The development of the pharyngeal apparatus into the craniofacial region, thymus, parathyroid and cardiac outflow tract (OFT) involves complex gene interaction, making them highly sensitive to genetic and environmental insults. Velo-cardio-facial syndrome/DiGeorge syndrome is associated with hemizygous 22q11 deletions and characterized by defects in the derivatives of the pharyngeal apparatus. Tbx1, a T-box transcription factor, and the gene for VCFS/DGS in humans, is associated with defects in the same structures when inactivated in the mouse. The most serious defect in patients with the syndrome is OFT defects. Tbx1 is expressed in the multiple tissues in the pharyngeal apparatus including the second heart field (SHF) mesenchyme required for OFT development. The mouse serves as an excellent model organism to identify genes for OFT development because many aspects of its morphogenesis are shared with humans. To identify Tbx1 regulated genetic pathways in the SHF, we performed gene expression profiling of the caudal pharyngeal region in Tbx1-/- and wild type mouse embryos. Isl1, a key marker for the SHF, among selected others, were downregulated in Tbx1-/- mutants, while genes required for posterior specification of the first heart field (FHF), such as Raldh2, Gata4, and Tbx5, as well as a subset of atrial-specific muscle contractile genes were ectopically expressed. Opposite expression changes concomitant with SHF-derived cardiac defects occurred in TBX1 gain-of- function mutants. Based upon these data, we hypothesize that Tbx1 positively regulates SHF cell proliferation and restricts premature differentiation in the caudal pharyngeal mesenchyme. Several genes important for the retinoic acid pathway, transcription factors and novel genes were found in the microarrays. We propose to characterize the most biologically interesting genes in order to build the genetic pathway downstream of Tbx1 in the SHF using null, conditional loss- and gain-of-function mutant mouse embryos. To identify direct downstream target genes of Tbx1, reporter systems will be used in mammalian cells. Tissue interactions are required for neural crest cell (NCC) migration and OFT septation, defective in VCFS/DGS and mouse mutants. Recently, Crkl, another gene deleted on 22q11.2, was shown to mediate Fgf8 signaling downstream of Tbx1. To test this hypothesis in the SHF and in NCCs, embryos with pan-mesodermal and neural crest inactivation of Crkl, respectively, will be analyzed for altered expression of Fgf responsive genes and SHF genes characterized. Using genes identified by microarray studies and conditional mutants we will be able to dissect the genetic pathway of Tbx1 in the SHF and NCCs for OFT development from the pharyngeal apparatus. Narrative: The relevance of this research to public health is that this program will enable us to find genes that cause birth defects. By taking genetics approaches in model organisms, we can obtain insights that would otherwise not be possible in humans.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL088698-01A2
Application #
7372750
Study Section
Development - 2 Study Section (DEV2)
Program Officer
Schramm, Charlene A
Project Start
2009-07-01
Project End
2011-06-30
Budget Start
2009-07-01
Budget End
2010-06-30
Support Year
1
Fiscal Year
2009
Total Cost
$415,000
Indirect Cost
Name
Albert Einstein College of Medicine
Department
Genetics
Type
Schools of Medicine
DUNS #
110521739
City
Bronx
State
NY
Country
United States
Zip Code
10461
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Freyer, Laina; Nowotschin, Sonja; Pirity, Melinda K et al. (2013) Conditional and constitutive expression of a Tbx1-GFP fusion protein in mice. BMC Dev Biol 13:33
Simrick, Subreena; Szumska, Dorota; Gardiner, Jennifer R et al. (2012) Biallelic expression of Tbx1 protects the embryo from developmental defects caused by increased receptor tyrosine kinase signaling. Dev Dyn 241:1310-24
Monks, Dennis C; Morrow, Bernice E (2012) Identification of putative retinoic acid target genes downstream of mesenchymal Tbx1 during inner ear development. Dev Dyn 241:563-73
Aggarwal, Vimla S; Carpenter, Courtney; Freyer, Laina et al. (2010) Mesodermal Tbx1 is required for patterning the proximal mandible in mice. Dev Biol 344:669-81