Transcriptome analysis to compare control and Ahed-deficient hematopoietic stem and progenitor cells (HSPC) and erythroblasts [RNA-Seq]

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. Overall design: Lin- Sca1+ c-Kit+ (LSK) CD48- HSC-enriched fractions and Lin- CD71Hi Ter119-/Lo pro-erythroblasts were sorted from fetal liver (FL) and adult bone marrow (BM) of control or Ahed-deficient mice; Ahedfl/+; Vav1-cre (control) and Ahedfl-/-; Vav1-cre (cKO) in E14.5 FL and Rosa26-CreERT2 (R26CE) Ahedfl/+ (control) mice and R26CE Ahedfl/fl (cKO) in BM. We conducted RNA-seq analyses to compare the gene expression features of haematopoietic cells derived from the FL and adult BM of control or Ahed-deficient mice.