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Muriel PERRON, Pauline Cabochette, Guillermo Vega-Lopez, Juliette Bitard, Karine Parain, Christel Masson, Marie Hedderich, Kristine Henningfeld, Morgane Locker, Odile Bronchain; Maintenance of retinal stem cells: the role of YAP, a downstream effector of the Hippo pathway, in DNA replication timing and genomic stability. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1687.
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© ARVO (1962-2015); The Authors (2016-present)
The adult frog retina retains a reservoir of active neural stem cells in the ciliary margin that contribute to life-long growth of the eye as well as to regeneration in case of injury. It thus represents an ideal model for dissecting molecular cues underlying retinal stem cell (RSC) properties in vivo. Such knowledge is essential for the development of innovative therapeutic strategies based on the mobilization of endogenous RSCs for tissue repair to cure retinal dystrophies. In this context, we focused our interest on the effector of the Hippo pathway, YAP, which recently emerged as a crucial player of adult tissue homeostasis. Since its function in adult neural stem cells is presently unknown, we investigated whether YAP is involved in the maintenance of an active pool of RSCs in the continuously growing frog eye.
To address the function of YAP in post-embryonic RSCs, we set up in Xenopus an inducible knock-down technique using photo-cleavable Morpholinos. We then undertook powerful experimental approaches dedicated to the dynamic exploration of cell proliferation in vivo (mitotic index, percentage of labeled mitosis and EdU cumulative labeling measurements). Replication foci, DNA damage and cell death were explored using immunolabeling/TUNEL assay and confocal microscopy. Impact on cell cycle gene expression was investigated using the NanoString technology. The interaction between YAP and a potential novel partner, PKNOX1, was examined by two-hybrid assay, co-immunoprecipitation and bimolecular fluorescent complementation (BiFC) assay.
We found that YAP is specifically expressed in RSCs and required for post-embryonic retinal growth. YAP knock-down leads to an accelerated S-phase, a phenotype likely mediated by upregulation of c-Myc. This is associated with an increased occurrence of DNA damage and eventually p53-p21 pathway-mediated cell death. Finally, we identified PKNOX1, a transcription factor involved in the maintenance of genomic stability, as a functional and physical interactant of YAP.
Our work suggests a novel role for YAP in stem cell proliferation regulation. We indeed propose a model where YAP would ensure DNA quality control and maintain genomic stability in RSCs by regulating the temporal firing of replication origins throughout S-phase.
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