Project Summary Current understanding of the molecular mechanisms underlying hematopoietic stem cell specification (HSCs) to generate induced pluripotent stem cells (iPS cells) is still limited. In this proposal, we seek to understand the role of calcium signaling, acting through CaMKII, in HSC specification in zebrafish embryos. Suppression of one of the seven transcriptionally active CaMKII genes, camk2g1, leads to increased expression of ezh2, the functional enzymatic component of the polycomb repressive complex 2 (PRC2), inhibiting HSC specification. We first seek to characterize the role of camk2g1 in HSC specification through use of genetic knockdown techniques and pharmacological inhibitors. We then seek to determine how increased expression of ezh2 in camk2g1 deficient embryos alters downstream signaling important for HSC specification. Preliminary results suggest increased ezh2 expression inhibits downstream expression of target genes (tgfbr2 and jag1a) resulting in apoptosis of HSCs in the ventral wall of the dorsal aorta. The results from this proposal will provide novel insight into the calcium-dependent signaling pathways necessary to generate all lineages of HSCs from iPS cells.