Induction and Patterning of Pre-Hemato-Vascular Mesoderm by the Mouse Mix Homeodomain Protein.

Members of the Xenopus and zebrafish Mix/Bix family of paired class homeodomain proteins play determining roles in both mesoderm and endoderm development and are induced by members of the TGFbeta/BMP family of signaling molecules. A single Mix gene has been identified in mouse, humans and chick. Prior to gastrulation, the mouse Mix (mMix) gene is expressed in the visceral endoderm and later in the primitive streak and nascent mesoderm, where it overlaps, in part, with T. Mix expression in ES-derived embryoid bodies is early and transient, overlapping partially with Flk1 activation around the time of formation of hemangioblasts. Both mMix mRNA and protein are found in a FACS-sorted population of T+Flk1+ cells from ES cell-derived embryoid bodies (EBs) which contains hemangioblasts. A complex embryonic lethal phenotype has been reported for Mix deficient embryos, including defects in allantoic (vascular) and cardiogenesis. Mesoderm forms in these embryos but is not patterned properly. Embryonic lethality occurs around E10.5–11, presumably as a result of the cardiovascular defects. We have generated inducible ES cell lines in which expression of Mix protein is responsive to doxycycline. Ectopic expression of Mix in EBs results in premature, enhanced expression of hemangioblast, angioblast and hematopoietic stem cell markers (mRNA and FACS analyses) and increased formation of stem/progenitor cells in clonogenic assays in methylcellulose. Together, the expression analyses, knockout phenotype, and gain-of-function studies in ES cells suggest that mMix functions early in induction and patterning of mesoderm, including formation of hematopoietic and endothelial lineages. Potential mMix target genes are being identified by microarray analyses of the inducible Mix ES lines. To examine mMix activities in vivo, we have generated null and conditional mMix knockout mice from several independently targeted ES cell lines. Analysis of these animals is in progress. Like Xenopus Mix.1, mouse Mix may represent an important connection between the TGFbeta/BMP pathway and hematopoietic/vascular development in the embryo.