Mechanisms of Hematopoietic stem cell development during fetal life - Summary: In the mouse, multipotent erythro-myeloid progenitors are produced through an endothelial-to- hematopoietic transition (EHT) process, in the vasculature of the yolk sac, between E8-8.5. Starting at E9, a subset of endothelial cells in the intra-embryonic vasculature undergoes EHT, spanning for around 48 hours and resulting in the emergence of the first lympho-myeloid progenitors that enter the circulation and home to the fetal liver (FL). It is in this environment that multipotent progenitors give rise to the hematopoietic stem cell (HSC) compartment starting at E10.5, but it is only after E12 that HSCs can be identified through the acquisition of characteristic surface markers such as SLAMF1 (CD150) and Sca1. For a period of 1.5 days, from E10.5 to E12, HSCs can only be detected indirectly through transplantation experiments, a gap in knowledge we will address in Aim 1. The signals promoting the development of HSCs in the FL remain incompletely defined. Stem cell factor (SCF) and Thrombopoietin (THPO) activate receptor tyrosine kinases (RTK) that play pivotal roles in HSC proliferation and survival, but also orchestrate the intricate process of hematopoietic multipotent progenitor cell differentiation into various cell lineages, including lymphoid, myeloid, and megakaryocyte/erythrocyte cells. We hypothesized that other signals must exist to allow HSC expansion during fetal life while counterbalancing the pro-differentiation effects of RTK signaling induced by SCF and THPO. GPCRs signal through heterotrimeric G proteins composed of alpha, beta and gamma subunits, with the alpha subunit conferring pathway specificity. GPCR signaling via Ga12/13 proteins potently inhibits PI3K activation such that genetic deficiency in Ga12 or Ga13 results in lymphoproliferative disease and lymphomagenesis. In recent and unpublished studies, we uncovered a critical role for the Ga12/13-coupled GPCRs GPR56 and GPR97 in HSCs. Specifically, we demonstrate that deficiency in both receptors is lethal due to a complete loss of HSCs during embryogenesis, while induced deficiency in both receptors in HSCs of adult mice leads to the complete loss of HSCs and adult hematopoiesis. Our findings provide the foundation for a new paradigm in which HSC homeostasis is controlled by a balance between stimulatory (RTKs) and inhibitory (GPCRs) pathways. The central goal of this grant proposal is to precisely define the role of GPR56 and GPR97 in HSCs. In Aim 1 we will test if Gpr56 protein expression marks the birth of the first HSC in early embryonic stages (starting at E9). In Aim 2, we will directly test if GPR56 and GPR97 counter-balance cKit signaling using a combination of in vitro and in vivo experiments that will modulate cKit signaling intensity. We will also study the transcriptional hubs controlled by GPR56 and GPR97 in fetal liver HSCs by bulk and scRNAseq. Together, the experiments described in these aims will reveal novel mechanisms and fundamental new principles of HSC homeostasis and multilineage differentiation.
