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- Insights into birth defects from mice
- Signaling wars in worms
- Double danger for tadpoles
- The Secrets of Limb Developments in Flies
- New view of Phage Phylogeny
- Mouse genetics uncovers multiple roles for CtBP
- A New Look at Specificity
- Modified Proteins Found in Tumors

Pittsburgh Bacteriophage Institute

Dr. Deborah Chapman
 

Mr. Philip White

The Chapman lab is interested in how paraxial mesoderm is initially established and patterned during embryonic development. Paraxial mesoderm develops first as presomitic mesoderm and later as the somites, which will differentiate to give rise to the skeletal muscle of the back and body wall, the dermis, vertebrae and ribs. The specification of this presomitic mesoderm involves the T-box transcription factor, Tbx6. Our previous mutational studies in mice (Chapman and Papaioannou 1998) revealed that Tbx6 was required for formation of paraxial mesoderm posterior to the forelimb bud. Embryos with Tbx6 null mutations form extra neural tubes in place of somites in the trunk and tail region of the embryo, but later die at midgestation due to problems in blood vessel formation.

In White et al. 2003, the Chapman lab shows that in addition to its role in the specification of presomitic mesoderm, Tbx6 is also required for proper somites patterning. These studies demonstrate that reducing Tbx6 expression levels below those found in heterozygous embryos ultimately results in fusions of ribs and vertebrae and improper segmentation of skeletal muscle precursors.

Figure 1. Tbx6 transgenic embryo at 9.5 days of development. The Tbx6 regulatory elements drive expression of the reporter (blue stain) in the primitive streak (tip of the tail), presomitic mesoderm and somites.

A major focus of the Chapman lab is to identify the cis-acting regulatory regions of Tbx6, and ultimately the proteins responsible for controlling Tbx6 expression. During these studies, we identified a Tbx6 genomic fragment that was capable of driving the expression of reporter gene in a Tbx6-specific manner (Figure 1, right). As this transgene contained the entire Tbx6 coding region along with regulatory sequences, transgenic lines were used to rescue the Tbx6 mutant phenotype.

Although somite formation was rescued in these embryos, patterning of the somites was defective and resulted in fusions of ribs and vertebrae in the developing embryo. Incomplete rescue of the Tbx6 mutant phenotype was due to low levels of Tbx6 expression from the transgenes. The degree of rescue, as assessed by how well the ribs and vertebrae formed, was dependent on the level of Tbx6 expression (Figure 2, below).

In White et al. 2003, we also demonstrate that the classic mouse mutant rib-vertebrae is a mutation in Tbx6 that lowers the level of Tbx6 expression, such that in a homozygous rib-vertebrae mutant mouse, expression levels fall below those of a heterozygote. This mutation results in fusions of ribs and vertebrae At the other extreme, the Tbx6 heterozygous embryos (Tbx6 +/-) have normal ribs and vertebrae (Figure 2, below), and improper segmentation of skeletal muscle precursors.

Figure 2. Series of skeletal phenotypes resulting from insufficient expression levels of Tbx6. The level of Tbx6 expression ranges from 0 in the Tbx6 null embryo (Tbx6 -/-) to heterozygous levels (Tbx6 +/) and is shown along the x-axis. Cartoon of a transverse section of the Tbx6 null embryo with no Tbx6 expression shows the formation of two extra neural tubes in place of the somites. At the other extreme, Tbx6 heterozygous embryos (Tbx6 +/-) form normal ribs and vertebrae. The skeletal phenotypes of embryos expressing various levels of Tbx6 between these two extremes are also shown. Reducing Tbx6 expression below heterozygous levels results in fusions of ribs and vertebrae and was observed in both the incomplete transgenic rescued embryos and in the rib-vertebrae (rv/rv) homozygous embryos.

Tbx6 linked to the Notch signaling pathway
Features of the embryos described above are similar to those of Notch signaling mutants in mice and humans. We examined the possible link between Tbx6 and Notch signaling, by mating mice bearing a single mutation in Tbx6 with mice bearing a single mutation in Dll1, which encodes a Notch ligand. While mice heterozygous for either mutation are normal, mice heterozygous for both mutations (double heterozygotes) have fusions of vertebrae that lead to a kinky tail phenotype in the animals (Figure 3, below). This strong genetic interaction between Tbx6 and Dll1, together with the absence of Dll1 expression in Tbx6 null mutant embryos, suggests that Tbx6 is upstream of Dll1 in the pathway leading to somite formation and patterning.

Figure 3. Mutant phenotypes. The genetic interaction between Tbx6 and Dll1 is observed in the Tbx6 +/- Dll1 +/- animals, which have fusions of tail vertebrae that result in a kinky tail.

Link to human birth defects
The similarity of phenotypes described in White et al. 2003 with those 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. Whereas null mutations in mouse Tbx6 lead to early embryonic lethality, mutations that lower Tbx6 expression levels lead to perinatal lethality or birth defects depending on the Tbx6 expression level achieved.

In humans, it is likely that a null mutation in TBX6 would similarly lead to early embryonic lethality and finally a miscarriage; however, mutations that reduce the level of TBX6 expression or activity during human embryonic development might lead to human birth defects like those observed in the Tbx6 hypomorphic mutants described in White et al. 2003. Current studies in the Chapman lab focus on dissecting the genetic pathway leading to somite formation and patterning using genetics, transgenic technology, and molecular and biochemical techniques. It is through these studies that we hope to understand how these tissue form in during human development.

 
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