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 通过大规模运动产生刻板形状变化仍然知之甚少。细胞的一种方式 相互作用是通过机械耦合。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。高度可重复的开发计划和 果蝇强大的遗传工具包使果蝇腹沟成为研究此类疾病的理想系统 网络。在沟槽形成过程中，细胞协调脉冲收缩以产生组织范围的弯曲。组织 具有在折叠之前完全形成的动态肌球蛋白网络。然而，鲜为人知的是，如何 网络中的机械信息引导集体收缩。该提案将解决如何 建立机械连接网络来驱动组织折叠。首先，2D网络如何 细胞间连接促进上皮折叠的建立。新颖的方法，调整方法 天文学和网络理论的数学将绘制以前无法量化的肌球蛋白 网络跨越发育组织中的数百个细胞。初步研究已确定初步增长 阶段和随后的网络收缩阶段。成长阶段：我们假设持续 当相邻细胞同时经历脉冲肌球蛋白时建立网络连接 积累。为了检验这一假设，肌球蛋白募集的位置和时间将与 创建或重组网络连接。胚胎注射抑制肌球蛋白脉冲将测试 网络形成的脉冲要求。收缩阶段：我们假设网络中的签名 几何引导定型组织折叠。组织范围的连接将与区域相关 协调的细胞收缩。激光切割将测试组织折叠连接模式的重要性 有选择地切断网络中的配置。我们的方法可以确定一个新的合作单位 细胞和组织尺度之间的细胞同步。二、RhoA信号如何影响 将研究跨组织的细胞相互作用。 Rho 相关卷曲螺旋激酶 (ROCK) 可以激活 肌球蛋白直接通过磷酸化或间接通过肌球蛋白磷酸酶的抑制性磷酸化 （议员）。为了检验 ROCK 和 MP 之间的平衡决定肌球蛋白网络连接的假设， MP 将在不同水平上被组成性激活，使其活性与 ROCK 调节脱钩。这个 技术产生了一种表型状态，其中网络受到不同严重程度的破坏。全球议员 肌球蛋白网络调节所需的 ROCK 活性，以及 ROCK 在保护肌球蛋白中的局部作用 拆卸时产生的细丝将得到解决。这两个目标结合起来将形成一个基本框架 了解控制细胞如何相互作用以可重复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.