Regeneration from three cellular sources and ectopic mini-retina formation upon neurotoxic retinal degeneration in Xenopus.

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.

Uveitic glaucoma-like features in Yap conditional knockout mice.

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.

Awakening adult neural stem cells: NOX signalling as a positive regulator of the quiescence-to-proliferation transition in the Xenopus retina.

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.

CRISPR/Cas9-Mediated Models of Retinitis Pigmentosa Reveal Differential Proliferative Response of Müller Cells between Xenopus laevis and Xenopus tropicalis.

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.

Update on Müller glia regenerative potential for retinal repair.

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.

In Vivo Assessment of Neural Precursor Cell Cycle Kinetics in the Amphibian Retina.

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.

Linking YAP to Müller Glia Quiescence Exit in the Degenerative Retina.

Contrasting with fish or amphibian, retinal regeneration from Müller glia is largely limited in mammals. In our quest toward the identification of molecular cues that may boost their stemness potential, we investigated the involvement of the Hippo pathway effector YAP (Yes-associated protein), which is upregulated in Müller cells following retinal injury. Conditional Yap deletion in mouse Müller cells prevents cell-cycle gene upregulation that normally accompanies reactive gliosis upon photoreceptor cell death. We further show that, in Xenopus, a species endowed with efficient regenerative capacity, YAP is required for their injury-dependent proliferative response. In the mouse retina, where Müller cells do not spontaneously proliferate, YAP overactivation is sufficient to induce their reprogramming into highly proliferative cells. Overall, we unravel a pivotal role for YAP in tuning Müller cell proliferative response to injury and highlight a YAP-EGFR (epidermal growth factor receptor) axis by which Müller cells exit their quiescence state, a critical step toward regeneration.

Müller glial cell reactivation in Xenopus models of retinal degeneration.

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.

YAP controls retinal stem cell DNA replication timing and genomic stability.

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.

Ascl1 as a novel player in the Ptf1a transcriptional network for GABAergic cell specification in the retina.

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.

Shaping the eye from embryonic stem cells: Biological and medical implications.

Organogenesis is regulated by a complex network of intrinsic cues, diffusible signals and cell/cell or cell/matrix interactions that drive the cells of a prospective organ to differentiate and collectively organize in three dimensions. Generating organs in vitro from embryonic stem (ES) cells may provide a simplified system to decipher how these processes are orchestrated in time and space within particular and between neighboring tissues. Recently, this field of stem cell research has also gained considerable interest for its potential applications in regenerative medicine. Among human pathologies for which stem cell-based therapy is foreseen as a promising therapeutic strategy are many retinal degenerative diseases, like retinitis pigmentosa and age-related macular degeneration. Over the last decade, progress has been made in producing ES-derived retinal cells in vitro, but engineering entire synthetic retinas was considered beyond reach. Recently however, major breakthroughs have been achieved with pioneer works describing the extraordinary self-organization of murine and human ES cells into a three dimensional structure highly resembling a retina. ES-derived retinal cells indeed assemble to form a cohesive neuroepithelial sheet that is endowed with the intrinsic capacity to recapitulate, outside an embryonic environment, the main steps of retinal morphogenesis as observed in vivo. This represents a tremendous advance that should help resolving fundamental questions related to retinogenesis. Here, we will discuss these studies, and the potential applications of such stem cell-based systems for regenerative medicine.

Hes4 controls proliferative properties of neural stem cells during retinal ontogenesis.

The retina of fish and amphibian contains genuine neural stem cells located at the most peripheral edge of the ciliary marginal zone (CMZ). However, their cell-of-origin as well as the mechanisms that sustain their maintenance during development are presently unknown. We identified Hes4 (previously named XHairy2), a gene encoding a bHLH-O transcriptional repressor, as a stem cell-specific marker of the Xenopus CMZ that is positively regulated by the canonical Wnt pathway and negatively by Hedgehog signaling. We found that during retinogenesis, Hes4 labels a small territory, located first at the pigmented epithelium (RPE)/neural retina (NR) border and later in the retinal margin, that likely gives rise to adult retinal stem cells. We next addressed whether Hes4 might impart this cell subpopulation with retinal stem cell features: inhibited RPE or NR differentiation programs, continuous proliferation, and slow cell cycle speed. We could indeed show that Hes4 overexpression cell autonomously prevents retinal precursor cells from commitment toward retinal fates and maintains them in a proliferative state. Besides, our data highlight for the first time that Hes4 may also constitute a crucial regulator of cell cycle kinetics. Hes4 gain of function indeed significantly slows down cell division, mainly through the lengthening of G1 phase. As a whole, we propose that Hes4 maintains particular stemness features in a cellular cohort dedicated to constitute the adult retinal stem cell pool, by keeping it in an undifferentiated and slowly proliferative state along embryonic retinogenesis.

Antagonistic cross-regulation between Wnt and Hedgehog signalling pathways controls post-embryonic retinal proliferation.

