Retinal stem cells activity in neural tissue growth and repair – EYE-STEM

Studying the retina in Xenopus allows in vivo experimental approaches, which are much more delicate to perform in mammals. Of note, the cell organisation and the embryology of the Xenopus retina is very similar to that of all other vertebrates. We plan to undertake mainly experimental embryology approaches coupled to cellular and molecular biology techniques to uncover the impact of gain or loss of function of a given gene on retinal stem cell behaviour. Another approach we have chosen relies on transgenesis techniques. Xenopus transgenic models that we will generate will allow us to monitor the cellular events that take place during retinal tissue repair. We will then study the influence of various signalling pathways on regeneration efficiency.

We have already uncovered the role of several signalling pathways, as well as their interactions, in retinal stem cell proliferation control, in a physiological context. This work contributes to a better understanding of the molecular network underlying retinal stem cell behaviour and therefore constitutes important knowledge to set up rational therapeutic strategies.

In addition, our large scale studies led us to propose a « genetic signature » of retinal stem cells, thanks to the identification of 18 novel specific markers of these cells. This constitutes precious tools to discriminate stem and progenitor cells in vivo.

We have undertaken a functional study of one of these genes and showed that it confers stemness properties to retinal stem cells. We also highlighted some links between this gene and the signalling network mentioned above. This therefore also contributes to a better understanding of the molecular network controlling retinal stem cell potential.

Finally, we have generated Xenopus transgenic animals with induced retinal degeneration. We now plan to study the impact of the signalling network mentioned above, this time in a pathological context, and thereby investigate its importance for retinal regeneration process.

The discovery of quiescent retinal stem cells in human retina has opened new avenues for regenerative medicine and the fight against blindness. A better knowledge of genetic interactions governing retinal stem cell proliferation and potential is a prerequisite for safe therapeutic application. Our work on retinal stem cells in a species that is able to regenerate its retina is thus of prime importance. Our future work should continue to increase our knowledge on the molecular mechanisms governing the activity of these cells.

During the first part of the project we published 3 articles on our results in international peer reviewed journals (Development, Stem Cells et Developmental Neurobiology). We have also written two book chapters aiming at reviewing the scientific literature in the field.

The recent identification of neural stem cells in mammals has raised the possibility that cell-based therapies might be efficient strategies to treat a wide range of neurological disorders. Comprehensive analysis of stem cell properties is also of utmost importance in cancerology due to their high similarities with some types of tumour cells, the cancer stem cells. However, the successful therapeutic exploitation of these cells primarily requires achievements in fundamental research. Indeed, key questions regarding the molecular mechanisms controlling retinal stem cell behaviour remain to be answered. In this context, our research program focuses on neural stem cells in Xenopus retina as a model. The amphibian retina contains a population of neural stem cells, which, in contrast to the mammalian situation, are active, allowing continuous tissue growth throughout the animal life, as well as regeneration following retinal damage. In addition, amphibian retinal stem cells reside in a defined niche localized at the margin of the retina. Last but not least, Xenopus is highly suitable for in vivo analyses and large-scale strategies. Thus, our model offers an exceptional tool to study in vivo the complex genetic network operating in neural stem cells.

First, we will contribute to the cellular and molecular characterization of retinal stem cells. Their embryonic origin remains to be investigated. Their cell cycle parameters are unknown. Finally, only very few specific markers have been identified so far, precluding their comprehensive in vivo analysis. Thus, we decided to fill these gaps through (i) a lineage analysis of adult retinal stem cells using transgenic animals; (ii) an in vivo estimation of their cell cycle length and duration of their G1, S and G2/M phases; (iii) the identification of numerous novel markers through a large-scale in situ screen; and (iv) a global analysis of the transcript profile of these cells through a deep sequencing strategy.

Besides, the identification of intrinsic and extrinsic cues regulating retinal stem cells maintenance and proliferation is our prime concern. During the past few years, our laboratory and others have made significant progress to decipher the role of individual signalling pathways in retinal stem cell behaviour. However, how these signalling pathways work in concert to finely tune the balance between quiescence, proliferation and differentiation is still poorly understood. Our current work on the interactions between Wnt and Hedgehog led us to propose a model in which antagonistic relationships between these signalling pathways control the rate of neurogenesis in the post-embryonic retina. We now wish to investigate the underlying molecular mechanisms linking these pathways. In addition, we will extend our work to the BMP and Notch pathways to acquire a global view of the signalling network governing adult neural stem cell properties.

Another goal is to perform functional analyses of intrinsic factors retrieved from our large-scale approaches. We will also evaluate their interactions with key signalling pathways, in order to dissect the global regulatory network sustaining retinal stem cell maintenance and activity. We wish to prioritise genes encoding transcription factors but also RNA binding proteins, given our long lasting interest in this field. This part of the project should lead to original data on post-transcriptional regulations involved in neural stemness.

Finally, we plan to establish a model for retinal neuro-degenerative diseases based on conditional targeted cell ablation in transgenic animals. This model should allow to uncover the molecular cues influencing stem cell behaviour in the context of neural repair.