Tbx1 and canonical Wnt signaling in the second heart field

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 DESCRIPTION (provided by applicant): Haploinsufficiency of TBX1, encoding a T-box transcription factor is largely responsible for heart defects in DiGeorge syndrome/velo-cardio-facial syndrome/22q11.2 deletion syndrome (22q11DS). Approximately 70% of 22q11DS patients have cardiovascular anomalies, mostly of the conotruncal type, caused by abnormalities in the formation of the cardiac outflow tract (OFT). Tbx1 is expressed throughout the mesoderm of the pharyngeal apparatus during early mouse development. Inactivation of one allele of Tbx1 in mice results in mild defects with low penetrance, but inactivation of both alleles of Tbx1 results in a persistent truncus arteriosus (PTA) with complete penetrance. Gene profiling of the distal pharyngeal apparatus containing the anterior second heart field (aSHF) mesoderm, from E8.5-10.5, suggested that Tbx1 promotes cell proliferation and restricts premature differentiation. We inactivated Tbx1 in the aSHF using the Mef2c-AHF-Cre driver and found that all embryos died at birth with a PTA. Gene profiling revealed the same set of genes as for Tbx1 null mutant mouse embryos. In contrast to Tbx1, canonical Wnt signaling in the aSHF promotes cell differentiation. To test whether there are opposing effects between Tbx1 and canonical Wnt signaling, we inactivated both alleles of Tbx1 and one allele of ß-catenin in the aSHF and found significant rescue of the heart defects. Based upon this, we hypothesize that there is a negative feedback mechanism that exists between Tbx1 and canonical Wnt/ß-catenin signaling in the aSHF. To test this, we will define the epistatic relationship between Tbx1 and ß-catenin, evaluate cell proliferation versus differentiation, as well as perform ChIP-seq to identify direct transcriptional target genes. Interestingly, some of the genes increased in expression, such as Wnt2 and Gata6, are specific to the posterior SHF (pSHF) and are required to form the cardiac inflow tract. Since the microarrays could not distinguish between gene expressions versus cell population changes, we hypothesize that loss of Tbx1 in the aSHF, in addition to having tissue autonomous functions, may cause an expansion of the surrounding pSHF cells, also affecting heart development. This hypothesis will be tested by pSHF lineage tracing and genetic studies of Tbx1 with Wnt2 and Gata6. The 22q11DS patients show variable phenotypic expressivity of cardiovascular anomalies. Some of the genes characterized in this program may serve as genetic modifiers. Moreover, because heart defects are rescued by altering Wnt signaling in Tbx1 mutant mice, a better mechanistic understanding of this process may be translated in the future to novel drug therapies on 22q11DS patients.