Positional cloning of gene that regulates proliferation and differentiation of embronic ectoderm.

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Mice homozygous for the tw5 allele arrest at gastrulation stage from defects associated with embryonic ectoderm proliferation/differentiation. The mutated gene mapped very close to H-2K gene in mouse MHC. To positionally clone the mutated gene, a BAC contig spanning 〜1000 kb of the corresponding genomic region was constructed. Combining our own sequencing analysis data of those BAC clones and genomic data available from public database, it was determined that the size of the tw5 critical region is 750 kb. EST database search, gene prediction program analysis and an experimental expression analysis suggest that there are possibly 36 genes within this region. Based on their expression profiles, this region is extremely rich in embryonically active genes, which are candidates for the tw5 embryonic lethality.To understand the function of tclw5 gene product, we tried to determine the primary site of tclw5 gene action. Histological examination demonstrated that homozygotes of t^<w5> died at the gastrulation stage due to extensive death of the embryonic ectoderm cells while the extraembryonic ectoderm and the visceral endoderm were less affected in these embryos. By aggregation with diploid wild-type embryos, t^<w5>/t^<w5> cells were capable of contributing to all three germ layers. Thus, it is likely that tclw5 is not cell autonomous lethal. Furthermore, the hypothesis that tissues other than the embryonic ectoderm are the primary sites of tclw5 action was supported by the rescue of t^<w5>/t^<w5> embryos by aggregation with tetraploid embryos. Therefore, it is probable that the product of tclw5 is essential for the function of extraembryonic tissues.