Maintenance of tissue integrity during organ growth, development and repair

在组织发育、生长和修复过程中，几乎所有的新血管都是由血管生成产生的。此外，血管生成的不平衡导致许多疾病状态，包括失明和癌症。我们最近的研究表明，血管生成是由不对称内皮细胞（EC）分裂引导的，这种分裂产生了协调分支过程的不同“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关键特征，但它也是分裂和分离子细胞的固有破坏性过程。因此，子细胞-细胞连接的快速重建对于维持维管组织的完整性和集体运动至关重要。然而，子细胞如何在分裂后快速重建细胞间连接以防止组织破坏尚不清楚。我们最近未发表的研究使用高时空分辨率的血管发育活体成像研究表明，细胞-细胞连接的有丝分裂后重组涉及连接重组位点的肌动蛋白的快速重塑。利用一种新的斑马鱼转基因系，在脉管系统中表达gfp标记的联系，我们发现这种肌动蛋白调节因子在EC分裂后动态地迁移到新生连接形成的位点。特别是，内层堆积发生在体中部细胞质分裂后立即发生，在子细胞-细胞连接的显著“拉链化”之前，重建组织完整性。此外，一旦子细胞-细胞连接重新组装，这种接触积累就会分散。这些观察结果导致有丝分裂后连接重组的模型，其中子细胞界面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。因此，有丝分裂后的活化和接触蛋白的重新定位，以及随后的局部肌动蛋白重塑，可能对器官形成过程中组织完整性的维持至关重要。为了确定有丝分裂后肌动蛋白重塑与组织完整性维持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收缩中的作用：利用现有的斑马鱼转基因工具在高时空分辨率下监测活体肌动球蛋白动力学，我们将首先定义有丝分裂后连接重组过程中肌动蛋白重塑位点的时间分辨率理解。我们还将创建一个新的体内发色团辅助光失活（CALI）光遗传学工具工具包，该工具将独特地实现有丝分裂后关键肌动球蛋白组分的精确时空失活。将这些光遗传学工具与体内活细胞成像相结合，我们将确定肌动蛋白重塑的扰动如何破坏有丝分裂后连接重组。(2)确定启动有丝分裂后连接“拉链”的信号和机械线索：为了确定触发有丝分裂后连接激活、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初始钙粘蛋白在中体附近的接合和激活是否触发了这一过程。其次，使用工程微模式，我们将确定子细胞的空间限制是否对连接重组至关重要。(3)定义连接拉链在维持组织完整性中的作用：使用上述方法干扰有丝分裂后连接重组，我们将定义这种现象在维持维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导致有丝分裂后子细胞的脱离，无序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cells rapidly rebuild their cell-cell junctions following division to prevent tissue disruption is unclear.Our recent unpublished work using high spatiotemporal resolution in vivo live-imaging studies of vascular development indicates that post-mitotic reassembly of cell-cell junctions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.