Discrete Notch signaling requirements in the specification of hematopoietic stem cells

Hematopoietic stem cells (HSCs) are generated during embryonic development and possess the ability to reconstitute all adult blood lineages and self-renew for the life of an organism. Despite efforts to generate HSCs in vitro in a manner that recapitulates embryonic development, all attempts to date have failed indicating that our knowledge of this process is incomplete. Cell signaling pathways are crucial for the formation of HSCs during embryonic development. Understanding the requirements for cell signaling are complicated by the fact that there is crosstalk between pathways during development, necessitating a clear understanding of these relationships. Notch signaling is essential for HSC formation, as evidenced by the fact that many Notch pathway proteins are required for this process including the specific Notch1 receptor that functions cell- autonomously in mouse. Additionally, recent data from the zebrafish indicate that Notch signaling downstream of the non-canonical Wnt protein Wnt16 is also required in the somitic environment for HSC and sclerotome specification, suggesting that these two developmental events are related. We asked if the remaining Notch receptors perform any role in HSC emergence, here we present evidence that Notch3 is required in zebrafish. Tissue and temporal- specific activation of Notch intracellular domain (NICD) rescue experiments in Notch3 knockdown embryos indicate that Notch3 is required in somitic tissues for the generation of HSCs and sclerotome well before establishment of the HSC program, conversely Notch1 homologues Notch1a and Notch1b are required in the endothelium for dorsal aorta (DA) and HSC specification just before HSCs are generated. We identified that the function of Notch3 lies downstream of Wnt16, Dlc, and Dld in a linear genetic pathway. In contrast, endothelial expression of Notch1b requires the activity of Pdgfra receptor in a distinct signaling cascade from Wnt16- Notch3. Collectively, these findings demonstrate that multiple inputs from the Notch pathway are required at different times and places during development, with distinct inputs regulated by specific signaling pathwa