Cordon-bleu, a novel murine developmental marker gene isolated by a gene trap approach

grantor: University of TorontoGene trap (GT) vectors which can act as both a reporter and a mutagen have been previously used to isolate new genes that are essential for mouse development. I used gene trap GT vectors and embryonic stem (ES) cell chimeras to screen for insertions of the lacZ reporter gene into transcription units that are spatially and temporally regulated during early mouse embryogenesis. 308 G418$\sp{\rm R}$ ES cell clones expressing the lacZ reporter gene were individually injected into blastocysts to screen for expression in vivo, in ES cell/embryo chimeras. The expression of the reporter gene in chimeric embryos was analyzed at embryonic day 8.5 (E8.5). The characterization of one GT insertion permitted the cloning of a novel gene named cordon-bleu (cobl) because of its expression in E8.5 embryos. In embryos carrying the GtC101 insertion, $\beta$-Gal activity was first detected at E7.5 in the node region and at E8.5, expression was restricted to axial structures, namely gut endoderm, notochord and the floor plate of the neuro-epithelium. Cobl's open reading frame shows no homology to any known protein or protein domain. No phenotype was detected in mice homozygous for the insertion and mapping of the mouse and human syntenic loci did not reveal any mutation for which cobl could be a candidate gene. The insertion may not have completely disrupted cobl function and cobl's potential role in vertebrate axis formation or patterning remains a conundrum. Finally, I have used the viable GtC101/lacZ insertion into cobl as a molecular marker and studied $\beta$-Gal expression in Brachyury (T) and Danforth's short-tail (Sd) mutants. Mutations at both loci are semi-dominant and affect the normal development of the notochord. I generated mouse lines carrying the GtC101 marker and one of the $T,\ T\sp{wis}$ or Sd alleles. For the three alleles, cobl expression in axial mesoderm revealed the earliest defects to date in heterozygous and homozygous mutant embryos. Other domains of cobl expression also provide evidence for patterning defects in the somites and the tail bud resulting from abnormal organogenesis of the notochord.Ph.D