Definitive Erythro-Myeloid Progenitors (EMPs) Emerge in the Myb-/- Embryo and Retain the Capacity to Differentiate into Macrophages

In the adult, the proto-oncogene Myb critically regulates both the maintenance of hematopoiesis and the differentiation of several hematopoietic lineages.  Myb^-/- mouse embryos die by embryonic day (E) 15 with severe anemia due to the absence of definitive erythropoiesis (Mucenski et al., Cell, 1991).  Similarly, zebrafish embryos lacking myb do not express adult globin genes, have a reduction in other mature hematopoietic lineages by 48 hours post fertilization, and maintain a bloodless phenotype through adulthood (Soza-Ried et al., PNAS, 2010).  These and other data have led to the concept that Myb^-/- embryos entirely lack definitive hematopoiesis.

In both mouse and zebrafish embryos, the first definitive hematopoietic potential arises as a hematopoietic stem cell (HSC)-independent wave of erythro-myeloid progenitors (EMPs).  EMPs emerge in the murine yolk sac beginning at E8.25, partially overlapping with an earlier wave of primitive hematopoietic progenitors.   We previously demonstrated that EMPs are multipotent progenitors, and are the major source of definitive erythroid potential in the early fetal liver, prior to the colonization of adult-repopulating HSCs (McGrath et al., Blood, 2011).  Recently, we identified a unique cell surface phenotype that facilitates the prospective isolation of murine definitive EMPs, distinguishing them from primitive hematopoietic progenitors and maturing populations of megakaryocytes and macrophages in the yolk sac (McGrath et al., Cell Reports, 2015).  We detected expression of Myb in sorted EMPs, suggesting that Myb may regulate the emergence and/or differentiation of EMPs.  We tested this hypothesis by assessing the emergence, hematopoietic potential and expansion capacity of EMPs, compared with other maturing primitive hematopoietic lineages, in Myb^-/- mouse embryos.  Consistent with the proposed Myb-independence of the earlier wave of primitive progenitors, we observed normal numbers of maturing macrophages in E9.5 Myb^-/- yolk sacs.  Interestingly, E9.5 Myb^-/- yolk sacs also contained normal numbers of immunophenotypic EMPs.  These EMPs were present in hemogenic endothelial-derived clusters expressing Runx1, similar to littermate controls, suggesting that Myb is dispensable for EMP emergence from hemogenic endothelium.  

We next assessed the differentiation capability of Myb^-/- EMPs in vitro.  E9.5 Myb^-/- yolk sacs lacked high proliferative colony-forming potential (HPP-CFC), a hallmark of immature definitive hematopoietic progenitors.  In addition, both definitive erythroid and granulocyte colony-forming potential were absent in methylcellulose cultures of sorted Myb^-/- EMPs, in contrast to littermate controls.  Surprisingly, however, sorted Myb^-/- EMPs gave rise to macrophage progenitors in colony-forming assays, and CD11b⁺F4/80⁺ macrophages in differentiation cultures.   These data indicate that Myb is not required for the differentiation of primary definitive EMPs into macrophages.  Analysis of Myb^-/- fetal livers also confirmed the presence of F4/80+ macrophages.  While these fetal liver macrophages have been previously proposed to be of primitive hematopoietic origin, our data raise the possibility that they may also be derived from EMPs.

Further analysis of in vitro differentiation cultures demonstrated an inability of sorted Myb^-/- EMPs to proliferate when compared with normal littermates, although these cultures still generated small numbers of macrophages.   It is not yet clear whether this reduction in proliferation is due solely to the loss of differentiation of multiple hematopoietic lineages, or is also due to defective maintenance or expansion of EMPs.  However, consistent with a role for Myb in continued emergence and/or expansion of EMPs, we observed a reduction in the total number of EMPs by E10.5 in yolk sacs of Myb^-/- embryos compared with normal littermates.  Taken together, these data indicate that Myb is a critical regulator not only of HSCs, but also of HSC-independent definitive hematopoietic progenitors (EMPs).  While Myb is dispensable for the initial emergence of EMPs, it is required for their subsequent differentiation into erythroid and granulocyte lineages.  Surprisingly, the persistence of EMPs, while reduced, may provide a source of definitive macrophages in Myb^-/- embryos.