> Stages de M2 > stages proposés pour l’année 2019-2020 > Functional analysis of Shroom proteins during nephric morphogenesis in (...)

Functional analysis of Shroom proteins during nephric morphogenesis in Xenopus

proposé par Mélanie Paces-Fessy, Sorbonne Université – IBPS UMR7622 Laboratoire de Biologie du développement 75005 PARIS

Projet de stage : Morphogenesis of xenopus pronephros is currently poorly understood, especially the initial condensation and epithelialization stages. Shrooms belong to a family of four genes all expressed in the developing pronephros from specification of the pronephric field to tubule differentiation. The M2 project described below is organized in two main axis. Axis 1) Detailed expression profiles In order to explore subcellular dynamics of the proteins and their potential polarized expression, tagged shroom1-4 will be expressed in the developing pronephros during condensation and epithelialization steps (Chu et al., 2018). Cellular expression of the tagged Shrooms proteins will be compared to apico-basal epithelial cell polarity and adherent junctions markers using immunofluorescence staining. In parallel, we will look for antibodies cross-reacting with the xenopus Shroom proteins in order to localize the endogenous proteins. Axis 2) Functional analysis of Shrooms The roles of shroom proteins will be studied by loss and gain of function approaches with a set of tools (dominant negative form, morpholinos, tagged proteins…) already available. Effects on condensation and epithelialization of the pronephric field will be evaluated by visualization of cells along with staining of cytoskeletal components, adherent junctions markers, ECM proteins in order to analyze cell polarity, and cell shape. Preliminary results show an alteration of cell shape in mesenchyme with mislocalized laminin expression, resulting in an abnormal nephron. These preliminary results strongly support the feasibility of the project. This project will bring new knowledge on how Shroom proteins contribute at the cellular level to the establishment of kidney shape and function.

Techniques mises en œuvre par le stagiaire :
  visualization of cells and Shroom localization after injection of mRNA-tag in embryo (tagged Shrooms proteins, expression of GFP targeted to the plasma membrane and of H2b-mCherry in nuclei)
  immunofluorescence staining against apico-basal epithelial cell polarity proteins and adhérent junctions markers (ZO1, Ecad, aPKC, Rab11, cat, -tubulin…), cytoskeletal components ( and  tubulin, pMLC, MyoII, F-actin) and ECM proteins (laminin, fibronectin, collagen…).
  Injection of morpholino and dominant negative form in 4 cells stage embryos
  In situ hybridization against specialized nephric markers at different stages of development.

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