Unlike mammals, some nonmammalian species recruit Müller glia for retinal regeneration after injury. Identifying the underlying mechanisms may help to foresee regenerative medicine strategies. Using a Xenopus model of retinitis pigmentosa, we found that Müller cells actively proliferate upon photoreceptor degeneration in old tadpoles but not in younger ones. Differences in the inflammatory microenvironment emerged as an explanation for such stage dependency. Functional analyses revealed that enhancing neuroinflammation is sufficient to trigger Müller cell proliferation, not only in young tadpoles but also in mice. In addition, we showed that microglia are absolutely required for the response of mouse Müller cells to mitogenic factors while negatively affecting their neurogenic potential. However, both cell cycle reentry and neurogenic gene expression are allowed when applying sequential pro- and anti-inflammatory treatments. This reveals that inflammation benefits Müller glia proliferation in both regenerative and nonregenerative vertebrates and highlights the importance of sequential inflammatory modulation to create a regenerative permissive microenvironment.

A growing wealth of data suggest that reactive oxygen species (ROS) signalling might be crucial in conferring embryonic or adult stem cells their specific properties. However, how stem cells control ROS production and scavenging, and how ROS in turn contribute to stemness, remain poorly understood. Using the Xenopus retina as a model system, we first investigated the redox status of retinal stem cells (RSCs). We discovered that they exhibit higher ROS levels compared with progenitors and retinal neurons, and express a set of specific redox genes. We next addressed the question of ROS functional involvement in these cells. Using pharmacological or genetic tools, we demonstrate that inhibition of NADPH oxidase-dependent ROS production increases the proportion of quiescent RSCs. Surprisingly, this is accompanied by an apparent acceleration of the mean division speed within the remaining proliferating pool. Our data further unveil that such impact on RSC cell cycling is achieved by modulation of the Wnt/Hedgehog signalling balance. Altogether, we highlight that RSCs exhibit distinctive redox characteristics and exploit NADPH oxidase signalling to limit quiescence and fine-tune their proliferation rate.

Nous constatons chez les étudiant·es de 1er cycle universitaire en sciences de la vie une passivité en cours comme en travaux dirigés (TD) et un apprentissage trop surfacique. Dans le but de les engager davantage et stimuler un apprentissage en profondeur, nous avons déployé un dispositif de classe inversée dans lequel les exposés magistraux ont été remplacés par un apprentissage du cours en autonomie (distanciel asynchrone), et les TD modifiés pour introduire du travail collaboratif. Nous avons ensuite interrogé les effets de ce dispositif sur l’engagement de nos étudiant·es. Afin de mener une analyse comparative contrôlée, le dispositif a été testé sur la moitié de la promotion (cohorte d’intérêt ; n=137) ; l’autre moitié a reçu un enseignement au format traditionnel (cohorte contrôle ; n=180). L’engagement comportemental, émotionnel, agentique et cognitif des étudiants a été mesuré via un questionnaire auto-rapporté, en considérant l’unité d’enseignement dans son ensemble, ou en distinguant deux situations très différentes : le cours et les TD. Nos données révèlent un bénéfice global du dispositif sur l’engagement émotionnel et cognitif des étudiant·es. En situation d’apprentissage de cours, l’engagement est accru dans les quatre dimensions interrogées. En TD, l’impact positif ne porte que sur la dimension émotionnelle.

In this paper, we present a feedback of active pedagogy with flipped teaching, carried out with undergraduate students in Life Sciences (second year of bachelor's degree; University Paris-Saclay, France). In a teaching context where biology programs are very busy, our students are subjected to significant cognitive overload, associated with difficulties to sort out and mobilise information, and a global lack of scientific methodological training. How combining the necessary assimilation of knowledge, vocabulary and basic concepts and the acquisition of higher-level cognitive skills (to describe, analyse, interpret, represent, model, link facts together ...)? More generally, how can we motivate students, empower them in their learning and boost their work? Facing these questions, our educational team has been experimenting for several years with active and reverse learning at Master level. We wanted to assay its applicability in a bachelor's degree, where the success of the greatest number of students is a major issue for the university, while constituting one of its main challenges (Annoot, 2012). With the aim of reinforcing motivation, volition and cognition (Houart, 2017), we designed and deployed for two consecutive years, a hybrid pedagogical device based on the alternation of distant assimilation phases and face-to-face applications (Charlier et al., 2006). Analysis of its benefits, carried out through questionnaires submitted to students, indicates a very strong support on their part to the process, increased motivation to work and a feeling of methodological progress.

