| In the developing embryo, the first wave of hematopoiesis is the transient embryonic (primitive) wave, which, in the mouse, commences in the yolk sac at embryonic day 7.5. Here, bipotential cells called hemangioblasts form the two types of cells found in the yolk sac blood islands, consisting of hematopoietic progenitors surrounded by angioblasts. The hematopoietic progenitors then differentiate to form primitive hematopoietic cells, including erythrocytes, macrophages, and megakaryocytes, while angioblasts generate endothelial cells. The primitive wave provides essential early hemato-vascular components during embryonic growth, but it is transient and under tight temporal control involving genetic pathways.;One key regulator is the bone morphogenic protein (BMP) signaling pathway, which plays an important role in specifying and patterning ventral mesoderm and subsequent hematovascular development. However, there are major gaps in understanding the downstream mediators of BMP signaling that regulate the specification of hemangioblasts from the ventral mesoderm.;Using an inducible ESC line that allows for temporal expression of exogenous Smad1, a main effector of the BMP signaling pathway, it was shown that continuous induction of Smad1 expression in embryoid body (EB) cultures from day 2-5 did not perturb hematopoietic development. However a 6 hour pulse of Smad1 expression in EBs between day 2 and day 2.25 was sufficient to significantly increase formation of BL-CFCs, the in vitro equivalent of hemangioblasts, subsequent erythroid progenitors (eryPs), and other hematopoietic lineages.;To further investigate the downstream mechanism for how SMAD1 specifies hemangioblasts, ChIP-seq experiments were performed that identified Notch1 as a direct target of SMAD1 binding. To analyze if this had functional consequences, I found that, upon SMAD1 induction, levels of cleaved (active) NOTCH1 were decreased. Furthermore, inhibition of NOTCH signaling, via addition to EB cultures of the chemical inhibitor DAPT between day 2 and day 2.25, recapitulated the increase in formation of BL-CFCs, eryPs, and other hematopoietic lineages, as seen by induction of Smad1 at the same stage. This inhibition of NOTCH signaling corresponds with an upregulation of Wnt3 and Wnt3a transcripts. Additionally, a gradual increase in WNT inhibitors was seen upon reversal of NOTCH inhibition.;Together, the data reveals that specification of the hemangioblast population from mesoderm involves transient inhibition of NOTCH1 through repression by SMAD1, resulting in upregulation of WNT signaling. When SMAD1 expression is relieved, this reactivates NOTCH signaling, and WNT inhibitors are expressed, restricting the window of WNT signaling, and thereby regulating the transient nature of hemangioblast specification. |
