Hox transcription factors in hematopoiesis

Abstract: Hematopoiesis is a lifelong, dynamic process in which a small number of pluripotent hematopoietic stem cells (HSC) residing in the bone marrow (BM) give rise to billions of mature blood cells of both myeloid and lymphoid origin each day. This enormous capacity of HSC has been utilized in BM transplantations for 4 decades and is also very important for future gene therapy. Understanding what molecular mechanisms control regulation of HSC and primitive hematopoietic progenitors is crucial for optimal manipulation of these cells, such as ex vivo expansion or viral mediated gene transfer. Amongst the transcription factors that are important for this regulation are Hox homeobox proteins. We have chosen to analyze further the role of three Hox genes, HOXA10, Hoxb3 and Hoxb4, in hematopoiesis by generating novel animal models where the expression of these genes is deregulated. We demonstrate that expression of HOXA10 in a transgenic mouse model, based on the tetracycline transactivator system, is inducible and reversible. High expression of HOXA10 alters myeloid differentiation in vitro, leading to formation of blast like cells and enhanced megakaryocytic generation. Induced HOXA10 expression increased the numbers of hematopoietic cells with CFU-S ability in a reversible manner, suggesting a direct role for HOXA10 in the regulation of these primitive myeloid progenitors. To analyze the role of Hoxb3 and Hoxb4 in early hematopoiesis, two gene targeting models were generated using the Cre/loxP system, one model deficient in Hoxb4 and one deficient in Hoxb3 and Hoxb4. Homozygous null mutants of both models were born at normal Mendelian ratios and appear healthy and fertile. Analysis of the hematopoietic system revealed similar phenotypes for both models although the penetrance was stronger in the double knockout model. A significant reduction in cellularity numbers was observed in bone marrow and spleen of these mice with a more marginal reduction in red blood cell counts and hemoglobin values. Progenitor numbers were mildly reduced, however cell cycle kinetics and lineage distribution were unaffected in endogenous hematopoiesis. In vitro proliferation recruitment studies and in vivo transplantation experiments demonstrated a significant defect in the proliferative response of HSC, resulting in lower expansion and reduced reconstitution capacity in recipient mice. This proliferative defect is detectable already during development, resulting in reduced fetal liver stem cell pool at day 14.5. These studies demonstrate that Hoxb4 and Hoxb3 are necessary for maximum proliferation response of HSC while their role in steady state hematopoiesis is less prominent.