Dll1 is a downstream target of Tbx6 in the paraxial mesoderm.

Tbx6 is a member of the T-box family of transcription factors. In the mouse, Tbx6 is expressed in the primitive streak, tail bud, and presomitic mesoderm and is essential for the specification of posterior paraxial mesoderm; in its absence, posterior somites are replaced by ectopic neural tubes. Analysis of embryos expressing reduced levels of Tbx6 also revealed that it is required for the correct patterning of the somites as well as their initial specification. As a first step toward identifying downstream targets of Tbx6, we examined the DNA binding properties of Tbx6 and identified a Tbx6 consensus binding site. Previously, we have shown that expression of Dll1, which encodes a Notch ligand, is lost in the Tbx6 mutant and that Tbx6 and Dll1 genetically interact, indicating that Dll1 may be a direct target of Tbx6 in the paraxial mesoderm. We uncovered four putative Tbx6 binding sites within a Dll1 paraxial mesoderm enhancer and show that Tbx6 can bind two of these sites in vitro. Altogether, these results lend further support for Dll1 being a direct target of Tbx6 in the presomitic mesoderm.

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.