Abstract

The establishment of the circulatory system including blood, vasculature, and the heart, is highly coordinated and a prerequisite for embryonic development. Recent studies have demonstrated that the receptor tyrosine kinase FLK-1 expressing (FLK-1⁺) mesodermal cells contribute to these cell lineages. However, signals/factors that regulate the generation of FLK-1⁺ cells and their subsequent differentiation are not clearly defined. To understand FLK-1⁺ mesoderm generation and differentiation, the in vitro serum-free differentiation model of embryonic stem (ES) cells and genetic approaches were employed. First, I showed that Bone Morphogenetic Protein (BMP)-4 is critical for induction of Brachyury⁺ mesoderm from ES cells and FLK-1⁺ cells from Brachyury⁺ cells. In combination with Vascular Endothelial Growth Factor (VEGF), BMP-4 significantly enhanced the production of the transcription factor SCL (Stem Cell Leukemia)⁺ cells, which represent hematopoietic precursors. In addition, BMP-4 activated the SMAD1/5 pathway and inhibition of the SMAD1/5 pathway reduced the generation of FLK-1⁺ cells. VEGF mediated expansion of hematopoietic and endothelial cell progenitors was inhibited by TGF-β1, but was augmented by Activin A. These results suggest that hematopoietic commitment from the mesoderm occurs via BMP-4 mediated signals and that expansion and/or differentiation of such progenitors is achieved by an interplay of VEGF, TGF-β1, and Activin A signaling. Second, I inactivated Alk-3 (BMP receptor 1A) in FLK-1⁺ cells by crossing Alk-3^{floxed/floxed} and Flk-1^+/CreAlk-3 ^+/floxed mice and investigated the function of BMP receptor signaling in the generation of blood, vessel and smooth muscle cells from FLK-1⁺ mesoderm. Alk-3 conditional knockout embryos exhibited normal development of blood cells, but Smad4 conditional knockout embryos displayed impaired hematopoiesis. This suggests that a family of TGF-β growth factors contribute to hematopoietic development. Absence of ALK-3 signaling resulted in embryonic lethality due to defects in vessel remodeling and vessel integrity. In addition, defects in the endocardial cushion of the atrioventricular canal were found in the Alk-3 conditional knockout mice. These findings indicate a critical role for ALK-3 in vessel remodeling, vessel integrity, and endocardial cushion formation during the development of the circulation system. Collectively, this study has revealed novel and specific roles for BMP/BMP receptor signaling in early embryogenesis.