Abstract
Hematopoietic stem cells (HSC) are responsible for life-long maintenance and regeneration of the adult vertebrate blood system. The first HSCs arise from intra-aortic hematopoietic cluster cells (IAHC) in the mouse aorta at embryonic day (E)10.5 through a transdifferentiation process called endothelial-to-hematopoietic-transition (EHT). Previously we showed that the heptad factor Gata2, one of a group of 7 (heptad) transcription factors pivotal to definitive hematopoiesis, is required for HSC generation and survival. Gata2-/- embryos suffer lethality at E10.5 and Gata2+/- HSCs are qualitatively defective. Utilizing our Gata2Venus reporter mouse for time-lapse imaging of EHT in vivo, we found rapid pulsatile Gata2 expression level changes in single transitioning cells, implicating transcriptional instability during establishment of hematopoietic fate. Flow cytometric analysis of IAHCs revealed cells with varying levels of Venus (Vmedium, Vhigh). Hematopoietic progenitors (HPC) were highly enriched and HSCs exclusively found in the Vmed fraction. From these results, we hypothesized that fate choice is based on stochastic expression (levels and combinations) of the pivotal heptad factors. Here we test this by single cell transcriptomics and functional assays. Single-cell RNA sequencing of index-sorted Vmed cells shows surprising heterogeneity in heptad gene expression and has facilitated further HSC enrichment, defining intensity levels of known markers and providing additional markers. These new markers were functionally validated and their expression visualized by immunohistochemistry. Single-cell HPC assays also allowed for enrichment of immature HPCs. Further sorting and sequencing reiterations will refine the HSC transcriptomic profile and provide information on whether transcriptome heterogeneity correlates with and plays a role in generating both HSCs and HPCs.
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