Ectopic expression of T in the paraxial mesoderm disrupts somite maturation in the mouse.

T is the founding member of the T-box family of transcription factors; family members are critical for cell fate decisions and tissue morphogenesis throughout the animal kingdom. T is expressed in the primitive streak and notochord with mouse mutant studies revealing its critical role in mesoderm formation in the primitive streak and notochord integrity. We previously demonstrated that misexpression of Tbx6 in the paraxial and lateral plate mesoderm results in embryos resembling Tbx15 and Tbx18 nulls. This, together with results from in vitro transcriptional assays, suggested that ectopically expressed Tbx6 can compete with endogenously expressed Tbx15 and Tbx18 at the binding sites of target genes. Since T-box proteins share a similar DNA binding domain, we hypothesized that misexpressing T in the paraxial and lateral plate mesoderm would also interfere with the endogenous Tbx15 and Tbx18, causing embryonic phenotypes resembling those seen upon Tbx6 expression in the somites and limbs. Interestingly, ectopic T expression led to distinct embryonic phenotypes, specifically, reduced-sized somites in embryos expressing the highest levels of T, which ultimately affects axis length and neural tube morphogenesis. We further demonstrate that ectopic T leads to ectopic expression of Tbx6 and Mesogenin 1, known targets of T. These results suggests that ectopic T expression contributes to the phenotype by activating its own targets rather than via a straight competition with endogenous T-box factors.

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 in vitro We investigated the functional similarities and differences of T and Tbx6 DNA binding and transcriptional activity in vitro and their interaction genetically in vivo We show that at one target, Dll1, the T-domains of T and Tbx6 have different affinities for the binding sites present in the mesoderm enhancer. We further show using in vitro 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 in vivo First, we generated embryos that express Tbx6 at greater than wild-type levels embryos and show that these embryos have short tails, resembling the T heterozygous phenotype. Next, using the dominant-negative TWis allele, we show that Tbx6+/- TWis/+ embryos share similarities with embryos homozygous for the Tbx6 hypomorphic allele rib-vertebrae, 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 T null-like phenotypes in chimeric embryos generated with ES cells heterozygous for a Tbx6 knockin at the T 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 in vivo.

Kinked tail mutation results in notochord defects in heterozygotes and distal visceral endoderm defects in homozygotes.

Proper formation of the anterior-posterior (AP) axis in the developing embryo is critical for the correct patterning and often survival of the organism. In the mouse, an initial step in axis establishment is the specification and migration of the distal visceral endoderm (DVE). We have identified a semi-dominant spontaneous mutation in mouse, named kinked tail (knk), which when heterozygous results in a kinky tail phenotype due to fusions and dysmorphology of the tail vertebrae. Vertebral fusions appear to be a secondary effect of notochord thickening and branching in the tail region. Homozygosity for knk results in early embryonic lethality by embryonic day 8.5 due to improper timing of DVE specification and migration, and subsequent failure to establish the AP axis.

Regulation of Tbx6 expression by Notch signaling.

Somites are the first overt sign of segmentation in the vertebrate embryo and form from bilateral strips of paraxial mesoderm. Paraxial mesoderm arises from the primitive streak; it then migrates laterally and comes to lie on both sides of the neural tube. In the mouse, the T-box transcription factor Tbx6 is required for both somite formation and patterning. Tbx6 expression corresponds both temporally and spatially to somite formation, with expression in the primitive streak and presomitic mesoderm. Its expression in the latter could simply be explained by maintenance following its initial activation in the primitive streak. Alternatively, its expression in the presomitic mesoderm may be contributed by separate regulatory elements possibly under the control of different signals. We have begun to investigate how Tbx6 expression is controlled during development using a transgenic approach to identify the cis-acting regulatory regions. We show that it is possible to separate an element required for presomitic mesoderm expression from that driving expression in the primitive streak. Further, we show that a binding site for the Notch transcription factor RBP-Jkappa is necessary for Tbx6 presomitic mesoderm enhancer activity, indicating that Notch signaling is upstream of Tbx6 in the pathway directing somite formation and patterning.

Defective somite patterning in mouse embryos with reduced levels of Tbx6.

During vertebrate embryogenesis, paraxial mesoderm gives rise to somites, which subsequently develop into the dermis, skeletal muscle, ribs and vertebrae of the adult. Mutations that disrupt the patterning of individual somites have dramatic effects on these tissues, including fusions of the ribs and vertebrae. The T-box transcription factor, Tbx6, is expressed in the paraxial mesoderm but is downregulated as somites develop. It is essential for the formation of posterior somites, which are replaced with ectopic neural tubes in Tbx6-null mutant embryos. We show that partial restoration of Tbx6 expression in null mutants rescues somite development, but that rostrocaudal patterning within them is defective, ultimately resulting in rib and vertebral fusions, demonstrating that Tbx6 activity in the paraxial mesoderm is required not simply for somite specification but also for their normal patterning. Somite patterning is dependent upon Notch signaling and we show that Tbx6 genetically interacts with the Notch ligand, delta-like 1 (Dll1). Dll1 expression, which is absent in the Tbx6-null mutant, is restored at reduced levels in the partially rescued mutants, suggesting that Dll1 is a target of Tbx6. We also identify the spontaneous mutation rib-vertebrae as a hypomorphic mutation in Tbx6. The similarity in the phenotypes we describe here and that of some human birth defects, such as spondylocostal dysostosis, raises the possibility that mutations in Tbx6 or components of this pathway may be responsible for these defects.