Hippo pathway-mediated regulation of micropyle formation by microRNA 202 (miR-202) in the fish oocyte – MicroHippo
In medaka (Oryzias latipes), a model fish species for reproduction, we have recently showed that the lack of miR-202 results in a dramatic decrease of female fertility; a remarkably strong phenotype that is associated with reduced fertilization rate in mutant eggs. While underlying mechanisms are currently unknown, we have gathered preliminary evidence indicating that reduced fertilization rate is associated with abnormal micropyle (a particular structure that functions as a channel to guide sperm to the oocyte during fertilization). While micropyle is present in mutant mir-202 -/- eggs, scanning electron microscopy data have showed that it is not functional and not fully “open” to allow sperm entry. In fish, micropyle formation has been traced back to a single cell of the granulosa cell layer (i.e. somatic cells surrounding the oocyte within the ovarian follicle), the micropylar precursor cell (MPC), that differentiates during oogenesis at the animal pole of the oocyte. MPC differentiation is under the control of the Hippo pathway and results from the accumulation of high levels of transcriptional co-activator Taz, the Hippo pathway effector, in the nucleus. Recent genome editing studies in fish have shown that Taz is required for MPC differentiation and micropyle formation. We have also obtained preliminary evidence showing that several genes of the Hippo pathway, including taz, mst1 (the vertebrate ortholog of drosophila hippo) and mob1b, are dysregulated in mir-202 -/- ovary. Several members of the Hippo pathway, including nf2b (the vertebrate ortholog of drosophila merlin) are also in silico predicted to be targeted by miR-202.
The objective of the project is therefore to understand how miR-202 regulates MPC differentiation (and ultimately micropyle formation) through the modulation of the Hippo pathway. Our working hypothesis is that miR-202 is targeting directly or indirectly the Hippo pathway ultimately leading to dysregulation of MPC differentiation and abnormal micropyle formation.
The project will consist in 4 overlapping tasks. We will first further describe the “closed micropyle” phenotype using scanning electron microscopy and serial block-face scanning electron microscopy and perform a thorough histological analysis of abnormal MPC differentiation in growing ovarian follicles in the mir-202 -/- model. A comprehensive a molecular 3D phenotyping of MPC differentiation will be carried out in mir-202 -/- mutants using both whole mount in situ hybridization and fluorescent immunodetection to analyze Hippo pathway players, including Taz, as well as specific proteins involved in MPC differentiation in fish. In order to identify new players involved in the regulation of MPC differentiation, a spatially resolved transcriptomic analysis will be performed.
In order bridge the gap between miR-202 and MPC differentiation, we will aim at identifying the direct target(s) of miR-202, which is the most difficult and critical point when investigating the in vivo action of a specific miRNA. We will identify genes that are in silico predicted to be targets of miR-202 and have a low inter-individual expression variability in the MPC region. Gene exhibiting these features are expected to be credible targets of miR-202, given that in vivo action of miRNAS only results in modest differences in mRNA levels. The in vivo relevance of identified target(s) will subsequently be validated using a genome-editing based strategy aiming at disrupting miR-202 binding sites in the 3’ UTR region, that is considered the most definitive approach for isolating and confirming the importance of a particular target. Disrupting miR-202 target site of direct biological targets is expected to result in a phenotype similar to mir-202 knock out. Finally, information obtained in the MicroHippo project will be used to provide a working model of Hippo pathway mediated miR-202 regulation of MPC differentiation and micropyle formation.
Monsieur Julien BOBE (Laboratoire de Physiologie et Génomique des Poissons)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
IGH Institut de Génétique Humaine
LPGP Laboratoire de Physiologie et Génomique des Poissons
Help of the ANR 406,339 euros
Beginning and duration of the scientific project: December 2021 - 48 Months