Hematopoietic stem cells (HSCs) produce all mature blood cells throughout the life of an organism. Following hematologic injuries that greatly reduce blood cell numbers, such as chemotherapy, a slow HSC response can result in life threatening complications from infection, bleeding, and anemia. Furthermore, malfunctioning HSCs can also result in bone marrow failure disorders or malignancies. Mounting evidence shows that proper gene expression control in HSCs is essential for maintenance of hematopoietic homeostasis. Most prior work centered on studying transcriptional control in HSCs. Recent human genomics studies of patients with hematologic diseases uncovered splicing as a putative regulator of HSCs, but how or why mutated spliceosomal components lead to aberrant hematopoiesis is unclear. In previous work studying gene expression in murine HSCs, we observed a change in splicing dynamics between resting and injury-activated HSCs. This process of HSC activation can be modulated by stimulation of the Wnt signal transduction pathway, which has been implicated in splicing regulation. Moreover, in developing zebrafish, we and others have data demonstrating that mutations in several spliceosomal components result in diminished production of hematopoietic stem and progenitor cells (HSPCs). Together these data suggest splicing regulation is important for HSCs during development, regeneration, and disease. As spliceosomal components are ubiquitously expressed, in this proposal we plan to test the hypothesis that external signals and intrinsic elements drive cell- specific outcomes of splicing i HSCs. Aim 1 will examine how the Wnt signaling pathway alters splicing in HSCs. We will perform RNA-sequencing experiments in purified murine HSCs in the presence and absence of Wnt pathway activation and identify which transcripts and exons are differentially spliced. Specific inhibition of various steps of the Wnt pathway will then be performed to define which step is important for the effects on splicing. Aim 2 will address which epigenetic factors are central for the splicing defects observed in HSPC formation in zebrafish. We will execute a synthetic lethal screen where we knockdown the levels of select epigenetic factors in zebrafish carrying mutations in spliceosomal components and then look for alterations in HSPC levels. We will then employ biochemical and genomics techniques to determine the mechanism through which the epigenetic and splicing components interact. This work will uncover elements of cell-type specific splicing regulation in HSCs.