Abstract

Congenital amegakaryocytic thrombocytopenia with radio-ulnar synostosis is associated with mutations in the HOXA11 gene, suggesting that HoxA11 may play a role in megakaryocytic lineage commitment or differentiation. The HOX genes encode transcription factors that are involved in cellular differentiation in embryonic as well as adult tissues. Numerous studies have identified HOX genes as important regulators of various aspects of hematopoiesis including self-renewal, proliferation, differentiation and leukemogenesis. Our initial studies failed to identify the expression of HoxA11 in platelets, TPO-induced CD34+ umbilical cord stem cells or normal bone marrow. More recently our lab has detected a small amount of HoxA11 mRNA in cells isolated from unfractionated human cord blood, suggesting the expression of HoxA11 may occur in a small subset of early hematopoietic or stromal cells. To test this hypothesis we have employed a murine embryonic stem (ES) cell culture system. Co-culture of ES cells and the bone marrow stromal cell line, OP9, can give rise to primitive as well as definitive hematopoietic progenitors in the absence of leukemia inhibitory factor (LIF). By day 6, ES cells on OP9 can differentiate into mesodermal colonies, which contain a bi-potential progenitor known as the hemangioblast. The hemangioblast can further differentiate into either a hematopoietic or endothelial lineage. To determine when HoxA11 is expressed we have employed this model using green fluorescent protein (GFP) expressing ES cells grown on OP9 and differentiated into hematopoietic precursors in the absence of LIF. Nested RT-PCR revealed that HoxA11 mRNA is highly expressed in ES cells following 6 days (D6) on OP9. HoxA11 expression was restricted to D6 ES cells, as HoxA11 mRNA was not found in OP9 cells alone or ES cells differentiated on OP9 for 0, 3, or 9 days. RT-PCR revealed HoxA11 mRNA expression coincided with the expression of flk-1, a marker for the hemangioblast. Since HoxA11 expression is concurrent with hemangioblast differentiation, we sought to determine if the hemangioblast is the cell that expressed HoxA11. Using flow cytometery and fluorescence activated cell sorting (FACS) analysis we separated D6 ES cells into flk-1 positive (flk-1+) and negative (flk-1−) populations and investigated which population expressed HoxA11. Nested RT-PCR revealed that HoxA11 mRNA expression is found in both the flk-1+ and flk-1- fractions. We further analyzed these fractions by RT-PCR for SCL/Tal-1. SCL/Tal-1 is a transcription factor that plays a critical role in the commitment of mesoderm into hematopoietic progenitor cells. We find SCL/Tal-1 mRNA also expressed in both flk-1+ and flk-1- fractions, which parallels HoxA11 mRNA expression. These data suggest HoxA11 expression occurs in the flk-1+ hemangioblast but also possibly in a flk-1-/SCL+ hematopoietic precursor cell population. Current studies are underway to determine the cell fate and role of the HoxA11 expressing progenitor cell. Taken together, these data are the first findings of HoxA11 expression in early progenitor cells as well as the first evidence of controlled HoxA11 regulation during early hematopoietic development.

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