Role of Rhophilin2, a RhoB GTPase effector, in zebrafish reproduction

Supervisor: Dr. Isabelle Pirson
Collaborator: Dr. Pascale Lybaert

RhoGTPases regulate cytoskeleton dynamics and vesicular trafficking and play key roles early embryo development. Rhophilin-2 (RHPN2), a RhoB effector, is overexpressed in various human cancers and promotes their survival but less is known about its molecular mechanisms of action in normal physiology. Expression of RHPN2 mRNAs has been initially described in mouse brain, thyroid, testis, ovary and lung and in secretory epithelia in human. We have shown that RHPN2 has a link with vesicular trafficking and identified proteic partners involved in extracellular vesicles synthesis. Interestingly, we recently identified a tetraspanin, involved in exosome generation, and a protein playing an important role in apoptosis as new Rhpn2 proteic partners. Zebrafish has emerged as a major model for studying developmental processes.

Zebrafish KO lines were established by CRISPR/Cas9 and two stable mutant lines were raised. During generation of our CRISPR/Cas9 lines, we observed that rhpn2-/- individuals obtained by heterozygous in-cross, were in mendelian ratio, grew till adulthood without any apparent defects but were affected in their spawning success and eggs development. Young rhpn2-/- adult females presented with a severe abdomen hyperplasia due to abnormal egg accumulation early in age, an interesting trait that could be related to laying efficiency or oocytes proliferation. The objective of the present project is, using zebrafish as a model, to evaluate the role of RHPN2 in gonads and eraly embryo development.

We propose:

  • To perform H/E staining to assess the morphology of paraffin-embedded ovaries in KO and wt females (1-5 mpf) and to quantify the oocytes at the four stages of zebrafish oogenesis. Histology of testis of KO and wt males will also be performed.
  • To analyze gene expression modulation during gonads development and oogenesis will be measured by bulk RNAseq analysis on fish gonads at different ages to determine which pathway is affected by Rhpn2 deficiency. We could further obtain See-thru-Gonad fish (Bodø, Norway) to purify primordial germ cells thanks to fluorescence form younger fish or observe the impact of Rhpn2 deficiency on fluorescent gonads development.
  • To study a detailed distribution profile of gene expression in vivo in specific cell populations during developmental stages and in adult fish using an already generated rhpn2 reporter transgenic line expressing a fluorescent gene (dTomato) under the control of the minimal promoter (1.5kb) of rhpn2. These data will be compared with qPCR results that will be obtained on fish organs or with single-cell RNA seq databeses. In case the expression of the reporter or the transmission rate is not successful (too low expression due to incomplete promoter), we will take advantage of our very efficient CRISPR guides (>95%) to generate KI fish. In these KI zebrafish lines the coding sequence of rhpn2 gene will be replaced by a GFP reporter gene. This will be done using the small homology directed repair system (Gene-Weld) giving high efficiency recombination.

We will perform immunostaining analysis of PAF-fixed KO eggs compared to wt, using antibodies against F-actin, β-catenin, dynamin, clathrin, Rab11 to unfold the potential implication of Rhpn2 in cytoskeleton organization of the cleavage furrow in the dividing egg to analyze early embryonic development.

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