TISSUE INTERACTIONS IN PALATAL SHELF CLOSURE

这项研究的长期目标是了解细胞和 决定正常次级腭形态发生的分子因素。 构成腭的细胞外基质（ECM）分子 间质基质和基底膜对该过程很重要。 腭间质 ECM 排列为支架，由 主要由纤连蛋白 (Fn)、胶原蛋白 III(Col ]III) 和 透明质酸 (Ha)，在预期体内提前一天发挥作用 腭架重新定向。 如果架子被移植到器官培养中 此时，它们会重新定位并显示 ECM 的伴随变化 分布。 IV 型胶原蛋白 (Col IV) 和分布的变化 上皮基底膜局部区域的层粘连蛋白（Ln）和 Syndecan(Sn) 在间充质中的分布立即发生变化 这些区域下方也伴随着架子的改造。 显然， 一旦这些 ECM 结构建立起来，腭架就 能够重塑以实现重新定位。 试点数据表明 必要的改变可能涉及金属蛋白酶（MMP）和 纤溶酶原激活剂（PA）。 现有的工作还表明，特别是 在发育过程中，ECM、MMP、PA 及其激活剂之间的相互作用 抑制剂决定了 ECM 的特性。 然而，迄今为止还没有 关于这些酶在次级味觉中的作用的工作已经完成 形态发生。 根据目前的知识，有以下假设 提出：Ha介导的货架扩张导致重新定位 是由临时组织结构的改变引导的 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 间质紧邻其下方。 这些变化涉及 蛋白酶介导的 ECM 分子降解。 三具体 提出的目标是检验这一假设： (1) 描述 腭架 ECM 支架的时空发展 从上颌突到腭架出现的时间 结构具有功能性； (2) 识别 MMP、PA 及其 腭架中存在的抑制剂 体内和体外形态发生并确定其时间序列 外观和空间分布； (3) 确定效果 抑制或激活上颚基质降解蛋白酶 形态发生以及间质的组成和组织 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 结合图像处理和分析进行免疫组织化学分析 形态测量学将用于实现这些目标。 结果 这些研究将增加我们对 ECM 和 上颚形态发生过程中的基质降解酶。 这样一个 理解可能会导致更准确地识别可以 破坏该过程以及那些可能逆转或补偿的过程 破坏性因素或缺陷基因的影响。

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.