Extrinsic Regulation of Hematopoietic Stem Cell Formation and Function

Abstract

Hematopoietic stem/progenitor cells (HSPCs) are formed during development from hemogenic endothelium in the ventral wall of the dorsal aorta (VDA). The transcriptional programs that promote HSPC production are increasingly understood, but the extrinsic factors and niche-derived signals that initiate these intracellular processes remain largely uncharacterized. Matrix metalloproteinase (MMP) enzymes remodel extracellular matrix (ECM) and regulate signaling pathways. Inhibiting Mmp2 function in zebrafish embryos retained runx1/cmyb+ HSPCs in the VDA through accumulation of fibronectin-rich ECM, delaying their colonization of the caudal hematopoietic tissue (CHT) and thymus. In contrast, Mmp9 was dispensable for HSPC budding, being instead required for their migration and CHT colonization. These migratory defects were phenocopied by over-expression and blocked by knockdown of the chemokine Cxcl12, suggesting that Mmp9 controls CHT homeostasis through chemokine regulation. Mmp9 is also required downstream of the regulatory factor prostaglandin-E2 (PGE2) for normal HSPC maturation. Co-treatment with PGE2 and an Mmp9-inhibitor retained HSPCs in the CHT, preventing their colonization of adult niches. This prolonged CHT residence promoted myeloid differentiation, establishing the CHT as a myeloproliferative niche. In addition to regulating Cxcl12, Mmp9 is known to proteolytically activate several growth factors. Targeted inhibition of one such factor, Tgfb, phenocopied Mmp9 inhibition, causing HSCs to adopt myeloid identity at the expense of their own maintenance and lymphoid/erythroid differentiation potential; this identified a novel pathway that maintains HSC stemness in the CHT. Finally, prior studies demonstrated that HSC formation is tied to the biophysical forces of blood flow, which include wall shear stress (WSS) and circumferential stress (CS). WSS works through nitric oxide signaling, but how CS impacts HSC formation is undetermined. In vitro, CS increased expression of the HSC marker RUNX1 by activating YAP signaling. This was conserved in the zebrafish, where Yap overexpression increased HSPC production. Rho GTPase activation rescued HSPC production in the absence of flow in a Yap-dependent manner, identifying Rho GTPases as intermediaries between CS and Yap activity. Together, these findings establish several novel mechanisms for the extrinsic regulation of HSPC production, and describe functional intersections between cytokines, inflammatory mediators, and biophysical forces in the onset and maintenance of developmental hematopoiesis.