Functionally distinct roles for T and Tbx6 during mouse development

The mouse T-box transcription factors T and Tbx6 are co-expressed in the primitive streak and have unique domains of expression; T is expressed in the notochord, while Tbx6 is expressed in the presomitic mesoderm. T-box factors are related through a shared DNA binding domain, the T-domain, and can therefore bind to similar DNA sequences at least We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity and their interaction genetically We show that at one target, , the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using assays that T and Tbx6 differentially affect transcription with Tbx6 activating expression tenfold higher than T, that T and Tbx6 can compete at target gene enhancers, and that this competition requires a functional DNA binding domain. Next, we addressed whether T and Tbx6 can compete First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the heterozygous phenotype. Next, using the dominant-negative allele, we show that embryos share similarities with embryos homozygous for the hypomorphic allele , specifically fusions of several ribs and malformation of some vertebrae. Finally, we tested whether Tbx6 can functionally replace T using a knockin approach, which resulted in severe null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a knockin at the locus. Altogether, our results of differences in affinity for DNA binding sites and transcriptional activity for T and Tbx6 provide a potential mechanism for the failure of Tbx6 to functionally replace T and possible competition phenotypes .