Amino terminal tyrosine phosphorylation of human MIXL1

Seven members of the Mix family of paired-type homeoproteins regulate mesoderm/endoderm differentiation in amphibians. In mammals, the MIXL1 (Mix. 1 homeobox [Xenopus laevis]-like gene 1) gene is the sole representative of this family. Unlike the amphibian Mix genes that encode an open reading frame of >300 amino acids, mammalian MIXL1 encodes a smaller protein (~230aa). However, mammalian MIXL1 contains a unique proline-rich domain (PRD) with a potential to interact with signal transducing Src homolgy 3 (SH3) domains. Notably, human MIXL1 also contains a unique tyrosine residue Tyr20 that is amino-terminal to the PRD. Here we report that mammalian MIXL1 protein is phosphorylated at Tyr20 and the phosphorylation is dramatically reduced in the absence of PRD. Our findings are consistent with Tyr20 phosphorylation of MIXL1 being a potential regulatory mechanism that governs its activity.     

Laminin α1 domains LG4-5 are essential for the complete differentiation of visceral endoderm

The heterotrimeric basement membrane protein laminin-111 is essential for early mouse embryogenesis. Its β1 and γ1 chains are crucial for endoderm differentiation and for the formation of basement membranes, whereas α1 chain null mice only lack the extraembryonic Reichert’s membrane. Nevertheless, mice deficient in the cell-binding α1 globular domains 4-5 (LG4-5) have a more severe phenotype than animals devoid of the whole α1 chain, as these domains are required for the formation of a polarized ectoderm. However, the influence of the α1LG4-5 domains on endoderm differentiation is unclear. We have used microarray analysis to compare the expression profiles of normal and α1LG4-5-deficient embryoid bodies and show that genes encoding secreted plasma proteins and proteins involved in endocytosis are reduced in α1LG4-5-deficient embryoid bodies, indicating incomplete differentiation of the visceral endoderm. Moreover, mice lacking α1LG4-5 display endoderm disorganization and a defective expression of the endoderm marker Dab2. We hypothesize that α1LG4-5 domains provide an autocrine signal necessary for the complete differentiation of a functional visceral endoderm and vital signals for the polarization of the epiblast.     

Mix.1, a homeobox mRNA inducible by mesoderm inducers, is expressed mostly in the presumptive endodermal cells of Xenopus embryos

In frogs, mesoderm presumably derives from presumptive ectoderm by induction under the control of diffusible substances produced by the endoderm. To analyze the early phase of mesoderm induction, I have isolated cDNA copies of mRNAs induced in presumptive ectoderm by mesoderm inducing factor secreted by the XTC cell line. One of the inducible mRNAs encodes a homeodomain-containing protein that is likely to play a regulatory role in development. Mix.1 behaves as an immediate early response to induction, and its kinetics of expression suggest a major role for MBT in the control of inducible gene expression. Unexpectedly, Mix.1 is expressed mostly in the future endoderm, suggesting that endoderm may be formed by induction in a similar way as mesoderm.