3152 – IDENTIFYING SELECTIVE REGULATORS OF HEMATOPOIETIC STEM CELLS IN DEVELOPMENT

Abstract

Hematopoietic stem cells (HSCs) can sustain the entire adult blood system due to their multipotency and self-renewal properties. In contrast, embryonic HSC-independent progenitor cells (HPCs) have limited regeneration and restricted differentiation. Understanding what distinguishes HSCs from HPCs could reveal the key to multipotency and self-renewal. To dissect early differences between these cells during embryonic development, we established a temporally-regulated lineage tracing assay in zebrafish that allowed us to distinguish HSCs from HPCs based on distinct differentiation kinetics. We determined that T-cell and granulocyte production from HSCs was significantly delayed compared to HPCs. Using this system, we performed a focused reverse genetic screen for factors that modulate the timing of HSC-derived T-cell production. As a control, we demonstrated that loss of Runx1, a master transcription factor for definitive hematopoiesis, diminished both HSC and HPC-derived T-cells. We then targeted 30 novel genes using CRISPR/Cas9-mutagenesis and screened for acceleration or further delay in HSC differentiation to T-cells, identifying one positive and two negative HSC regulators. Mutagenesis of pa2g4b (proliferation-associated factor 4b) led to increased HSC-derived T-cells, but no change in HPC differentiation, suggesting it is an HSC-selective negative regulator. Beyond T-cells and granulocytes, we delineated the full differentiation repertoire of embryonic HSCs and HPCs using scRNA-seq of lineage-traced cells and found HPCs could contribute to all major blood lineages in larval zebrafish suggesting they might possess a broader differentiation potential. Identifying factors that regulate HSC's ability to differentiate and self-renew in development can aid the efforts for generating HSCs in vitro from alternative sources such as patient-derived pluripotent stem cells. Hematopoietic stem cells (HSCs) can sustain the entire adult blood system due to their multipotency and self-renewal properties. In contrast, embryonic HSC-independent progenitor cells (HPCs) have limited regeneration and restricted differentiation. Understanding what distinguishes HSCs from HPCs could reveal the key to multipotency and self-renewal. To dissect early differences between these cells during embryonic development, we established a temporally-regulated lineage tracing assay in zebrafish that allowed us to distinguish HSCs from HPCs based on distinct differentiation kinetics. We determined that T-cell and granulocyte production from HSCs was significantly delayed compared to HPCs. Using this system, we performed a focused reverse genetic screen for factors that modulate the timing of HSC-derived T-cell production. As a control, we demonstrated that loss of Runx1, a master transcription factor for definitive hematopoiesis, diminished both HSC and HPC-derived T-cells. We then targeted 30 novel genes using CRISPR/Cas9-mutagenesis and screened for acceleration or further delay in HSC differentiation to T-cells, identifying one positive and two negative HSC regulators. Mutagenesis of pa2g4b (proliferation-associated factor 4b) led to increased HSC-derived T-cells, but no change in HPC differentiation, suggesting it is an HSC-selective negative regulator. Beyond T-cells and granulocytes, we delineated the full differentiation repertoire of embryonic HSCs and HPCs using scRNA-seq of lineage-traced cells and found HPCs could contribute to all major blood lineages in larval zebrafish suggesting they might possess a broader differentiation potential. Identifying factors that regulate HSC's ability to differentiate and self-renew in development can aid the efforts for generating HSCs in vitro from alternative sources such as patient-derived pluripotent stem cells.