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UBC Theses and Dissertations

Smad2 overexpression rescues the tgf-β3 null mutant mice cleft palate by increased apoptosis Almegbel, Abdullah M.


Objectives: Cleft lip/palate is a common birth defect. It occurs in about one in 700 live births worldwide. In non-syndromic cleft lip/palate, a linkage to TGF-β3 has been shown. Signaling of TGF-β3 is mediated in the cell through the Smad2 protein. During secondary palate fusion TGF-β3 signaling leads to the disappearance of the epithelial midline seam and the confluence of the palatal mesenchyme. TGF-β3 null mice are born with a cleft in the secondary palate, a phenotype that has been rescued by targeted overexpression of Smad2 in the MEE. The goal of this research was to understand the mechanism of palatal fusion in the rescue mice. Methods: The heads of embryos of four different mice models (wild-type, rescue, K14-Smad2 overexpression and TGF-β3 null) were collected at gestational age E14.5 genotyped, fixed and embedded in paraffin. Serial sections were studied for detection of apoptosis and epithelial mesenchymal transition using immunofluorescence. Images were captured with confocal laser microscopy. Results: TGF-β3 null mice developed a cleft in the secondary palate while mice that had both the TGF-β3 null and overexpression K14-Smad2 genotypes had fusion of the secondary palate. The medial edge epithelium of the rescue mice had a much higher ratio of cells with cleaved caspase (31.7% anterior, 33% middle and 35.6% posterior), than in the wild-type mice (0.0% anterior, 5.31% middle and 0.0% posterior). The K14-Smad2 overexpression genotype mice had an increased number of apoptosis positive MEE when compared to the wild-type mice (13.7% anterior, 10.1% middle and 17.6% posterior). The increase in apoptosis was correlated with increased p-Smad2 in the MEE. Conclusions: Smad2 overexpression may have rescued the cleft in the secondary palate by increasing apoptosis in the medial edge epithelium. Thus, the mechanism of rescue is not identical to the events that occur normally during palatal fusion.

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