RESUMEN
Single-cell transcriptional profiling has rapidly advanced our understanding of the embryonic hematopoiesis; however, whether and what role RNA alternative splicing (AS) plays remains an enigma. This is important for understanding the mechanisms underlying splicing-associated hematopoietic diseases and for the derivation of therapeutic stem cells. Here, we used single-cell full-length transcriptome data to construct an isoform-based transcriptional atlas of the murine endothelial-to-hematopoietic stem cell (HSC) transition, which enables the identification of hemogenic signature isoforms and stage-specific AS events. We showed that the inclusion of these hemogenic-specific AS events was essential for hemogenic function in vitro. Expression data and knockout mouse studies highlighted the critical role of Srsf2: Early Srsf2 deficiency from endothelial cells affected the splicing pattern of several master hematopoietic regulators and significantly impaired HSC generation. These results redefine our understanding of the dynamic HSC developmental transcriptome and demonstrate that elaborately controlled RNA splicing governs cell fate in HSC formation.
RESUMEN
In the aorta-gonad-mesonephros (AGM) region of mouse embryos, pre-hematopoietic stem cells (pre-HSCs) are generated from rare and specialized hemogenic endothelial cells (HECs) via endothelial-to-hematopoietic transition, followed by maturation into bona fide hematopoietic stem cells (HSCs). As HECs also generate a lot of hematopoietic progenitors not fated to HSCs, powerful tools that are pre-HSC/HSC-specific become urgently critical. Here, using the gene knockin strategy, we firstly developed an Hlf-tdTomato reporter mouse model and detected Hlf-tdTomato expression exclusively in the hematopoietic cells including part of the immunophenotypic CD45- and CD45+ pre-HSCs in the embryonic day (E) 10.5 AGM region. By in vitro co-culture together with long-term transplantation assay stringent for HSC precursor identification, we further revealed that unlike the CD45- counterpart in which both Hlf-tdTomato-positive and negative sub-populations harbored HSC competence, the CD45+ E10.5 pre-HSCs existed exclusively in Hlf-tdTomato-positive cells. The result indicates that the cells should gain the expression of Hlf prior to or together with CD45 to give rise to functional HSCs. Furthermore, we constructed a novel Hlf-CreER mouse model and performed time-restricted genetic lineage tracing by a single dose induction at E9.5. We observed the labeling in E11.5 AGM precursors and their contribution to the immunophenotypic HSCs in fetal liver (FL). Importantly, these Hlf-labeled early cells contributed to and retained the size of the HSC pool in the bone marrow (BM), which continuously differentiated to maintain a balanced and long-term multi-lineage hematopoiesis in the adult. Therefore, we provided another valuable mouse model to specifically trace the fate of emerging HSCs during development.
RESUMEN
OBJECTIVE: To analyze the dynamic molecular expression characteristics of single cell RNA binding proteins (RBPs) in the development of mouse embryonic hematopoitic stem cells (HSCs), and obtain the functional research target RNA splicing factor--Mbnl1, to clarify the function of Mbnl1 involved in regulating mouse embryonic HSC development. METHODS: Bioinformatics was used to analyze the single-cell transcriptome data of mouse embryos during HSC development, and the single-cell RBP dynamic molecular expression maps in HSC development was obtained. Mbnl1 was obtained by combining differential analysis and literature research screening. The Mbnl1-knockout mouse model was constructed by the CRISPER/Cas9 technology. Aorta-gonad-mesonephros (AGM) and yolk sac (YS) tissue in two genotype embryos of Mbnl1+/+ and Mbnl1-/- at E11.5 were digested into single cells, and then a methylcellulose semi-solid culture system was used to perform an in vitro CFU-C of hematopoietic cells. The number and type of hematopoietic cell colonies in the two hematopoietic tissues in Mbnl1-knockout mice after 7 days were calculated. Furthermore, the whole AGM tissue cells of E11.5 Mbnl1+/+ and Mbnl1-/- donor mice were transplanted via tail vein injection respectively, and the function of HSC in AGM region of mice after Mbnl1 knockout was evaluated through the chimerism rate of peripheral blood in recipient mice at 4w and 8w and the kinetic experiment of hematopoietic system reconstruction. RESULTS: The in vitro CFU-C experiment of hematopoietic cells preliminarily indicated that there was no significant difference in the number of cell colonies in AGM region and YS transformed by the two genotypes of Mbnl1+/+ and Mbnl1-/- at E11.5, which suggested that HPC function in AGM and YS tissues had no abnormality after Mbnl1 knockout. Single cell transplantation in AGM region vin tail vein showed no significant difference in the hematopoietic reconstruction chimeric rate between Mbnl1+/+ and Mbnl1-/- mice, suggesting that there was no abnormality in HSC function in AGM tissues after Mbnl1 knockout. CONCLUSION: Through functional experiments in vivo and in vitro, it has been confirmed that knockout of the RNA splicing factor--Mbnl1 does not affect the development of HSPC in AGM region of mouse embryo.
