Maintenance of tissue integrity during organ growth, development and repair

血管生成在组织发育、生长和修复过程中产生几乎所有的新血管。此外，血管生成的失衡导致了许多疾病状态，包括失明和癌症。我们最近的工作揭示了血管生成是由不对称的内皮细胞(EC)分裂引导的，它产生了协调分支过程的不同的“领导者”和“追随者”身份的子细胞。因此，细胞分裂是血管形态发生的一个重要特征，但它也是一个固有的分裂和分离子细胞的破坏性过程。因此，子代细胞-细胞连接的快速重建对于维持血管组织的完整性和集体运动至关重要。然而，子代细胞如何在分裂后快速重建其细胞-细胞连接以防止组织分裂尚不清楚。我们最近未发表的工作使用高时空分辨率的活体血管发育成像研究表明，细胞-细胞连接的有丝分裂后重组涉及连接重组部位肌动蛋白的快速重塑。利用一种新型的斑马鱼转基因品系，在血管系统中表达GFP标记的皮质肌动蛋白，我们发现这种肌动蛋白调节因子在EC分裂后动态地重新定位到新生连接形成的位置。特别是，皮质素的积累在中体细胞胞质分裂后立即发生，并先于重建组织完整性的子细胞-细胞连接的显著“拉链”。此外，一旦子代细胞-细胞连接重新组装，这种皮质蛋白的积聚就会分散。这些观察结果导致了一个有丝分裂后连接重组的模型，根据该模型，子细胞界面上的局部肌动蛋白重塑驱动极化突起，迫使子细胞之间接触并促进连接组装。为了确定有丝分裂后肌动蛋白重塑和维持组织完整性之间的精确相互关系，本项目将：(1)确定肌动蛋白重塑在有丝分裂后连接拉链中的作用：使用现有的斑马鱼转基因工具以高时空分辨率监测活体肌动蛋白的动态变化，我们将首先定义对连接重组过程中有丝分裂后肌动蛋白重塑位置的时间分辨的理解。我们还将创建一个新的工具包，体内生色团辅助的光失活(CALI)光遗传工具，将独特地使关键的肌动肌球蛋白成分在有丝分裂后的时空失活成为可能。将这些光遗传学工具与活体细胞成像相结合，我们将确定肌动蛋白重塑的扰动如何扰乱有丝分裂后连接重新组装。(2)识别启动有丝分裂后连接拉链的信号和机械信号：为了定义触发有丝分裂后皮质蛋白激活、招募和/或肌动蛋白重塑以启动连接拉链的关键信号，我们将探索两个假设。首先，我们将使用cdh5 CRISPR突变体和/或干扰钙粘蛋白功能的药理学工具来确定最初的钙粘蛋白参与和中体附近的激活是否触发了这一过程。其次，使用工程微模式，我们将确定子代细胞的空间限制是否对连接重组至关重要。(3)明确连接拉链在维持组织完整性中的作用：使用上面建立的方法干扰有丝分裂后连接重组，我们将确定这一现象在维持血管组织完整性中的重要性。特别是，我们将确定肌动蛋白重塑的扰动是否会导致有丝分裂后的子细胞分离、无组织的集体运动和新血管形成的破坏。

Angiogenesis generates almost all new blood vessels during tissue development, growth and repair. Furthermore, imbalances in angiogenesis contribute to numerous disease states, including blindness and cancer. Our recent work has revealed that angiogenesis is directed by asymmetric endothelial cell (EC) divisions, which generate daughter cells of the differential 'leader' versus 'follower' identity that coordinates the branching process. As such, cell division is a critical feature of vascular morphogenesis, but it also an inherently disruptive process that splits and separates daughter cells. As such, rapid reestablishment of daughter cell-cell junctions is critical to maintain vascular tissue integrity and collective movement. However, how daughter cells rapidly rebuild their cell-cell junctions following division to prevent tissue disruption is unclear.Our recent unpublished work using high spatiotemporal resolution in vivo live-imaging studies of vascular development indicates that post-mitotic reassembly of cell-cell junctions involves rapid remodelling of actin at sites of junction reassembly. Using a novel zebrafish transgenic line that expresses GFP-tagged cortactin in the vasculature, we find that this actin-regulator is dynamically relocated to sites of nascent junction formation following EC division. In particular, cortactin accumulation occurs immediately after cytokinesis at the midbody and precedes a notable 'zippering' of daughter cell-cell junctions that re-establishes tissue integrity. Moreover, as soon as daughter cell-cell junctions have reassembled, this cortactin accumulation disperses. These observations lead to a model for post-mitotic junction reassembly whereby local actin remodelling at the interface of daughter cells drives polarised protrusions that force contacts between daughter cells and facilitate junction assembly. As such, the post-mitotic activation and relocation of cortactin, followed by local actin remodelling, may be essential to the maintenance of tissue integrity during organ formation.To define the precise interrelationships between post-mitotic actin remodelling and maintenance of tissue integrity this project will:(1) Define the role of actin remodelling in post-mitotic junction zippering: Using existing zebrafish transgenic tools to monitor actomyosin dynamics live in-vivo at high spatiotemporal resolution, we will first define a time-resolved understanding of the sites of post-mitotic actin remodelling during junction reassembly. We will also create a novel toolkit of in vivo chromophore-assisted light inactivation (CALI) optogenetic tools that will uniquely enable precise post-mitotic spatiotemporal inactivation of key actomyosin components. Combining these optogenetic tools with the in vivo live cell imaging we will then determine how perturbation of actin remodelling disrupts post-mitotic junction reassembly.(2) Identify signals and mechanical cues that initiate post-mitotic junction zippering: To define the key signals that trigger post-mitotic cortactin activation, recruitment and/or actin remodelling to initiate junction 'zippering', we will explore two hypotheses. First, we will use cdh5 CRISPR mutants and/or pharmacological tools that perturb cadherin function to determine if initial cadherin engagement and activation near the midbody triggers this process. Second, using engineered micropatterns we will determine if spatial confinement of daughter cells is critical to junction reassembly. (3) Define the role of junction zippering in the maintenance of tissue integrity: Using approaches established above to perturb post-mitotic junction reassembly, we will define the importance of this phenomenon in maintaining vascular tissue integrity. In particular, we will determine if perturbation of actin remodelling leads to detachment of post-mitotic daughter cells, disorganised collective movement and disruption of new blood vessel formation.