Maintenance of tissue integrity during organ growth, development and repair

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Our recent unpublished work using high spatiotemporal resolution in vivo live-imaging studies of vascular development indicates that post-mitotic reassembly of cell-cell junctions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.