Deciphering ARAP3 Functions in Hematopoiesis and Hematopoietic Stem Cells

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Doctor of Philosophy (PhD)
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Cell & Molecular Biology
hematopoietic stem cells
HSC niche
mouse models
Developmental Biology
Molecular Biology
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ARAP3 is a GTPase-activating protein that inactivates Arf6 and RhoA GTPases. ARAP3 deficiency in mice causes a sprouting angiogenic defect resulting in embryonic lethality by E11. Mice with an ARAP3 R302,303A mutation (KI/KI) that prevents activation by PI3K have a similar angiogenic phenotype, although rare animals survive to adulthood. Although ARAP3 was first discovered in porcine leukocytes, it remains largely unstudied in hematopoiesis and hematopoietic stem cells (HSCs). In this thesis, we aim to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in hematopoiesis and HSCs using several conditional knockout (CKO) transgenic mouse models in addition to the KI/KI mutant mouse model. Here, we report that HSCs from surviving adult KI/KI bone marrow (BM) are compromised in their ability to self-renew and reconstitute recipient mice. To decipher the possible mechanisms of the KI/KI mutation, we utilize our genetic CKO models to conditionally delete Arap3 in hematopoietic cells and in several cell types within the HSC niche. Excision of Arap3 in hematopoietic cells using Vav-Cre does not alter the ability of ARAP3-deficient HSCs to provide multi-lineage reconstitution and to undergo self-renewal, suggesting ARAP3 does not play a cell-autonomous role in HSCs. Deletion of Arap3 in osteoblasts and mesenchymal stromal cells using Prx1-Cre resulted in no discernable phenotypes in hematopoietic development or HSC homeostasis in adult mice. However, reverse transplantation into Arap3;Prx1 CKO mice resulted in an expanded phenotypic HSC compartment, suggesting ARAP3 plays a crucial role in the BM niche to maintain and regulate HSC self-renewal. In contrast, deletion of Arap3 using VEC-Cre resulted in embryonic lethality, yet HSCs from surviving adult mice were largely normal and reverse transplantation into Arap3;VEC CKO mice revealed HSC frequencies and functions comparable to control mice. Taken together, this thesis work suggests that despite a critical role for ARAP3 in embryonic vascular development, its loss in endothelial cells minimally impacts HSCs in adult BM; however ARAP3 may play a pivotal role in the mesenchymal and osteoblastic BM niche to regulate HSC functions.

Wei Tong
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