The broad goal of this project is to understand the genetic regulation of heart morphogenesis. The T-box family of transcription factors were first identified a splaying an important role in this process because mutations in one member of the family Tbx-5, are responsible for Holt-Oram syndrome, a human congenital disorder which includes atrial septal defects (ASD). This is a dominant mutation and it appears that the phenotype is produced by a half-dose of the gene product (haploinsufficiency) in individuals heterozygous for a loss-of-function mutation. The homozygous phenotype is not yet known in humans or in animal models, thus the gene may play an even more profound role in heart morphogenesis. Other members of this gene family are also expressed at early stages of heart development. These genes also appear to play important roles in limb development, offering a second very well characterized system in which to evaluate their molecular activities. The chick embryo system is ideal for investigating their functions. The egg affords continuous direct access to the developing heart throughout embryogenesis, allowing both experimental manipulations and physiological measurements. In addition the chick system allows easy and rapid gene mis- expression using retroviral vectors, a method the investigators pioneered and have extensive experience with, including in studying left-right asymmetry during heart morphogenesis. The investigators will use this system to test the roles of different T- box genes in heart morphogenesis and limb patterning. More specifically, the aims are: (1) Examine the roles of T-box genes in heart development by: (a) further defining the spatial and temporal expression patterns of Tbx-2, 3, 4, and 5 during heart morphogenesis (preliminary data shows that they are expressed early and regionally restricted); (b) mis-expressing wild-type copies of the Tbx-2, 3, 4, and 5 genes throughout the early cardiac primordium, and assessing heart morphology; (c) mis-expressing forms of these genes designed to act as dominant-negative repressors of normal target transcription and assessing heart morphogenesis; and (d) utilize the Physiology Core to determine whether there is an alteration in physiological parameters (such as heart pressure changes) as a result of the viral mis-expression of wild-type and mutant Tbx genes.
Specific Aim 2 will test the roles of theses genes in limb development by (a) misexpression of the various wild-type genes utilizing the same viral vectors designed for the fear studies and (b) mis-expressing the dominant- negative variants of the genes.
Specific Aim 3 will test whether the Tbx genes are induced in the cardiac primordium by BMP proteins and/or crescent, utilizing a combination of delivering of purified BMP proteins on carrier beads and virally-mediated mis-expression of crescent, in collaboration with SCOR member Andrew Lassar.
Specific Aim 4 will test whether the human mutation identified by the laboratory of SCOR member Christine Seidman are null alleles, hypomorphs or dominant-negative variants by assessing phenotypes after viral mis-expression in both the heart and the limb, allowing the variable expressivity of different mutations to be addressed.
Specific Aim 5 will test whether the related Tbx genes are functionally redundant or are providing distinct positional information in the developing embryo, taking advantage of the availability of a powerful limb-specific promoter for transgenic mice. Colleagues on the SCOR grant from the Seidman laboratory are knocking out different Tbx genes (Project II). These will be cross to transgenic Tbx mice to test whether the same and/or different Tbx genes can complement the limb defect produced by the knockout.
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