Master-2 Project 2018-2019 : Functional analysis of Shroom proteins during pronephros morphogenesis in Xenopus

Chronic kidney disease (CKD) affects 750 million people around the world. Mutation of Shroom 3 has been identified in Human and mice associated with CKD but also neural tube closure defect. Shroom3 encodes an intracellular protein located at adherent junctions where it generates contractile forces by controlling the distribution of Myosin II downstream of the PCP pathway. It participates in the reorganization of actomyosin network during apical constriction, cell elongation and cell intercalation, and is therefore a central player in cellular remodeling of polarized epithelia. The role of Shroom3 during renal development is still poorly understood. The aim of this internship is to understand the molecular mechanisms involved in renal cell shape changes taking place during nephron morphogenesis using the xenopus pronephros as a model. Shrooms belong to a family of four genes all expressed in the developing pronephros. A set of molecular tools including morpholinos, dominant-negative, constitutive and tagged proteins has been previously used to study Shroom-dependent cellular processes during neural tube closure in xenopus and are available to study Shroom function during pronephros morphogenesis.

Morphogenesis of xenopus pronephros is currently poorly understood, especially the initial condensation and epithelialization stages. Thus, we propose to analyse cell polarity, cytoskeleton and cell shape by visualization of cells membrane, along with staining of cytoskeletal components, ECM proteins, cell polarity and adherent junctions markers. In parallel, we will perform a functional analysis of the role of Shrooms by a strategy of loss of function obtained by overexpression a truncated form of Shrooms corresponding to the actin binding domain (dominant negative form) or the use of morpholinos. Preliminary results show an alteration of cell shape in the condensed mesenchyme with mislocalized laminin expression, resulting in an abnormal nephron with increased proximal tubule size and interruptions in intermediate and distal tubule. These preliminary results strongly support the feasibility of the project.

Team Signalling et Morphogenesis, UMR7622, Laboratory of Developmental Biology, Institute of Biology Paris-Seine (IBPS)

Contact : Mélanie Paces-Fessymelanie.paces-fessy@upmc.fr , +33 (0)1 44 27 36 90.