Single-cell dissection of long non-coding RNAs pivotal for embryonic hematopoietic stem cell development

In mammalian embryos, the stepwise generation of hematopoietic stem cells (HSCs) from endothelial precursors and pre-hematopoietic stem cells (pre-HSCs) is stringently modulated by signaling pathways and transcription factors. Nevertheless, the understanding of regulatory strength and mechanism by non-coding RNA (ncRNA) remains entirely lacking. Taking advantage of our capacity to capture rare single pre-HSCs and HSCs in vivo and accordingly generate the single-cell transcriptome datasets, here we presented the first long non-coding RNA (lncRNA) landscape of HSC development at single-cell resolution. Combining bioinformatics analysis and functional screening, we identified 6 lncRNAs showing influence on hematopoietic progenitor generation in vitro. Moreover, endothelial disruption of H19 in vivo resulted in defective emergence of functional pre-HSCs and HSCs in aorta-gonad-mesonephros region, albeit with unperturbed development of erythro-myeloid progenitors in yolk sac. Single-cell RNA sequencing revealed that H19 deficient pre-HSCs (The DMR region of H19 was knocked out in the "KO" samples) possessed similar molecular pattern with endothelial cells, suggesting their severe blockade during hematopoietic transition. Methylation sequencing of pre-HSCs further demonstrated hyper methylation on promoter region of several master hematopoietic transcription factors, including Runx1 and Spi1, by H19 deficiency. Taken together, our study identified H19 as the first non-coding gene indispensable for endothelial-to-HSC transition, and the single-cell lncRNAomics map should be valuable for exploring epigenetic control of HSC generation and regeneration.