The morphogenesis of actin-based structures in mucosal epithelia

描述（由申请人提供）：粘膜上皮形成许多敏感组织的外部界面。这些上皮细胞的外表面显示糖蛋白花萼，并吸附粘蛋白形成一层粘液，保护这些组织免受磨损并保持其水合作用。例如，在角膜中，黏液层保留着泪膜，使我们的眼睛保持湿润。人类角膜粘膜层缺陷引起干眼病（DED）。这些情况是常见的，痛苦的，并随着年龄的增长而发展。DED常伴有口干，提示其潜在原因影响粘膜上皮的共同特性。其中一个特性是这些上皮细胞表面存在复杂的基于肌动蛋白的结构，称为微褶和微脊。这些结构很少被研究，但可能对粘膜上皮的功能特性做出重要贡献，通过增加糖萼的表面积，从而最大限度地发挥其水合组织的能力。大多数关于DED病理学的研究都集中在泪液的产生上，但考虑到粘膜上皮在维持黏液层和泪液膜方面的关键作用，上皮形态发生的缺陷很可能也导致了这些疾病。研究微晶片和微晶片形态发生的主要障碍之一是缺乏可接近的模型系统。我们开发了幼体斑马鱼皮肤作为研究粘膜上皮发育的模型。斑马鱼幼虫的整个表面被单层粘膜上皮包裹，称为周皮。外周细胞的顶端表面覆盖着微褶和微脊，它们与人类角膜表面的结构非常相似。这些细胞非常容易获得转基因标记和共聚焦成像，使其有可能在活体动物中可视化脊的形成。此外，斑马鱼系统对复杂的遗传和转基因操作的适应性将使揭示微脊形成的潜在分子机制成为可能。在这个建议中，我们结合了描述性、假设驱动和基于发现的方法来剖析山脊形态形成的过程。具体来说，在目标1中，我们将使用实时成像来描述微脊的初始形成及其在细胞收缩期间的重组。在目标2中，我们将使用分子和成像方法来测试磷酸肌苷微域协调微脊形成的假设。最后，在Aim 3中，我们将使用RNA-Seq鉴定表皮中富集的BAR结构域蛋白和肌动蛋白调节因子，并测试所选候选蛋白是否在微脊发育中发挥作用。总之，这些研究将首次提供脊状突起形态发生的见解，并建立斑马鱼模型作为一个系统，不仅可以理解正常细胞中脊状突起的形成，还可以理解疾病（如DED）中脊状突起的病理原因。

DESCRIPTION (provided by applicant): Mucosal epithelia form the external interface of many sensitive tissues. The outer surface of cells in these epithelia displays a glycoprotein calyx and adsorbs mucins to create a mucus layer that protects those tissues from abrasion and maintains their hydration. In the cornea, for example, the mucus layer retains the tear film that keeps our eyes wet. Defects in the mucosal layer of the human cornea cause dry eye diseases (DED). These conditions are common, painful, and progress with age. DED is often accompanied by dry mouth, suggesting that its underlying causes affect properties common to mucosal epithelia. One such property is the presence of elaborate actin-based structures on the surface of these epithelial cells, known as microplicae and microridges. These structures have been little studied, but likely make a vital contribution to the functional properties of mucosal epithelia by increasin the surface area of the glycocalyx, thus maximizing their ability to hydrate tissues. Most studies of DED pathology have focused on tear production, but given the critical role of mucosal epithelia in maintaining the mucus layer and tear film, it is likely that defects in epithelial morphogenesis also contribute to these conditions. One of the main obstacles to studying the morphogenesis of microplicae and microridges has been the lack of an accessible model system. We have developed the larval zebrafish skin as a model for studying mucosal epithelial development. The entire surface of zebrafish larvae is wrapped in a single-layered mucosal epithelium known as the periderm. The apical surface of periderm cells is covered my microplicae and microridges that remarkably resemble structures on the surface of the human cornea. These cells are exceptionally accessible to transgenic labeling and confocal imaging, making it possible to visualize the formation of ridges in living animals. Moreover, the amenability of the zebrafish system to sophisticated genetic and transgenic manipulations will make it possible to uncover the underlying molecular mechanisms of microridge formation. In this proposal we combine descriptive, hypothesis-driven, and discovery-based approaches to dissect the process of ridge morphogenesis. Specifically, in Aim 1 we will use live imaging to describe the initial formation of microridges and their re-organization during cellular contraction In Aim 2 we will use molecular and imaging approaches to test the hypothesis that phosphoinositide microdomains orchestrate the formation of microridges. Finally, in Aim 3 we will use RNA-Seq to identify BAR domain proteins and actin regulators enriched in periderm and test whether select candidate proteins play roles in microridge development. Together these studies will provide the first insights into ridge morphogenesis and establish the zebrafish model as a system for understanding not only ridge formation in normal cells, but also the causes of their pathology in diseases, such as DED.