[Regulation of RNA Binding Protein Mbnl1 on Development of Mouse Embryonic Hematopoietic Stem Cells]

Abstract

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.