Project description:Mutations in genes intimately involved in the occurrence of haemotological cancers remains largely unknown. Here, we report a novel functional gene in hematopoiesis, which was discovered by screening mutant embryonic stem cells (ESCs). The gene, named “attenuated haematopoietic development” (Ahed), encoded an uncharacterised protein located in the nucleus. Ahed conditional knockout (cKO) mice were generated by mating with Vav1-cre transgenic mice. We found that Ahed cKO foetuses became anaemic after E14.5 and died before birth. Flow cytometric analysis showed that erythroid cells significantly decreased in E14.5 Ahed cKO livers. By contrast, the fetal liver contained a substantial number of lineage-Sca-1+c-KitHi (LSK) cells, whose phenotype corresponded to hematopoietic stem/progenitor cells. Transplantation experiments revealed that Ahed-deficient LSK cells were unable to reconstitute haematopoiesis in vivo. We confirmed the downregulation of expression levels of HSC-related genes, such as Gata2, Lmo2, and Runx1 in E14.5 FL LSK cells in Ahed cKO mice. In addition, upstream regulator analyses revealed that the GATA2 pathway was most significantly hampered in both FL and adult BM. Collectively, these data determined that Ahed is indispensable for functional haematopoietic stem/progenitor cells.
Project description:C/EBPalpha is a transcription factor critically involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor (LSK) cells, which express C/EBPalpha, we developed a mouse model expressing Cre recombinase from the Cebpa promoter and an inducible EYFP allele. We show that Cebpa/EYFP+ cells represent a significant subset of LSK cells, which predominantly give rise to myeloid cells in steady state hematopoiesis. C/EBPalpha induced a robust myeloid gene expression signature and downregulated E2A-induced regulators of early lymphoid development. In addition, Cebpa/EYFP+ cells comprise a fraction of early thymic progenitors (ETP) with robust myeloid potential. However, Cebpa/EYFP+ LSK and ETP cells retained the ability to develop into erythroid and T-lymphoid lineages, respectively. These findings support an instructive, but argue against a lineage restrictive role of C/EBPalpha in multipotent hematopoietic and thymic progenitors. We performed global gene expression profiling of double-sorted Cebpa/EYFP+ and Cebpa/EYFP- LSK cells of pooled Cebpa Cre/wt R26 EYFP reporter mice to identify differentially regulated genes in Cebpa+ versus Cebpa- LSK cells. RNA was isolated from three biological replicates of Cebpa/EYFP+ LSK cells and two biological replicates of Cebpa/EYFP- LSK cells. To determine if the identified genes were truly dependent on Cebpa expression, we also performed global gene expression profilling of Cebpa/EYFP+ and Cebpa/EYFP- fetal liver LSK cells of Cebpa Cre/fl R26 EYFP mice. Induction of Cebpa/Cre expression in these mice leads to Cre-mediated recombination of the floxed wt Cebpa allele resulting in a complete Cebpa knock-out. In this case, RNA was isolated from two biological replicates of either Cebpa/EYFP+ and Cebpa/EYFP- LSK cells. In addition, we included one biological replicate of Cebpa/EYFP+ and Cebpa/EYFP- fetal liver LSK cells of Cebpa Cre/wt R26 EYFP mice to determine the correlation of differentially regulated genes in bone marrow and fetal liver LSK cells.
Project description:To investigate the molecular mechanism underlying gender-specific regulation of hematopoiesis by Lxn. RNA sequencing was performed on LSK cells that were isolated from male and female WT and Lxn-/- mice.
Project description:The goal of this study is to identify gene expression changes upon SON overexpression in WT and Mettl3 cKO LSK (Lin-cKit+Sca1+) cells
Project description:The goal of this study is to identify gene expression changes upon SON overexpression in WT and Mettl3 cKO LSK (Lin-cKit+Sca1+) cells at single cell level
Project description:HIRA is a histone chaperone that deposits the histone variant H3.3 in transcriptionally active genes. HIRA is essential for mouse development, as the standard knockout (KO) results in early embryonic death. However, the role of HIRA in hematopoiesis is poorly understood. We investigated the effect of Hira KO on hematopoiesis using Vav-Cre Loxp system. We show that Hira KO dramatically reduces bone marrow LSK cells, resulting in anemia, thrombopenia and severe, combined immunodeficiency. To investigate the molecular mechanisms, RNA-seq and ATAC-Seq were performed using LSK cells isolated from WT and Hira conditional KO mice.
