Single-cell architecture and functional requirement of alternative splicing during hematopoietic stem cell formation

Hematopoietic stem cells (HSCs) are well known to generate from a rare hemogenic population of embryonic endothelia cells through a dynamic fate transition, accompanied by widespread changes in gene regulatory networks. Nevertheless, RNA splicing, a striking feature in shaping transcriptome and enabling the generation of vast transcripts and diverse proteins, has never been illustrated in this transient endothelial to HSC conversion. Here, we constructed an isoform-based transcriptome landscape during HSC development at single-cell resolution, which enables the identification of hemogenic signature isoforms, as well as stage-specific splicing events. Combining with functional screening, we showed that the inclusion of such hemogenic-specific exons was essential for hemogenic function in vitro. Moreover, their gradual appearance along with HSC specification from endothelia cells was orchestrated by the splicing regulator Srsf2. Therefore, early Srsf2 deficiency from endothelia cells dramatically impaired the HSC generation, which was accompanied by changes in splicing pattern of several master hematopoietic regulators. These results redefine our understanding of the dynamic transcriptome diversity and demonstrate that the elaborate control of splicing governs cell fate in HSC formation. Single-cell full-length RNA-sequencing of two HSC-associated cell populations, and RNA-sequencing of Srsf2 f/+ or Srsf2 f/f and Vec-Cre;Srsf2 f/f cells.

造血干细胞（Hematopoietic stem cells, HSCs）已知可通过动态命运转换过程，由罕见的生血型胚胎内皮细胞群体分化而来，该过程伴随基因调控网络的广泛重塑。然而，作为塑造转录组并产生海量转录本与多样蛋白质的核心特征，RNA剪接（RNA Splicing）在这一短暂的内皮细胞向造血干细胞转化过程中尚未被系统阐释。本研究构建了单细胞分辨率下造血干细胞发育过程中基于转录本亚型的转录组图谱，借此可鉴定生血特征性转录本亚型与阶段特异性剪接事件。结合功能筛选实验，本研究证实这类生血特异性外显子的保留对于体外生血功能不可或缺。此外，这类生血特异性外显子随内皮细胞向造血干细胞特化逐渐出现的过程，由剪接调控因子Srsf2精细调控。因此，内皮细胞中Srsf2的早期缺失会显著抑制造血干细胞的生成，该过程伴随多个核心造血调控因子的剪接模式改变。本研究结果重新定义了学界对动态转录组多样性的认知，并证实剪接的精细调控主导了造血干细胞形成过程中的细胞命运转换。本研究的测序数据集涵盖两类造血干细胞相关细胞群的单细胞全长RNA测序，以及Srsf2 f/+、Srsf2 f/f与Vec-Cre;Srsf2 f/f细胞的RNA测序。