Determination of the role and regulation of matrix metalloproteinase-25 during mouse secondary palate formation
Brown, Graham Douglas
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Development of the secondary palate (SP) is a complex event despite the small area it encompasses. Problems with SP development can lead to a cleft palate, which is one of the most common birth disorders. The matrix metalloproteinases (MMPs) are required for proper SP development, but a functional role for any one of them remains unknown. MMP-25 is a candidate MMP to have a functional role in SP formation as genetic scans of the DNA of human cleft palate patients indicate a common mutation at a region upstream of the Mmp-25 gene. The purpose of this thesis is to investigate gene expression of Mmp-25 in the developing mouse SP, whether it has a functional role in mouse SP development and begin to identify factors potentially upstream of Mmp-25 expression. Mmp-25 mRNA and protein is found at all SP developmental stages in mice with highest expression at embryonic day (E) 13.5 when analyzed by quantitative real-time PCR and western blotting. Immunohistochemistry localizes MMP-25 protein primarily to the plasma membranes of palate shelf epithelial cells with secondary expression in apical mesenchymal cells. Mmp-25 knockdown with siRNA in palatal cultures resulted in a significant decrease in palate shelf fusion and persistence of the medial edge epithelium in vitro. Mmp-25 mRNA and protein levels are significantly decreased in vitro when cultured palate shelves are incubated in growth medium with 5 ìg/ml of a TGFâ3-neutralizing antibody. Mmp-25 gene expression is highest at E12.5 and E13.5, which corresponds to increasing palate shelf growth downward alongside the tongue. Immunohistochemistry localized MMP-25 protein expression predominantly in the epithelium of the palate shelves, but also in areas of the mesenchyme that were immediately adjacent to the epithelium and apical in location. Knockdown of Mmp-25 expression resulted in palate shelf fusion being impaired and significant medial edge epithelium remaining in contacted areas. Bioneutralization of TGFâ3 resulted in a significant decrease in Mmp-25 gene expression. These data suggest a functional role for MMP-25 in mouse SP development by removing extra-cellular matrix barriers to increased palate shelf growth and place its expression downstream of TGF-â3 signaling. This is the first research to present a role for a single MMP in mouse SP development.
DegreeMaster of Science (M.Sc.)
DepartmentCollege of Pharmacy and Nutrition
ProgramCollege of Pharmacy and Nutrition
SupervisorNazarali, Adil J.
CommitteeKrone, Pat; Krol, Ed S.; Dobson, Roy T.