Hox transcription factors in hematopoiesis

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

Hoxb4-deficient mice undergo normal hematopoietic development but exhibit a mild proliferation defect in hematopoietic stem cells

Enforced expression of Hoxb4 dramatically increases the regeneration of murine hematopoietic stem cells (HSCs) after transplantation and enhances the repopulation ability of human severe combined immunodeficiency (SCID) repopulating cells. Therefore, we asked what physiologic role Hoxb4 has in hematopoiesis. A novel mouse model lacking the entire Hoxb4 gene exhibits significantly reduced cellularity in spleen and bone marrow (BM) and a subtle reduction in red blood cell counts and hemoglobin values. A mild reduction was observed in the numbers of primitive progenitors and stem cells in adult BM and fetal liver, whereas lineage distribution was normal. Although the cell cycle kinetics of primitive progenitors was normal during endogenous hematopoiesis, defects in proliferative responses of BM Lin(-) Sca1(+) c-kit(+) stem and progenitor cells were observed in culture and in vivo after the transplantation of BM and fetal liver HSCs. Quantitative analysis of mRNA from fetal liver revealed that a deficiency of Hoxb4 alone changed the expression levels of several other Hox genes and of genes involved in cell cycle regulation. In summary, the deficiency of Hoxb4 leads to hypocellularity in hematopoietic organs and impaired proliferative capacity. However, Hoxb4 is not required for the generation of HSCs or the maintenance of steady state hematopoiesis

Proliferation of primitive myeloid progenitors can be reversibly induced by HOXA10

Recent studies show that several Hox transcription factors are important for regulation of proliferation and differentiation in hematopoiesis. Among these is HOXA10, which is selectively expressed at high levels in the most primitive subpopulation of human CD34(+) bone marrow cells. When overexpressed, HOXA10 increases the proliferation of early progenitor cells and can lead to the development of myeloid leukemia. To study the effects of HOXA10 on primitive hematopoietic progenitors in more detail, transgenic mice were generated with regulatable HOXA10 expression. The transgenic mouse model, referred to as tetO-HOXA10, contains the HOXA10 gene controlled by a tetracycline-responsive element and a minimal promoter. Thus, the expression of HOXA10 is inducible and reversible depending on the absence or presence of tetracycline or its analog, doxycycline. A retroviral vector containing the tetracycline transactivator gene (tTA) was used to induce expression of the HOXA10 gene In bone marrow cells from the transgenic mice. Reverse transcription-polymerase chain reaction analysis confirmed regulatable HOXA10 expression in several transgenic lines. HOXA10 induction led to the formation of hematopoietic colonies containing blastlike cells and megakaryocytes. Moreover, the induction of HOXA10 resulted in significant proliferative advantage of primitive hematopoietic progenitors (spleen colony-forming units [CFU-S-12]), which was reversible on withdrawal of induction. Activation of HOXA10 expression in tet0-HOXA10 mice will therefore govern proliferation of primitive myeloid progenitors in a regulated fashion. This novel animal model can be used to identify the target genes of HOXA10 and better clarify, the specific role of HOXA10 in normal and malignant hematopoiesis

HOXA10 is a critical regulator for hematopoietic stem cells and erythroid/megakaryocyte development.

The Homeobox (Hox) transcription factors are important regulators of normal and malignant hematopoiesis because they control proliferation, differentiation, and self-renewal of hematopoietic cells at different levels of the hematopoietic hierarchy. In transgenic mice we show that the expression of HOXA10 is tightly regulated by doxycycline. Intermediate concentrations of HOXA10 induced a 15-fold increase in the repopulating capacity of hematopoietic stem cells (HSCs) after 13 days of in vitro culture. Notably, the proliferation induction of HSC by HOXA10 was dependent on the HOXA10 concentration, because high levels of HOXA10 had no effect on HSC proliferation. Furthermore, high levels of HOXA10 blocked erythroid and megakaryocyte development, demonstrating that tight regulation of HOXA10 is critical for normal development of the erythroid and megakaryocytic lineages. The HOXA10-mediated effects on hematopoietic cells were associated with altered expression of genes that govern stem-cell self-renewal and lineage commitment (eg, hepatic leukemia factor [HlF], Dickkopf-1 [Dkk-1], growth factor independent-1 [Cfl-1], and Gata-1). Interestingly, binding sites for HOXA10 were found in HLF, Dkk-1, and Gata-1, and Dkk-1 and Gfl-1 were transcriptionally activated by HOXA10. These findings reveal novel molecular pathways that act downstream of HOXA10 and identify HOXA10 as a master regulator of postnatal hematopoietic development. (C) 2007 by The American Society of Hematology

Reduced Proliferative Capacity of Hematopoietic Stem Cells Deficient in Hoxb3 and Hoxb4

Several homeobox transcription factors, such as HOXB3 and HOXB4, have been implicated in regulation of hematopoiesis. In support of this, studies show that overexpression of HOXB4 strongly enhances hematopoietic stem cell regeneration. Here we find that mice deficient in both Hoxb3 and Hoxb4 have defects in endogenous hematopoiesis with reduced cellularity in hematopoietic organs and diminished number of hematopoietic progenitors without perturbing lineage commitment. Analysis of embryonic day 14.5 fetal livers revealed a significant reduction in the hematopoietic stem cell pool, suggesting that the reduction in cellularity observed postnatally is due to insufficient expansion during fetal development. Primitive Lin(−) ScaI(+) c-kit(+) hematopoietic progenitors lacking Hoxb3 and Hoxb4 displayed impaired proliferative capacity in vitro. Similarly, in vivo repopulating studies of Hoxb3/Hoxb4-deficient hematopoietic cells resulted in lower repopulating capability compared to normal littermates. Since no defects in homing were observed, these results suggest a slower regeneration of mutant HSC. Furthermore, treatment with cytostatic drugs demonstrated slower cell cycle kinetics of hematopoietic stem cells deficient in Hoxb3 and Hoxb4, resulting in increased tolerance to antimitotic drugs. Collectively, these data suggest a direct physiological role of Hoxb4 and Hoxb3 in regulating stem cell regeneration and that these genes are required for maximal proliferative response