Camk2g1 regulates Notch signaling and histone methylation to specify hematopoietic stem cells

Hematopoietic stem and progenitor cells (HSPCs) are a self-renewing population that generates all blood lineages throughout life. Their specification requires a developmental niche that provides signals to establish hemogenic endothelium and drive endothelial-to-hematopoietic transition. We identify a previously unrecognized role for the Ca²⁺-dependent kinase, CaMKII, in this process. Pharmacological inhibition of CaMKII during zebrafish development results in a loss of definitive HSPCs, a defect reversible upon drug washout, indicating a defined developmental window of CaMKII activity. Among the seven paralogs, camk2g1 is highly expressed in tissues that give rise to HSPCs. Suppression of camk2g1 disrupts HSPC specification without altering arterial identity. Transcriptomic profiling of camk2g1 crispants reveals upregulation of apoptotic pathways, accompanied by increased cleaved caspase-3-positive endothelial cells in the dorsal aorta. Camk2g1 loss also reduces expression of the H3K27me3 demethylase kdm6ba and the Notch pathway components notch3 and jag1a, linking CaMKII activity to both epigenetic regulation and Notch signaling. Inhibition of the H3K27 methyltransferase Ezh2 restores HSPC marker expression, decreases apoptosis, and reduces tp53 expression, demonstrating that elevated H3K27me3 contributes to HSPC loss. Suppression of camk2g1 in tp53 heterozygous embryos restores T-cell marker gene expression, further supporting a tp53-mediated mechanism. Because Pkd2 functions upstream of arterial Notch signaling, we tested sub-effective suppression of pkd2 and camk2g1 individually and together. Dual knockdown synergistically reduces Notch signaling and HSPC formation, placing camk2g1 and pkd2 within a shared pathway. Together, these findings establish camk2g1 as a spatially and temporally restricted regulator that integrates chromatin state and Notch activity to enable HSPC specification.