C-Myb Influences the Proliferation and Commitment of Erythroid Progenitors.

The transcription factor c-Myb is involved in the regulation of proliferation, survival and differentiation of haemopoietic progenitors. Mouse embryos homozygous for a null mutation in the c-myb gene die of anaemia around 15 dpc, however, the reasons leading to the failure of erythropoiesis are unknown. In order to investigate the precise function of c-Myb in erythroid progenitors we manipulated c-Myb expression using targeted c-myb alleles. To study erythropoiesis with reduced c-Myb expression we used embryos heterozygous for a c-myb null allele (c-myb⁻) or homozygous for a previously described knockdown allele (c-myb^KD). Furthermore, we achieved complete inactivation of c-myb using the IFN-inducible transgene MxCre in erythroid progenitors cultured from foetal liver of embryos carrying a floxed c-myb allele (c-myb^F). We demonstrate that low levels of c-Myb lead to an accumulation in the foetal liver of CD71⁺Ter119⁻ erythroid cells expressing markers of uncommitted progenitors as well as low levels of c-Kit. To prove that this is the consequence of a block in commitment, we cultured foetal liver cells and demonstrated the outgrowth of immature cells unable to progress and terminally differentiate in response to Epo. A requirement for high levels of c-Myb in later stages of erythropoiesis was revealed by studying cultured foetal liver progenitors carrying one functional c-myb allele (c-myb^+/−). The number and size of CFU-Es was reduced, while BFU-Es were poorly haemoglobinised. Interestingly, we observed a tendency of cultured c-myb^+/− progenitors to spontaneously and rapidly exit the cell cycle and aberrantly differentiate. In agreement with this, Cre-mediated inactivation of c-myb in cultured cells led to a rapid decline in c-Kit expression and the induction of certain erythroid genes, although obvious terminal differentiation was absent. Strikingly, expression of the megakaryocytic marker CD41 emerged on a subpopulation of cells. In contrast to cells expressing low levels of c-Myb, complete inactivation resulted in cell cycle arrest. In order to systematically uncover molecular pathways regulated by c-Myb we have determined the global gene expression changes in response to induced c-myb inactivation using Affymetrix GeneChip arrays. Several differentially expressed genes were identified and are being further analysed as novel c-Myb target genes.

In conclusion, we provide evidence for a complex role for c-Myb in the regulation of several stages of erythroid development, including expansion, commitment and terminal differentiation. In particular, we demonstrate that high levels of c-Myb are required for the response of progenitors to erythropoietic stimuli as well as for ordered terminal differentiation. c-Kit, long suspected as a possible c-Myb-regulated gene but never before proven to be at the level of the endogenous gene in primary cells, has emerged as a key target for the actions of c-Myb in erythroid progenitors. Finally, a number of novel target genes have been identified as additional important potential effectors downstream of c-Myb in developing erythroid cells.