The homeobox gene HEX regulates proliferation and differentiation of hemangioblasts and endothelial cells during ES cell differentiation.

In this report we have investigated the role of the homeobox gene Hex in the development and differentiation of the blast colony-forming cell (BL-CFC), a progenitor with hemangioblast characteristics generated in embryonic stem (ES) cell-derived embryoid bodies (EBs). Molecular analysis showed that Hex is expressed in mesoderm, in populations that contain BL-CFCs, and in blast cell colonies, the progeny of the BL-CFCs. Hex(-/-) EBs displayed a defect in macrophage development but generated higher numbers of BL-CFCs than did wild-type EBs. In addition to differences in these progenitor populations, we also found that endothelial cells from the Hex(-/-) EBs showed enhanced proliferative potential compared with those from wild-type EBs. Forced expression of Hex at the onset of ES cell differentiation resulted in reduced EB cellularity, fetal liver kinase-1 (Flk-1) expression, and BL-CFC development. Taken together, these findings demonstrate that Hex functions at multiple stages of development within the differentiating EBs and uncover a novel role for this transcription factor as a negative regulator of the hemangioblast and the endothelial lineage.

Mouse Mix gene is activated early during differentiation of ES and F9 stem cells and induces endoderm in frog embryos.

In frog and zebrafish, the Mix/Bix family of paired type homeodomain proteins play key roles in specification and differentiation of mesendoderm. However, in mouse, only a single Mix gene (mMix) has been identified to date and its function is unknown. We have analyzed the expression of mouse Mix RNA and protein in embryos, embryoid bodies formed from embryonic stem cells and F9 teratocarcinoma cells, as well as several differentiated cell types. Expression in embryoid bodies in culture mirrors that in embryos, where Mix is transcribed transiently in primitive (visceral) endoderm (VE) and in nascent mesoderm. In F9 cells induced by retinoic acid to differentiate to VE, mMix is coordinately expressed with three other endodermal transcription factors, well before AFP, and its protein product is localized to the nucleus. In a subpopulation of nascent mesodermal cells from embryonic stem cell embryoid bodies, mMix is coexpressed with Brachyury. Intriguingly, mMix mRNA is detected in a population (T+Flk1+) of cells which may contain hemangioblasts, before the onset of hematopoiesis and activation of hematopoietic markers. In vitro and in vivo, mMix expression in nascent mesoderm is rapidly down-regulated and becomes undetectable in differentiated cell types. In the region of the developing gut, mMix expression is confined to the mesoderm of mid- and hindgut but is absent from definitive endoderm. Injection of mouse mMix RNA into early frog embryos results in axial truncation of developing tadpoles and, in animal cap assays, mMix alone is sufficient to activate expression of several endodermal (but not mesodermal) markers. Although these observations do not exclude a possible cell-autonomous function for mMix in mesendodermal progenitor cells, they do suggest an additional, non-cell autonomous role in nascent mesoderm in the formation and/or patterning of adjacent definitive endoderm.