The morphogenesis of actin-based structures in mucosal epithelia

描述(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一种糖蛋白花蕾，并吸附粘蛋白以形成粘液层，保护这些组织免受磨损并保持其水化。例如，在角膜中，粘液层保留了让我们的眼睛保持湿润的泪膜。人类角膜粘膜层的缺陷会导致干眼症(DED)。这些情况很常见，很痛苦，而且会随着年龄的增长而发展。DED通常伴有口干，这表明其潜在原因影响了粘膜上皮的共同特性。其中一个特性是在这些上皮细胞的表面存在复杂的基于肌动蛋白的结构，称为微皱纹和微脊。这些结构很少被研究，但可能对粘膜上皮的功能特性做出了重要贡献，因为它们增加了糖萼的表面积，从而最大限度地提高了它们水合组织的能力。大多数对DED病理学的研究都集中在泪液的产生上，但鉴于粘膜上皮在维持粘液层和泪膜方面的关键作用，上皮形态发生的缺陷很可能也是导致这些情况的原因。研究小脊线和小脊线的形态发生的主要障碍之一是缺乏可获得的模型系统。我们开发了斑马鱼幼体皮肤作为研究粘膜上皮发育的模型。斑马鱼幼体的整个表面被一层被称为周皮的单层粘膜上皮包裹着。周皮细胞的顶端表面覆盖着我的微皱纹和微脊，它们与人类角膜表面的结构非常相似。这些细胞特别容易被转基因标记和共聚焦成像，使其有可能在活着的动物身上可视化脊状结构的形成。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，我们结合了描述性的、假设驱动的和基于发现的方法来剖析脊形变过程。具体地说，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过程中的重组，在目标2中，我们将使用分子和成像方法来验证磷脂酰肌醇微域协调微脊形成的假设。最后，在目标3中，我们将使用RNA-Seq来鉴定富含周皮的bar结构域蛋白和肌动蛋白调节因子，并测试选定的候选蛋白是否在微脊发育中发挥作用。综上所述，这些研究将首次深入了解脊线形态发生，并将斑马鱼模型建立为一个系统，不仅可以理解正常细胞中的脊线形成，还可以了解疾病(如DED)中脊线形成的原因。

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.