The goal of this proposal is to experimentally assess a model of cleft-lip (CL) formation in which variation in midfacial cell proliferation (growth rate) affects the shape of the face and the incidence of CL in a predictable manner. The A/WySn strain mouse is a common model of human CL and has an increased incidence of facial clefting (20-25%) due to a defect in Wnt9b expression. Evidence suggests that members of the Wntsignaling pathway regulate cell proliferation in embryonic midfacial tissues during a critical period of facial outgrowth and fusion. Previous research on midfacial morphogenesis indicates that compared to normal mice the A/WySn strain has a normal-sized head but that the midfacial prominences are abnormally shaped and exhibit retarded growth. These findings implicate disruptions of Wnt-mediated regulation of midfacial cell proliferation as a causal factor in the etiology of non-syndromic CL. Here we propose to investigate the relationship of growth rate to midfacial shape and CL formation by first experimentally inducing variance in cell proliferation using retroviral expression of a Wnt-antagonist, Dkk1, and then comparing midfacial shape variation between groups using advanced landmark-based geometric morphometrics. Prior to facial formation (E9.5), embryos of both normal and A/WySn strain mice will be infected with an adenovirus expressing Dkk1 protein. Because Wnt-responsive cells are active primarily in the facial prominences, disruption of cell growth will affect only those regions involved in CL formation while leaving other regions of the head unaffected (e.g., brain growth). The model proposed here predicts that variation in midfacial growth will result in an axis of continuous shape change that is correlated with increased incidence of CL formation and bilateral trait expression. This hypothesis will be tested by quantifying and comparing the shape of the midface in infected mice with that of both uninfected controls and A/WySn mice at E12.5. Support for this model would indicate that the variance induced by genetic perturbations of cell proliferation are ontologically as important to the etiology of CL as the specific genes involved. For medical researchers, this model would help to link the effect of novel genetic or environmental disruptions of cell proliferation to a predictable phenotypic outcome. ? ? This study proposes to explore the role of variation in growth to both the shape of the face and the incidence of cleft lip formation, a common birth defect in humans. This research will enable doctors to better predict the effect of genetic mutations to growth on morphological outcomes like CL. In turn, this predictive power will help doctors plan more effective treatments of diseases associated with defects in growth. ? ? ?
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