Project Summary/Abstract Hematopoietic stem and progenitor cells (HSPCs) sustain lifelong hematopoiesis through self-renewal and differentiation into all blood cell types. They form during early embryogenesis through a regulated and conserved process termed the endothelial-to-hematopoietic transition (EHT). During the EHT, a subset of endothelial cells (ECs) adapt a hematopoietic transcriptional program to form the hemogenic endothelium (HE) followed by morphological changes to make HSPCs. As de novo production of HSPCs occurs solely during early embryonic development, HSPC formation has profound consequences on all embryonic and adult hematopoiesis. Defects in EHT regulators are prevalent in hematologic disorders, therefore lessons learned by studying EHT could inform the pathways driving these diseases. Studying EHT regulation is critical for understanding processes key to hematopoietic health from embryo to adult life. Though some factors controlling EHT are known, our knowledge of HE/HSPC regulators remains poorly understood. In prior studies, the Bowman lab determined that proper pre-mRNA splicing is required for EHT as HEs were largely absent in zebrafish mutants for the spliceosomal component splicing factor 3b, subunit 1 (sf3b1). These data indicate that splicing is important for HE formation, but the mechanisms regulating the splicing changes critical for EHT are largely unknown. To explore this process, I first defined the alternative splicing signature between embryonic zebrafish EC and HE/HSPC. Cis-acting regulatory elements within pre-mRNA guide splice isoform selection thus, to identify potential mechanisms controlling alternative splicing during EHT, I surveyed alternative splicing events between EC and HE/HSPC for differences in splicing regulatory sequence features. Through this preliminary analysis, I uncovered that the EHT alternative events were enriched for weaker splice sites and shorter intron length suggesting these features could have a regulatory function in dictating EHT specific-splice isoform choice. In addition to sequence-driven mechanisms, alternative splicing can be modified by transcriptional checkpoints such as promoter-proximal pausing. In pilot studies, I showed that pharmacological or genetic inhibition of promoter-proximal pausing factors can diminish HE/HSPC production. Based on my data and the literature, I will test the hypothesis that EHT-specific-splice isoform selection is guided by distinct cis-acting-regulatory elements (aim 1) and regulated by promoter proximal pausing factors (aim 2). These studies of cell-type specific splicing regulation in a complex, multicellular context will enable understanding of how splicing fine-tunes the EHT fate decision. Completion of this study will reveal critical regulation for the genesis of HSPC.