The role of elastic basement membrane pre-stress in Drosophila epithelial morphogenesis

组织的成形对于动物发育产生功能器官是重要的。组织形态发生依赖于力的产生和细胞的力学性质。机械力依赖于肌动球蛋白的细胞骨架。最近的工作也表明了细胞外基质（ECM）在组织成形中的重要作用。然而，目前，ECM在组织成形中的作用尚未完全了解。上皮细胞由称为基底膜的薄ECM层支撑。基底膜是一种致密、弹性、蛋白质材料，由于弹性变形，其可以处于机械预应力下。然而，这种弹性变形是如何产生的上皮细胞和这些变形如何有助于组织形态是未知的。果蝇翅盘是研究组织形态发生的一个有效模型系统。作为其形态发生的一部分，翅盘形成几个刻板的褶皱，包括一个位于预期铰链区的褶皱，称为H/H褶皱。我们先前发表的工作表明，H/H折叠的形成涉及前折叠细胞下方ECM的局部减少。我们最近获得的初步数据，原子力显微镜（AFM）显示，此外，翅盘的基底膜是在机械预应力，机械预应力取决于肌动球蛋白活性和基底膜成分（胶原IV）。建议的项目有四个目标：首先，我们的目标是量化基膜预应力在翼盘AFM测量在空间和时间与H/H折叠形成。其次，我们的目的是了解在地下室膜的机械预应力的起源。特别是，我们将通过实验测试i）细胞增殖，ii）静水压力和iii）横向细胞张力的变化如何影响机械预应力。同时，我们将确定这些改变如何改变H/H折叠的形成。第三，我们的目标是通过发展物理理论和计算机模拟，结合静水压力，侧向张力和前折叠细胞下ECM的局部减少的机械效应，提供对预应力产生及其对上皮折叠影响的机械理解。我们将使用成像和随后的粒子图像测速技术来量化在去细胞化过程中翅盘基底膜中的弹性预应变的量。我们期望这种跨学科的方法将获得对ECM在组织成形中的作用的新的机理认识，从而，将有助于更好地理解ECM改变引起的疾病，并为开发有效的组织工程方案铺平道路。

The shaping of tissues is important for animal development to generate functional organs. Tissue morphogenesis depends on the generation of force and the mechanical properties of cells. Mechanical force depends on the actomyosin cytoskeleton. Recent work also indicates an important role for the extracellular matrix (ECM) in shaping tissues. However, at present, the role of the ECM in shaping tissues is not fully understood. Epithelia are basally supported by a thin ECM layer termed basement membrane. The basement membrane is a dense, elastic, proteinaceous material that can be under mechanical prestress due to elastic deformations. However, how such elastic deformations are generated by an epithelium and how these deformations contribute to tissue morphogenesis is unknown. The Drosophila wing disc is a powerful model system to study tissue morphogenesis. As part of its morphogenesis, the wing disc forms several stereotypic folds, including a fold centred in the prospective hinge region called the H/H fold. Our previously published work showed that the formation of the H/H fold involves the local reduction of ECM beneath the pre-fold cells. Our recent preliminary data acquired by atomic force microscopy (AFM) shows, moreover, that the basement membrane of wing discs is under mechanical prestress, and that mechanical prestress depends on both actomyosin activity and basement membrane components (Collagen IV). The proposed project has four aims: Firstly, we aim to quantify basement membrane prestress in the wing disc by AFM measurements in space and time in correlation with H/H fold formation. Secondly, we aim to understand the origin of mechanical prestress in the basement membrane. In particular, we will test experimentally how changes in i) cell proliferation, ii) hydrostatic pressure, and iii) lateral cellular tension affect mechanical prestress. Concomitantly, we will identify how these alterations modify H/H fold formation.Thirdly, we aim to provide a mechanistic understanding of prestress generation and its influence on epithelial folding by developing a physical theory and computer simulations incorporating mechanical effects of hydrostatic pressure, lateral tension and local reduction of ECM beneath pre-fold cells.Fourth, we will quantify the amount of elastic prestrain in the basement membrane of wing discs during decellularization using imaging and subsequent particle image velocimetry.We expect that this interdisciplinary approach will gain new mechanistic insights into the role of ECM in shaping tissues and, thereby, will contribute to a better understanding of diseases caused by ECM alterations as well as pave the way to the development of efficient tissue engineering protocols.