ALPHA-CATENIN ROLES · Alpha-Catenin regulatory properties and functions during Zebrafish development

Alpha-catenin regulatory properties and functions during zebrafish development

A fundamental question in biology is how an embryo starting from one cell is able to organise the structure of the whole body. This process requires cell division, the specification of certain kind of cell and finally the organisation in functional tissue and organs. A critical role on building up the body in tissue and organ is played by protein involved in cell-cell adhesion that mediated the integrity of the embryonic and adult tissue. Regulation of cell-cell adhesion is also necessary to allow cells to migrate and reach a certain destination during development. In fact, it is very common the cells are generated in locations different from where they should carry out their function. Defects in cell-cell adhesion can lead to pathology like cancer where cancer cells lost contact with neighbouring cells and start to migrate generating metastatic tumours. Key molecular complexes that mediate cell-cell adhesion are the Adherens junction (AJ)s. The main components of the AJs are the classical cadherins. The classical cadherins are transmembrane proteins involved in homotypic interaction with cadherins exposed on opposed cells and with the cellular domain that connects the plasma membrane to the actin cytoskeleton at the cell cortex. The key regulator of the connection between AJs to the actin cytoskeleton is the alpha-catenin protein. Alpha-catenin was considered just few years ago as a static molecular bridge between the AJs and the actin cytoskeleton. In the last 5 years, however, many scientific reports highlighted additional role for alpha-catenin and a more dynamics role in the way it regulates the connection between AJs and the actin cytoskeleton. All these new aspects were described in vitro leaving as open question if alpha-catenin plays similar role in vivo. For these reason, we decided to dissect the role of alpha-catenin during zebrafish early development. Zebrafish is an ideal system to perform live imaging and manipulated gene expression at early stage of development. Moreover, zebrafish gastrulation (a very early stage of embryo development) is a model system to study cell-cell adhesion and cell migration AJs dependent. In order to dissect the role of alpha-catenin, we used a lost of function approach injecting specific morpholinos (antisense oligo targeting mRNA) that interfere with the translation of the alpha-catenin protein. As for other organisms, depletion of alpha-catenin was lethal at early stage. However, the alpha-catenin depleted embryo survived post-gastrulation allowing us to examine the role of alpha-catenin during gastrulation. We were able to describe a number of defects that correlate with defective cell-cell adhesion and cell migration. The fellow decided to focus on some of the defects in order to understand the cellular dynamics that caused it. Using live imaging at single cell resolution, the fellow was able to highlight two different cellular behaviours that caused anomaly during gastrulaition. Both cellular behaviours were observed during the Radial intercalation (RI) of the embryonic cells. The RI is a process in which the embryonic tissue dramatically reorganised itself from many cell layers to only two cell layers. Depletion of alpha-catenin, as the depletion of other members of the AJs, impaired this process due to embryonic cells not being able to adhere to each other properly. Moreover, depletion of alpha-catenin caused an additional defect respect that consists in persistent plasma membrane blebbing. Additional analyses, of this behaviour in different genetic backgrounds, highlighted the importance of alpha-catenin in the maintenance of the connection between the cell cortex and the plasma membrane during these dynamic cellular processes. This work was published in the international journal 'Development' (Schepis et al., 2012) and further explanations of the result were also published in a commentary in the journal 'Cell adhesion and migration' (Schepis and Nelson, 2012). Finally, during these studies, to better understand the role of alpha-catenin and the cell cortex, the fellow performed experiments focusing on genetic interaction between alpha-catenin and proteins involved in cell cortex regulation such as Ezrin and Septins. An interesting candidate was Septin-7 protein. The fellow performed an initial characterisation of the phenotypes caused by the depletion of the septin7 protein by antisense morpholinos. The fellow observed defects in gastrulation and in the left-right asymmetry of the body plan. All these defects were related with defective cilia morphology. Part of this work is included in a manuscript in preparation. In conclusion, the fellow was able to successfully terminate the project focusing on alpha-catenin role during zebrafish gastrulation revealing new function in vivo. During the return phase, the fellow had exposure to zebrafish model system in the laboratory of Professor Clarke where he could strengthen his skill and knowledge in zebrafish methodology. He had the opportunity to perform live imaging during neurogenesis and analysed fish mutant for genes that could be involved in regulation of cell-cell adhesion (Numb and Numb-like gene). Finally, he had the chance to supervise an undergraduate student. Overall the return phase at King's College was very positive in term of interactions in the Clarke laboratory and in the MRC for Developmental Neurobiology.

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