Human congenital heart disease, the most common form of heart disease in childhood, occurs in about 1% of live births and up to 10% of stillbirths. Presently, the most effective therapy for cardiac diseases is heart transplantation. However, due to the shortage of organs, cost and inaccessibility of treatment for most affected individuals this remains a limited therapeutic option. Alternative treatment is the administration of drugs that improve myocardial contractility, though this treatment is only effective as a short term therapy, with the 5-year survival rate using current agents being less than 60%. An alternative therapeutic option is to treat patients with cardiac progenitor cell populations that could infiltrate and repair damaged heart tissue. Thus, the ability to isolate and propagate cell populations that can differentiate into cardiomyocytes in vivo offers the opportunity to treat a wide range of cardiac diseases. To this end, our lab is interested in understanding the relationship between cardiac progenitor proliferation and the onset of cardiac differentiation focusing on the endogenous roles of the transcription factors TBX5 and CST and the protein phosphatase SHP-2.

A second human congenital human disease that shares overlapping clinical features with SHP-2 is the TBX5, a disease associated with mutations in the coding region of the transcription factor TBX5. Like Shp-2, TBX5 has also been demonstrated to be required for limb and heart development, suggesting the two proteins may function in the same cellular or molecular pathway. TBX5 is a member of the T-box genes that encodes a family of transcription factors that share a characteristic sequence similarity within the DNA-binding domain (T-domain). To date, eighteen different mammalian T-box genes have been identified, many of which have orthologues in a wide variety of multicellular organisms. The role of individual family members in early development and human disease is emphasized by clinical studies demonstrating that mutations in T-box genes are associated with numerous disease states in humans, including congenital diseases such as the DiGeorge and TBX5, and by the observation that T-box genes are amplified in a subset of cancers. A second major goal of our work is to identify and characterize the molecular relationship between the MAPK/SHP-2 pathway and specific members of the T-box gene family, and in particular TBX5. Consistent with the hypothesis that TBX5 and SHP-2 may function in the same cellular or molecular pathway, we have shown a direct link between the FGF/MAPK signal transduction pathway and TBX5 transcriptional activity. Moreover, we have shown that SHP-2 can lead to ectopic expression of TBX5. We are now investigating the pathways that link the two proteins. Through these approaches, we will be able to define and characterize the cellular and biochemical pathways through which SHP-2 functions both in normal development and in human disease.

The aim of Chris Showell's work in the lab is to establish a genotype-based reverse genetic screen to isolate Xenopus tropicalis individuals carrying mutations in specific genes of interest. The diploid genome and short generation time of X.tropicalis allows mutant lines to be established rapidly, while the availability of large numbers of embryos and its close morphological similarity to Xenopus laevis make it an ideal model for studying the control of vertebrate development. We have established conditions for raising X.tropicalis in sufficient numbers for genetic screening and are testing the efficacy of mutagenesis with two mutagens - N-ethyl N-nitrosourea (ENU) and the acridine mutagen ICR191. We are using denaturing high performance liquid chromatography (dHPLC) for high-throughput mutation screening of tadpoles derived from mutagenised parents. This technology allows the detection of mutations or polymorphisms in PCR products at single nucleotide resolution (see figure 1). By screening live F1 progeny carrying heterozygous mutations, founder animals can be immediately isolated following mutation detection and subsequently used to establish lines for the study of mutant phenotypes. Click here for X. tropicalis protocols.