Investigating Patterns of Cell Interactions During Epithelial Folding

正确的组织形状对于正常的组织功能至关重要，形态发生失调会导致 许多常见的先天性疾病。然而，数千甚至数百个细胞的群体如何协调 通过大规模运动来改变产量的刻板印象仍然知之甚少。细胞可以通过一种方式 相互作用是通过机械耦合实现的。事实上，细胞之间的肌动球蛋白连接的大规模网络 跨越各种模式生物的发育组织。高度可重复性的发展规划和 果蝇强大的遗传工具包使果蝇腹侧沟成为研究这种现象的理想系统 网络。在皱纹形成过程中，细胞协调脉冲式收缩以产生组织范围的弯曲。组织 具有在折叠前完全形成的动态肌球蛋白网络。然而，人们对此知之甚少。 网络中的机械信息引导集体建设。这项提案将解决如何 建立了机械连接网络来驱动组织折叠。首先，2D网络如何 细胞间的连接促进上皮折叠将被建立。一种新的方法，适应方法 从天文学和数学的网络理论将映射以前无法量化的肌球蛋白 在发育中的组织中跨越数百个细胞。初步研究已经确定了初步增长 在网络中的阶段和随后的收缩阶段。成长阶段：我们假设坚持不懈 当相邻细胞同时经历脉冲肌球蛋白时，就建立了网络连接 积累。为了验证这一假设，肌球蛋白招募的位置和时间将与 创建或重组网络连接。抑制肌球蛋白脉冲的胚胎注射将测试 脉冲对网络形成的要求。收缩阶段：我们假设网络中的签名 几何引导刻板组织折叠。组织范围的连通性将与以下区域相关 协调的细胞收缩。激光切割将测试连接模式对组织折叠的重要性 选择性地切断网络中的配置。我们的方法可以确定一种新的合作单位 在细胞和组织之间，细胞同步的尺度。第二，RhoA信号如何影响 我们将研究跨组织的细胞相互作用。Rho相关螺旋卷曲蛋白激酶(ROCK)可被激活 肌球蛋白直接通过磷酸化或间接通过抑制肌球蛋白磷酸酶的磷酸化 (MP)。为了测试ROCK和MP之间的平衡决定肌球蛋白网络连通性的假设， MP将在不同的水平上被结构性激活，使其活动与岩石监管脱钩。这 技术产生了一种表型制度，由此网络被破坏的程度各不相同。全局MP TO 肌球蛋白网络调节所需的岩石活性，以及岩石在屏蔽肌球蛋白方面的局部作用 长丝的拆卸，将得到解决。这两个目标加在一起将形成一个基本框架 了解控制细胞如何相互作用以可复制地改变组织形状的一般规则。

Correct tissue shape is essential for proper tissue function and morphogenetic dysregulation results in many common congenital disorders. Yet, how groups of thousands or even hundreds of cells coordinate to yield stereotypic shape change through large-scale movements is still poorly understood. One way for cells to interact is through mechanical coupling. In fact, largescale networks of actomyosin connections between cells span developing tissues across various model organisms. The highly reproducible developmental program and powerful genetic tool-kit of Drosophila makes the Drosophila ventral furrow an ideal system for studying such networks. During furrow formation cells coordinate pulsed constrictions to yield tissue-wide bending. The tissue possesses a dynamic myosin network which fully forms prior to folding. Little is known, however, how mechanical information in the network guides collective constriction. This proposal will address how a network of mechanical connections is established to drive tissue folding. First, how a 2D network of intercellular connections promotes epithelial folding will be established. A novel approach, adapting methods from both astronomy and the mathematics of network theory will map the previously unquantifiable myosin network across hundreds of cells in a developing tissue. Preliminary studies have identified an initial growth phase and a subsequent contractile phase in the network. Growth Phase: We hypothesize that persistent network connections are established when neighboring cells simultaneously undergo a pulsed myosin accumulation. To test this hypothesis the position and timing of myosin recruitment will be coupled with the creation or reorganization network connections. Embryo injections inhibiting myosin pulsing will test the requirement of pulsing for network formation. Contractile Phase: We hypothesize that signatures in network geometry guide stereotypic tissue folding. Tissue-wide connectivity will be correlated with regions of coordinated cell constriction. Laser cutting will test the importance of connectivity patterns for tissue folding by selectively severing configurations in the network. Our approach could identify a novel unit of cooperation between the cell and the tissue scale over which cells synchronize. Second, how RhoA signaling influences cell interactions across a tissue will be investigated. Rho-associated coiled-coil kinase (ROCK) can activate myosin directly through phosphorylation or indirectly via inhibitory phosphorylation of myosin phosphatase (MP). To test the hypothesis that the balance between ROCK and MP dictates myosin network connectivity, MP will be constitutively activated at varying levels uncoupling its activity from ROCK regulation. This technique yields a phenotypic regime whereby the network is disrupted with varying severity. The global MP to ROCK activity required for myosin network regulation, as well as the local role of ROCK in shielding myosin filaments from disassembly, will be addressed. Taken together the two aims will form a foundational framework to understand general rules that govern how cells interact to reproducibly change tissue shape.