Deletion of Hoxa Genes in Adult Mice Affects Hematopoietic Stem Cell and B Cell Progenitors

Functional compensation between homeodomain proteins has hindered the ability to unravel their role in hematopoiesis using single gene knock-outs. Although several Hox genes can expand hematopoietic stem cells (HSC) when overexpressed, it remains unclear whether these genes are required for proper adult hematopoiesis. Moreover, it has been shown that HoxB genes are dispensable for hematopoiesis, and that expression of most HoxA genes is ten-fold superior to genes from other Hox clusters in HSC enriched fetal liver populations (Bijl, 2006). Using a haploinsufficient mice for the entire HoxA cluster (HoxA+/−), we have shown that adult HSCs and progenitors are particularly sensitive to HoxA gene levels (Lebert-Ghali, 2010). Thus, we hypothesize that HoxA genes have a crucial function in definitive hematopoiesis.

To assess the role of HoxA genes in definitive hematopoiesis, we used a conditional mutant mouse model for the entire HoxA cluster in combination with an inducible Mx-Cre model. The functional effect of HoxA cluster deletion on hematopoietic cells was analysed by culture and repopulation assays.

Highly efficient excision of HoxA cluster was achieved by 7 doses of poly(I):poly(C) treatment (91–100%). Mice (control, n=3 and Mx-CreHoxAflox/flox, n=3) were sacrificed and analysed three days after the last injection. Immunophenotyping showed a 3 to 4 fold increase of CD150+/CD48-/CD244-/Sca+/c-kit+/Lin- hematopoietic stem cells. Despite the enhancement of the HoxA−/− HSC population, single cell cultures showed that their proliferative potential in response to growth factors was significantly reduced (p=0.036) as growth was observed only for 16.6 ± 14.4% of HoxA−/− compared to 42.4 ±10.3% of control HSCs after 3-weeks of culturing. In contrast, the number of multipotent progenitor (MPP) cells (CD34+/CD135+/Sca+/c-kit+/Lin-) was reduced, indicating a partial block from the short-term HSC (CD34+/CD135-/Sca+/c-kit+/Lin-) to the MPP transition. Colony forming cell assays showed a dramatic decrease of B-cell progenitors in the bone marrow (BM) (10-fold, p=0.0079), while myeloid progenitors were not affected by the deletion. Transplantation assays demonstrated that grafts composed of > 91% HoxA−/− HSCs have slower repopulation kinetics compared to control HSCs and strongly reduced long-term engraftment (37 ± 28% and 92 ± 6% for HoxA−/− and control, respectively, 20 weeks post-transplantation). Genotyping of engrafted donor cells is currently analyzed to confirm repopulation by HoxA−/− cells. Consistent with the observations in primary mice, peripheral blood analysis revealed also a dramatic reduction of B220+ B-cell population in mice transplanted with HoxA−/− BM cells compared to control (7.1 ± 8.5% and 56.2 ±4.8% respectively p=0,000002) Altogether, in vitro assays and transplantation assays revealed that the functions of HoxA−/− HSC seem to be impaired.

Together, these results show that HoxA cluster genes are required for both HSC function and B cell development, indicating that these genes are important regulators of adult hematopoiesis.

No relevant conflicts of interest to declare.