TISSUE INTERACTIONS IN PALATAL SHELF CLOSURE

这项研究的长期目标是了解细胞和 决定正常的继发性腭形态发生的分子因素。 组成腭部的细胞外基质（ECM）分子 间质基质和基底膜对于该过程是重要的。 腭间质ECM呈支架状排列， 主要是纤连蛋白（Fn）、胶原蛋白III（Col III）和 透明质酸（Ha），在体内预期前一天起作用 腭架重新定位 如果架子上放的是器官培养 此时，它们重新定向，并显示出ECM的伴随变化 分布 IV型胶原（Col IV）和 上皮基底膜局部区域的层粘连蛋白（Ln）， 间质中多配体蛋白聚糖（Sn）分布的变化立即 从属于这些区域也伴随着大陆架重塑。 很显然， 一旦这些ECM结构建立，腭架 能够重塑以实现重新定向。 试点数据显示， 必要的改变可能涉及金属蛋白酶（MMPs）， 纤溶酶原激活剂（PA）。 现有的工作也表明，特别是 在发育过程中，ECM、MMPs、PA及其激活剂之间的相互作用 而抑制剂决定ECM的特性。 然而，迄今为止， 已经对这些酶在次级腭中的作用进行了研究。 形态发生 根据目前的知识，以下假设 提出：HA介导的货架扩张，导致重新定位 是由颞叶组织结构的改变所引导的， Fn和Col III分布的空间局部变化， 间隙基质和Col III和IV，Ln和Sn在特定局部 覆盖上皮的基底膜区域和 间充质立即服从它。这些变化涉及 蛋白酶介导的ECM分子降解。 三个具体 目的是提出测试这一假设：（1）描述 腭架ECM支架的时空发育 从上颌突开始的腭架出现的时间， 结构是功能性的;（2）鉴定MMPs，PA及其 抑制剂，存在于腭架在腭 在体内和体外形态发生，并确定其时间顺序 外观和空间分布;（3）确定 抑制或激活腭上的基质降解蛋白酶 形态发生和间质的组成和组织 ECM结构和基底膜。 器官培养，SDS-PAGE， 酶谱分析、RNA表型分析、原位杂交和 免疫组织化学分析，图像处理和分析相结合 将使用形态测量来实现这些目标。 的结果 这些研究将增加我们对ECM相互作用的了解， 腭形态发生过程中的基质降解酶。 这样的 理解可能导致更精确地识别可以 破坏这一进程，以及那些可能逆转或补偿 破坏性因子或缺陷基因的影响。

The long-term goal of this research is to understand the cellular and molecular factors that determine normal secondary palate morphogenesis. The extracellular matrix(ECM) molecules that compose the palatal interstitial matrix and basement membranes are important to the process. The palatal interstitial ECM is arranged as a scaffolding, composed principally of fibronectin(Fn), collagen III(Col ]III) and hyaluronan(Ha), that is functional by one day before expected in vivo palatal shelf reorientation. If shelves are explanted into organ culture at this time, they reorient and show accompanying changes in ECM distribution. Changes in distribution of collagen IV(Col IV) and laminin(Ln) in local regions of the epithelial basement membrane and changes in syndecan(Sn) distribution in the mesenchyme immediately subjacent to these regions also accompany shelf remodelling. Apparently, once these ECM structures are established, the palatal shelves are capable of remodelling to achieve reorientation. Pilot data suggest the necessary alterations may involve the metalloproteinases(MMPs) and plasminogen activators(PAs). Existing work also suggests, particularly in development, the interplay between ECM, MMPs, PAs and their activators and inhibitors determine the character of an ECM. However, to date no work has been done on the role of these enzymes in secondary palate morphogenesis. In light of current knowledge, the following hypothesis is proposed: The Ha-mediated shelf expansion that leads to reorientation is guided by alterations in tissue architecture resulting from temporo- spatially localized changes in the distribution of Fn and Col III in the interstitial matrix and of Col III and IV, Ln and Sn in specific local areas of the basement membrane of the covering epithelium and the mesenchyme immediately subjacent to it. These changes involve proteinase-mediated degradation of the ECM molecules. Three specific aims are proposed to test this hypothesis: (1) Describe the temporo-spatial development of the palatal shelf ECM scaffolding from the time of emergence of the palatal shelves from the maxillary process until the structure is functional; (2) Identify the MMPs, PAs and their inhibitors that are present in the palatal shelves during palate morphogenesis in vivo and in vitro and determine their temporal sequence of appearance and spatial distribution; (3) Determine the effects of inhibiting or activating the matrix-degrading proteinases on palate morphogenesis and the composition and organization of the interstitial ECM structure and the basement membrane. Organ culture, SDS-PAGE, zymography, RNA phenotyping, in situ hybridization, and immunohistochemistry analyzed by image processing and analysis combined with morphometry will be used to achieve these aims. The results of these studies will increase our knowledge of the interactions of ECM and matrix-degrading enzymes in the process of palate morphogenesis. Such an understanding may lead to more precise identification of agents that can disrupt the process as well as those that might reverse or compensate for the effects of disruptive agents or defective genes.