Glaucoma is a multifactorial neurodegenerative disease characterized by the progressive and irreversible degeneration of the optic nerve and retinal ganglion cells. Despite medical advances aiming at slowing degeneration, around 40% of treated glaucomatous patients will undergo vision loss. It is thus of utmost importance to have a better understanding of the disease and to investigate more deeply its early causes. The transcriptional coactivator YAP, an important regulator of eye homeostasis, has recently drawn attention in the glaucoma research field. Here we show that Yap conditional knockout mice (Yap cKO), in which the deletion of Yap is induced in both Müller glia (i.e. the only retinal YAP-expressing cells) and the non-pigmented epithelial cells of the ciliary body, exhibit a breakdown of the aqueous-blood barrier, accompanied by a progressive collapse of the ciliary body. A similar phenotype is observed in human samples that we obtained from patients presenting with uveitis. In addition, aged Yap cKO mice harbor glaucoma-like features, including deregulation of key homeostatic Müller-derived proteins, retinal vascular defects, optic nerve degeneration and retinal ganglion cell death. Finally, transcriptomic analysis of Yap cKO retinas pointed to early-deregulated genes involved in extracellular matrix organization potentially underlying the onset and/or progression of the observed phenotype. Together, our findings reveal the essential role of YAP in preserving the integrity of the ciliary body and retinal ganglion cells, thereby preventing the onset of uveitic glaucoma-like features.

Regenerative abilities are not evenly distributed across the animal kingdom. The underlying modalities are also highly variable. Retinal repair can involve the mobilization of different cellular sources, including ciliary marginal zone (CMZ) stem cells, the retinal pigmented epithelium (RPE), or Müller glia. To investigate whether the magnitude of retinal damage influences the regeneration modality of the Xenopus retina, we developed a model based on cobalt chloride (CoCl2 ) intraocular injection, allowing for a dose-dependent control of cell death extent. Analyses in Xenopus laevis revealed that limited CoCl2 -mediated neurotoxicity only triggers cone loss and results in a few Müller cells reentering the cell cycle. Severe CoCl2 -induced retinal degeneration not only potentializes Müller cell proliferation but also enhances CMZ activity and unexpectedly triggers RPE reprogramming. Surprisingly, reprogrammed RPE self-organizes into an ectopic mini-retina-like structure laid on top of the original retina. It is thus likely that the injury paradigm determines the awakening of different stem-like cell populations. We further show that these cellular sources exhibit distinct neurogenic capacities without any bias towards lost cells. This is particularly striking for Müller glia, which regenerates several types of neurons, but not cones, the most affected cell type. Finally, we found that X. tropicalis also has the ability to recruit Müller cells and reprogram its RPE following CoCl2 -induced damage, whereas only CMZ involvement was reported in previously examined degenerative models. Altogether, these findings highlight the critical role of the injury paradigm and reveal that three cellular sources can be reactivated in the very same degenerative model.

Retinitis pigmentosa is an inherited retinal dystrophy that ultimately leads to blindness due to the progressive degeneration of rod photoreceptors and the subsequent non-cell autonomous death of cones. Rhodopsin is the most frequently mutated gene in this disease. We here developed rhodopsin gene editing-based models of retinitis pigmentosa in two Xenopus species, Xenopus laevis and Xenopus tropicalis, by using CRISPR/Cas9 technology. In both of them, loss of rhodopsin function results in massive rod cell degeneration characterized by progressive shortening of outer segments and occasional cell death. This is followed by cone morphology deterioration. Despite these apparently similar degenerative environments, we found that Müller glial cells behave differently in Xenopus laevis and Xenopus tropicalis. While a significant proportion of Müller cells re-enter into the cell cycle in Xenopus laevis, their proliferation remains extremely limited in Xenopus tropicalis. This work thus reveals divergent responses to retinal injury in closely related species. These models should help in the future to deepen our understanding of the mechanisms that have shaped regeneration during evolution, with tremendous differences across vertebrates.

Glaucoma is an optic neuropathy often referred to as “the silent thief of sight”, due to its late diagnosis, which is generally made when degeneration of the optic nerve and retinal ganglion cells is already well under way. It is thus of utmost importance to have a better understanding of the disease, and to investigate more deeply the early causes of glaucoma. The transcriptional coactivator YAP recently emerged as an important regulator of eye homeostasis and is drawing attention in the glaucoma research field. Here we show that Yap conditional knockout mice ( Yap cKO), in which the deletion of Yap is induced in both Müller glia ( i.e . the only retinal YAP-expressing cells) and the non-pigmented epithelial cells of the ciliary body, exhibit breakdown of the aqueous-blood barrier accompanied by progressive collapse of the ciliary body as we observed in human uveitic patients. In addition, aged Yap cKO mice harbor glaucoma features, including alteration of glutamate recycling, deregulation of key homeostatic Müller-derived proteins, retinal vascular defects, optic nerve degeneration, and retinal ganglion cell death. Together, our findings reveal the essential role of YAP in preserving the ciliary body and the retinal ganglion cells, thereby preventing the onset of glaucoma features.

