A Molecular Investigation of Growth and Shape Variation in Cleft-Lip Etiology

描述（由申请人提供）：本提案的目的是通过实验评估唇裂（CL）形成模型，其中面中部细胞增殖（生长速率）的变化以可预测的方式影响面部形状和CL发生率。A/WySn品系小鼠是人类CL的常见模型，并且由于Wnt 9 b表达缺陷而具有增加的面部裂开发生率（20-25%）。有证据表明，在面部生长和融合的关键时期，Wnt信号通路的成员调节胚胎面中部组织中的细胞增殖。先前关于面中部形态发生的研究表明，与正常小鼠相比，A/WySn品系具有正常大小的头部，但面中部的突起形状异常，并表现出生长迟缓。这些研究结果表明，Wnt介导的面中部细胞增殖调节的中断是非综合征性CL病因学的一个致病因素。在这里，我们建议调查的生长速度的关系，面中部的形状和CL的形成，首先实验诱导变异的细胞增殖使用逆转录病毒表达的Wnt拮抗剂，Dkk 1，然后比较面中部的形状变化组之间使用先进的地标为基础的几何形态测量。在面部形成（E9.5）之前，将用表达Dkk 1蛋白的腺病毒感染正常和A/WySn品系小鼠的胚胎。因为Wnt-反应性细胞主要在面部神经元中是活跃的，所以细胞生长的破坏将仅影响参与CL形成的那些区域，而头部的其他区域不受影响（例如，脑生长）。这里提出的模型预测，面中部生长的变化将导致一个轴的连续形状变化，与CL形成和双边性状表达的发病率增加。将通过定量和比较感染小鼠与未感染对照和A/WySn小鼠在E12.5时的面中部形状来检验该假设。支持这一模型表明，细胞增殖的遗传扰动引起的变异是本体论的重要性，作为特定的基因参与的CL的病因。对于医学研究人员来说，这种模型将有助于将细胞增殖的新遗传或环境干扰的影响与可预测的表型结果联系起来。 本研究旨在探讨生长发育变异对面部形状和人类常见出生缺陷唇裂发生率的影响。这项研究将使医生能够更好地预测基因突变对CL等形态学结果的影响。反过来，这种预测能力将帮助医生计划更有效的治疗与生长缺陷相关的疾病。

DESCRIPTION (provided by applicant): 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.