I am pleased to report that we have continued to make considerable progress on my CIRM New Investigator Award. In the past year, we have helped solve a long-standing controversy in the field of developmental hematopoiesis as to exactly where the first hematopoietic stem cells (HSCs) are born in the embryo. Using the strategies outlined in Aim 2 of this proposal, we demonstrated that HSCs are born from hemogenic endothelial cells lining the ventral floor of the dorsal aorta (Bertrand and Chi et al., Nature 464: 108-111). This new knowledge will be immensely helpful in continuing our genetic dissection of how HSCs are patterned from mesoderm, as we now know precisely when and where HSCs arise, and that a requisite step in their formation is through an endothelial intermediate. In addition, we have made excellent progress on Aims 3 and 4. Under Aim 3, we have recently demonstrated that, of the four independent waves of hematopoietic precursors that arise in the vertebrate embryo, Notch signaling plays a role only in HSC formation (Bertrand and Cisson, Blood, ePub 1/27/10). Furthermore the requirement for Notch signaling in HSCs is absolute, in that no HSCs are formed in the absence of all Notch ligands. These findings are guiding our studies in Year 3. Finally, under Aim 4, we have completed our first set of experiments on the role of Wnt16 in HSC formation and will submit a manuscript to Nature within the month. Wnt16 morphants lack HSCs, and transient induction of Notch signaling in Wnt 16 morphants rescues HSC development. We have demonstrated that Wnt16 lies genetically upstream of DllC and DllD, and shown, of the seven Notch ligands, that these two are necessary for HSC specification. These findings are important, since very little is known regarding the roles of Wnt signaling in HSC formation.