Molecular basis for Flk1 expression in hemato-cardiovascular progenitors in the mouse.

The mouse Flk1 gene is expressed in various mesodermal progenitor cells of developing embryos. Recent studies have shown that Flk1 expression marks multipotent mesodermal progenitors, giving rise to various hemato-cardiovascular cell lineages during development. Flk1 expression also marks hemato-cardiovascular cell lineages in differentiating embryonic stem (ES) cells, which may be used in transplantation decisions to treat cardiovascular diseases. Despite its developmental and clinical importance in cardiovascular tissues, the transcriptional regulatory system of Flk1 has remained unclear. Here, we report a novel enhancer of the mouse Flk1 gene directing early mesodermal expression during development as well as ES differentiation. The enhancer enriches various mesodermal progenitors, such as primitive erythropoietic progenitors, hemangioblast (BL-CFC) and cardiovascular progenitors (CV-CFC). The enhancer is activated by Bmp, Wnt and Fgf, and it contains Gata-, Cdx-, Tcf/Lef-, ER71/Etv2- and Fox-binding sites, some of which are bound specifically by each of these transcription factors. As these transcription factors are known to act under the control of the Bmp, Wnt and Fgf families, early Flk1 expression may be induced by cooperative interactions between Gata, Tcf/Lef, Cdx and ER71/Etv2 under the control of Bmp, Wnt and Fgf signaling. The enhancer is required for early Flk1 expression and for hemangioblast development during ES differentiation.

Runx1 is involved in the fusion of the primary and the secondary palatal shelves.

Runx1 is expressed in medial edge epithelial (MEE) cells of the palatal shelf. Conditionally rescued Runx1(-/-) mice showed limited clefting in the anterior junction between the primary and the secondary palatal shelves, but not in the junction between the secondary palates. In wild type mice, the fusing epithelial surface exhibited a rounded cobblestone-like appearance, while such cellular prominence was less evident in the Runx1 mutants. We also found that Fgf18 was expressed in the mesenchyme underlying the MEE and that locally applied FGF18 induced ectopic Runx1 expression in the epithelium of the palatal explants, indicating that Runx1 was induced by mesenchymal Fgf18 signaling. On the other hand, unpaired palatal explant cultures revealed the presence of anterior-posterior (A-P) differences in the MEE fates and fusion mechanism. Interestingly, the location of anterior clefting in Runx1 mutants corresponded to the region with different MEE behavior. These data showed a novel function of Runx1 in morphological changes in the MEE cells in palatal fusion, which is, at least in part, regulated by the mesenchymal Fgf signaling via an epithelial-mesenchymal interaction.

Characterization of GATA-1(+) hemangioblastic cells in the mouse embryo.

Hemangioblasts are thought to be one of the sources of hematopoietic progenitors, yet little is known about their localization and fate in the mouse embryo. We show here that a subset of cells co-expressing the hematopoietic marker GATA-1 and the endothelial marker VE-cadherin localize on the yolk sac blood islands at embryonic day 7.5. Clonal analysis demonstrated that GATA-1(+) cells isolated from E7.0-7.5 embryos include a common precursor for hematopoietic and endothelial cells. Moreover, this precursor possesses primitive and definitive hematopoietic bipotential. By using a transgenic complementation rescue approach, GATA-1(+) cell-derived progenitors were selectively restored in Runx1-deficient mice. In the rescued mice, definitive erythropoiesis was recovered but the rescued progenitors did not display multilineage hematopoiesis or intra-aortic hematopoietic clusters. These results provide evidence of the presence of GATA-1(+) hemangioblastic cells in the extra-embryonic region and also their functional contribution to hematopoiesis in the embryo.

Primitive erythropoiesis from mesodermal precursors expressing VE-cadherin, PECAM-1, Tie2, endoglin, and CD34 in the mouse embryo.

Vascular endothelial (VE) cadherin, PECAM-1 (platelet endothelial cell adhesion molecule-1, CD31), Tie2, CD34, and endoglin are established markers for adult and embryonic endothelial cells (ECs). Here, we report that the expression of these EC markers is initiated in the extraembryonic region at the late-streak stage (nominal stage E6.75). Immunohistochemical analysis shows that EC marker-positive cells arise in a subset of Flk1 (VEGF-R2) mesodermal cells. In contrast, GATA1, a marker for primitive erythropoietic progenitors, is expressed in a more restricted subset of Flk1-positive cells. Using flow cytometry, we observed that the GATA1-positive cell population existed as a subset of the EC marker-positive cell. Consistent with this notion, we showed with the primitive hematopoietic colony assay that primitive erythropoietic progenitors are enriched in PECAM-1- and Tie2-positive cells. These results suggest that primitive hematopoietic cells arise from EC marker-positive cells. Thus, VE-cadherin, PECAM-1, CD34, endoglin, and Tie2 are expressed not only in adult and embryonic ECs but in extraembryonic Flk1-positive cells during gastrulation. The latter cell population includes progenitors that give rise to primitive hematopoietic cells, suggesting that primitive and definitive hematopoietic cells in the mouse embryo arise from EC marker-positive cells.