![]() Three Mix-related genes have been identified to date in zebrafish (Alexander et al., 1999 Hirata et al., 2000) and are expressed in mesendoderm and/or in the yolk syncytial layer, a tissue believed to be the functional equivalent of mouse visceral endoderm (Ho et al., 1999). At later stages, expression is restricted to the marginal zone that will form mesoderm. Early in gastrulation, the Mix/Bix (mesoderm-induced or Brachyury-inducible homeobox) genes are expressed in the vegetal cells fated to form endoderm. In Xenopus, members of the Mix/Bix family of paired class homeodomain proteins play determining roles both in endoderm and mesoderm development and are induced by members of the transforming growth factor (TGF) β family of signaling molecules (Rosa, 1989 Huang et al., 1995 Chen et al., 1996, 1997 Mead et al., 1996, 1998 Vize, 1996 Ecochard et al., 1998 Henry and Melton, 1998 Lemaire et al., 1998 Tada et al., 1998). Homeobox genes control the multipotential state of stem cells as well as their specification to distinct cell lineages during the development of all eukaryotic organisms. In each of these cases, the respective tissue interactions result in activation of a set of transcription factors and, ultimately, different sets of target genes. Within the epiblast, interactions between ectoderm, mesoderm, and endoderm initiate the formation and patterning of the various organs and their constituent tissues (Rossant and Tam, 2002). During gastrulation, VE signaling functions in activation of extraembryonic hematopoiesis and vasculogenesis (Belaoussoff et al., 1998). It is now well established, for example, that signals from the anterior visceral endoderm (AVE) play a critical role in specification of the anterior–posterior axis and in limiting anterior extension of the primitive streak (for a review, see Lu et al., 2001). In the mouse, the extraembryonic (primitive or visceral) endoderm engages in inductive interactions with the underlying ectoderm and later the mesoderm. Morphogenesis and patterning in the developing embryo are mediated, in part, through interactions between neighboring tissues. 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. 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. In the region of the developing gut, mMix expression is confined to the mesoderm of mid- and hindgut but is absent from definitive endoderm. In vitro and in vivo, mMix expression in nascent mesoderm is rapidly down-regulated and becomes undetectable in differentiated cell types. 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 a subpopulation of nascent mesodermal cells from embryonic stem cell embryoid bodies, mMix is coexpressed with Brachyury. ![]() 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. Expression in embryoid bodies in culture mirrors that in embryos, where Mix is transcribed transiently in primitive (visceral) endoderm (VE) and in nascent mesoderm. 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. However, in mouse, only a single Mix gene ( mMix) has been identified to date and its function is unknown. In frog and zebrafish, the Mix/Bix family of paired type homeodomain proteins play key roles in specification and differentiation of mesendoderm.
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