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

Abstract

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. © 2003 Wiley-Liss, Inc.