing embryo (E7.75-E9.5) and was extinguished during the latter stages of development. Using a gene disruption strategy, we found that Etsrp71 mutant embryos lacked endocardial/endothelial lineages and were nonviable. Moreover, using transgenic technologies and transcriptional and chromatin immunoprecipitation (ChIP) assays, we further established that Tie2 is a direct downstream target of Etsrp71. Collectively, our results uncover a novel functional role for Nkx2-5 and define a transcriptional network that specifies an endocardial/endothelial fate in the developing heart and embryo.cardiac progenitor cells ͉ endocardium ͉ Etsrp71 ͉ Tie2 ͉ cardiac development

The Hedgehog signaling pathway is critical for a significant number of developmental patterning events. In this study, we focus on the defects in pharyngeal arch and cardiovascular patterning present in Sonic hedgehog (Shh) null mouse embryos. Our data indicate that, in the absence of Shh, there is general failure of the pharyngeal arch development leading to cardiac and craniofacial defects. The cardiac phenotype results from arch artery and outflow tract patterning defects, as well as abnormal development of migratory neural crest cells (NCCs). The constellation of cardiovascular defects resembles a severe form of the human birth defect syndrome tetralogy of Fallot with complete pulmonary artery atresia. Previous studies have demonstrated a role for Shh in NCC survival and proliferation at later stages of development. Our data suggest that SHH signaling does not act directly on NCCs as a survival factor, but rather acts to restrict the domains that NCCs can populate during early stages (e8.5-10.5) of cardiovascular and craniofacial development.

Members of the basic helix-loop-helix (bHLH) family of transcription factors regulate the specification and differentiation of numerous cell types during embryonic development. Hand1 and Hand2 are expressed by a subset of neural crest cells in the anterior branchial arches and are involved in craniofacial development. However, the precise mechanisms by which Hand proteins mediate biological actions and regulate downstream target genes in branchial arches is largely unknown. Here, we report that Hand2 negatively regulates intramembranous ossification of the mandible by directly inhibiting the transcription factor Runx2, a master regulator of osteoblast differentiation. Hand proteins physically interact with Runx2, suppressing its DNA binding and transcriptional activity. This interaction is mediated by the N-terminal domain of the Hand protein and requires neither dimerization with other bHLH proteins nor DNA binding. We observed partial colocalization of Hand2 and Runx2 in the mandibular primordium of the branchial arch, and downregulation of Hand2 precedes Runx2-driven osteoblast differentiation. Hand2 hypomorphic mutant mice display insufficient mineralization and ectopic bone formation in the mandible due to accelerated osteoblast differentiation, which is associated with the upregulation and ectopic expression of Runx2 in the mandibular arch. Here, we show that Hand2 acts as a novel inhibitor of the Runx2-DNA interaction and thereby regulates osteoblast differentiation in branchial arch development.

Forkhead box O1 (FoxO1), a member of the Forkhead box-containing O family of transcription factors, is a key regulator of numerous genes that govern a wide array of cellular functions, including differentiation, homeostasis, and survival. However, the role of FoxO1 in development remains elusive. Here, we describe an essential and previously undefined role for FoxO1 in placental development. We demonstrate that FoxO1-null embryos up to embryonic day 9.0 (E9.0) are indistinguishable, including their morphology, cardiovascular structure, and vascular gene expression, from wild-type (WT) littermates. However, FoxO1-nulls manifested a profoundly swollen/hydropic allantois, which failed to fuse with the chorion, a phenotype that leads to subsequent cardiovascular malformation, progressive apoptotic cell death, and embryonic lethality at E10.5. Quantitative RT-PCR analysis of genes involved in placental development revealed significant attenuation of VCAM1 expression in FoxO1-null embryos. Using immunohistochemical, transcriptional, and chromatin immunoprecipitation assays, we further discovered that FoxO1 is an essential upstream regulator of the VCAM1 gene. Collectively, our findings provide critical molecular insight into a unique FoxO1-VCAM1 axis that governs placental morphogenesis, a process that is essential for subsequent normal cardiovascular development and fetal life.