Retinal regeneration efficiency from Müller glia varies tremendously among vertebrate species, being extremely limited in mammals. Efforts towards the identification of molecular mechanisms underlying Müller cell proliferative and neurogenic potential should help finding strategies to awake them and ensure regeneration in mammals. We provide here an update on the most recent and original progresses made in the field. These include remarkable discoveries regarding (i) unprecedented cross-species comparison of Müller cell transcriptome using single-cell technologies, (ii) the identification of new strategies to promote both the proliferative and the neurogenic potential of mammalian Müller cells, (iii) the role of the epigenome in regulating Müller glia plasticity, (iv) miRNA-based regulatory mechanisms of Müller cell response to injury, and (v) the influence of inflammatory signals on the regenerative process.

Cell cycle progression is intimately linked to cell fate commitment during development. In addition, adult stem cells show specific proliferative behaviors compared to progenitors. Exploring cell cycle dynamics and regulation is therefore of utmost importance, but constitutes a great challenge in vivo. Here we provide a protocol for evaluating in vivo the length of all cell cycle phases of neural stem and progenitor cells in the post-embryonic Xenopus retina. These cells are localized in the ciliary marginal zone (CMZ), a peripheral region of the retina that sustains continuous neurogenesis throughout the animal's life. The CMZ bears two tremendous advantages for cell cycle kinetics analyses. First, this region, where proliferative cells are sequestered, can be easily delineated. Second, the spatial organization of the CMZ mirrors the temporal sequence of retinal development, allowing for topological distinction between retinal stem cells (residing in the most peripheral margin), and amplifying progenitors (located more centrally). We describe herein how to determine CMZ cell cycle parameters using a combination of (i) a cumulative labeling assay, (ii) the percentage of labeled mitosis calculation, and (iii) the mitotic index measurement. Taken together, these techniques allow us to estimate total cell cycle length (TC) as well as the duration of all cell cycle phases (TS/G2/M/G1). Although the method presented here was adapted to the particular system of the CMZ, it should be applicable to other tissues and developmental stages as well.

A striking aspect of tissue regeneration is its uneven distribution among different animal classes, both in terms of modalities and efficiency. The retina does not escape the rule, exhibiting extraordinary self-repair properties in anamniote species but extremely limited ones in mammals. Among cellular sources prone to contribute to retinal regeneration are Müller glial cells, which in teleosts have been known for a decade to re-acquire a stem/progenitor state and regenerate retinal neurons following injury. As their regenerative potential was hitherto unexplored in amphibians, we tackled this issue using two Xenopus retinal injury paradigms we implemented: a mechanical needle poke injury and a transgenic model allowing for conditional photoreceptor cell ablation. These models revealed that Müller cells are indeed able to proliferate and replace lost cells following damage/degeneration in the retina. Interestingly, the extent of cell cycle re-entry appears dependent on the age of the animal, with a refractory period in early tadpole stages. Our findings pave the way for future studies aimed at identifying the molecular cues that either sustain or constrain the recruitment of Müller glia, an issue of utmost importance to set up therapeutic strategies for eye regenerative medicine.

The adult frog retina retains a reservoir of active neural stem cells that contribute to continuous eye growth throughout life. We found that Yap, a downstream effector of the Hippo pathway, is specifically expressed in these stem cells. Yap knock-down leads to an accelerated S-phase and an abnormal progression of DNA replication, 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. Altogether, we propose that YAP is required in adult retinal stem cells to regulate the temporal firing of replication origins and quality control of replicated DNA. Our data reinforce the view that specific mechanisms dedicated to S-phase control are at work in stem cells to protect them from genomic instability.

The ability to regenerate damaged cells in the retina varies tremendously among species, being restricted for most of them to specific developmental stages. Regarding vertebrates, only the newt was thought to exhibit full regenerative capacity upon retinectomy in the adulthood. The recent discovery that the anuran amphibian Xenopus can regenerate its retina after metamorphosis opened new avenues to investigate the cellular and molecular mechanisms involved in this process. In this review, we provide an historical overview of regeneration studies in Xenopus. Particular emphasis is given to the cellular sources contributing to retinal replacement, the involvement of tissue interactions and the importance of the injury paradigm. We also describe recent progress and promises in the field brought by the development of 3D tissue culture methods and transgenic Xenopus models.

In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis.