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

描述(由申请人提供)：这项建议的目标是实验评估唇裂(CL)形成的模型，在该模型中，面中部细胞增殖(生长率)的变化以可预测的方式影响脸型和CL的发生率。A/WySn系小鼠是人类CL的常见模型，由于Wnt9b表达缺陷，面部裂伤的发生率增加(20%-25%)。有证据表明，在面部发育和融合的关键时期，Wnt信号通路的成员调控着面中部胚胎组织中的细胞增殖。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系小鼠有一个正常大小的头部，但面中部隆起形状异常，生长缓慢。这些发现表明，WNT介导的面中部细胞增殖调控的中断是非综合征CL的病因之一。在这里，我们建议研究生长速度与面中部形状和CL形成的关系，首先通过逆转录病毒表达Wnt拮抗剂Dkk1来实验诱导细胞增殖的差异，然后使用先进的基于里程碑的几何形态计量学比较不同组之间面中部形状的差异。在面部形成(E9.5)之前，正常小鼠和A/WySn系小鼠的胚胎都将被表达Dkk1蛋白的腺病毒感染。由于Wnt反应细胞主要活跃于面部隆起，细胞生长的中断只会影响参与CL形成的那些区域，而不会影响头部其他区域(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连续的形状变化的轴，这与CL形成的发生率和双侧性状表达的增加相关。这一假设将通过量化和比较感染小鼠与未感染对照组和E12.5的A/WySn型小鼠的面部中部形状来检验。支持这一模型将表明，细胞增殖的遗传扰动引起的差异在本体论上对CL的病因与涉及的特定基因一样重要。对于医学研究人员来说，这个模型将有助于将细胞增殖的新的遗传或环境干扰的影响与可预测的表型结果联系起来。 这项研究建议探索发育过程中的变异对脸型和唇裂形成的发生率的作用，唇裂是人类常见的出生缺陷。这项研究将使医生能够更好地预测基因突变对生长的影响，如CL等形态结果。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.