Continuous neurogenesis in the adult nervous system requires a delicate balance between proliferation and differentiation. Although Wnt/ß-catenin and Hedgehog signalling pathways are thought to share a mitogenic function in adult neural stem/progenitor cells, it remains unclear how they interact in this process. Adult amphibians produce retinal neurons from a pool of neural stem cells localised in the ciliary marginal zone (CMZ). Surprisingly, we found that perturbations of the Wnt and Hedgehog pathways result in opposite proliferative outcomes of neural stem/progenitor cells in the CMZ. Additionally, our study revealed that Wnt and Hedgehog morphogens are produced in mutually exclusive territories of the post-embryonic retina. Using genetic and pharmacological tools, we found that the Wnt and Hedgehog pathways exhibit reciprocal inhibition. Our data suggest that Sfrp-1 and Gli3 contribute to this negative cross-regulation. Altogether, our results reveal an unexpected antagonistic interplay of Wnt and Hedgehog signals that may tightly regulate the extent of neural stem/progenitor cell proliferation in the Xenopus retina.

A large scale screen for neural stem cell markers in Xenopus retina.

Neural stem cell research suffers from a lack of molecular markers to specifically assess stem or progenitor cell properties. The organization of the Xenopus ciliary marginal zone (CMZ) in the retina allows the spatial distinction of these two cell types: stem cells are confined to the most peripheral region, while progenitors are more central. Despite this clear advantage, very few genes specifically expressed in retinal stem cells have been discovered so far in this model. To gain insight into the molecular signature of these cells, we performed a large-scale expression screen in the Xenopus CMZ, establishing it as a model system for stem cell gene profiling. Eighteen genes expressed specifically in the CMZ stem cell compartment were retrieved and are discussed here. These encode various types of proteins, including factors associated with proliferation, mitotic spindle organization, DNA/RNA processing, and cell adhesion. In addition, the publication of this work in a special issue on Xenopus prompted us to give a more general illustration of the value of large-scale screens in this model species. Thus, beyond neural stem cell specific genes, we give a broader highlight of our screen outcome, describing in particular other retinal cell markers that we found. Finally, we present how these can all be easily retrieved through a novel module we developed in the web-based annotation tool XenMARK, and illustrate the potential of this powerful searchable database in the context of the retina.

Serotonergic 5-HT(2B) receptor controls tissue-nonspecific alkaline phosphatase activity in osteoblasts via eicosanoids and phosphatidylinositol-specific phospholipase C.

In previous studies, we observed that mice knocked out for the serotonin-2B receptor (5-HT(2B)R) show defects in bone homeostasis. The present work focuses on the downstream targets relaying the anabolic function of this receptor in osteoblasts. A functional link between the 5-HT(2B)R and the activity of the tissue-nonspecific alkaline phosphatase (TNAP) is established using the C1 osteoprogenitor cell line. During C1 osteogenic differentiation, both 5-HT(2B)R and TNAP mRNA translations are delayed with respect to extracellular matrix deposition. Once the receptor is expressed, it constitutively controls TNAP activity at a post-translational level along the overall period of mineral deposition. Indeed, pharmacological inhibition of the 5-HT(2B)R intrinsic activity or shRNA-mediated 5-HT(2B)R knockdown prevents TNAP activation, but not its mRNA translation. In contrast, agonist stimulation of the receptor further increases TNAP activity during the initial mineralization phase. Building upon our previous observations that the 5-HT(2B)R couples with the phospholipase A2 pathway and prostaglandin production at the beginning of mineral deposition, we show that the 5-HT(2B)R controls leukotriene synthesis via phospholipase A2 at the terminal stages of C1 differentiation. These two 5-HT(2B)R-dependent eicosanoid productions delineate distinct time windows of TNAP regulation during the osteogenic program. Finally, prostaglandins or leukotrienes are shown to relay the post-translational activation of TNAP via stimulation of the phosphatidylinositol-specific phospholipase C. In agreement with the above findings, primary calvarial osteoblasts from 5-HT(2B)R-null mice exhibit defects in TNAP activity.

Stemness or not stemness? Current status and perspectives of adult retinal stem cells.

Many retinal dystrophies are associated with photoreceptor loss, which causes irreversible blindness. The recent identification of various sources of stem cells in the mammalian retina has raised the possibility that cell-based therapies might be efficient strategies to treat a wide range of incurable eye diseases. A first step towards the successful therapeutic exploitation of these cells is to unravel intrinsic and extrinsic regulators that control their proliferation and cell lineage determination. In this review, we provide an overview of the different types and molecular fingerprints of retinal stem cells identified so far. We also detail the current knowledge on molecular cues that influence their self-renewal and proliferation capacity. In particular, we focus on recent data implicating developmental signaling pathways, such as Wnt, Notch and Hedeghog, both in the normal and regenerating retina in different animal models. Last, we discuss the potential of ES cells and various adult stem cells for retinal repair.

Canonical Wnt signaling controls proliferation of retinal stem/progenitor cells in postembryonic Xenopus eyes.

Vertebrate retinal stem cells, which reside quiescently within the ciliary margin, may offer a possibility for treatment of degenerative retinopathies. The highly proliferative retinal precursor cells in Xenopus eyes are confined to the most peripheral region, called the ciliary marginal zone (CMZ). Although the canonical Wnt pathway has been implicated in the developing retina of different species, little is known about its involvement in postembryonic retinas. Using a green fluorescent protein-based Wnt-responsive reporter, we show that in transgenic Xenopus tadpoles, the canonical Wnt signaling is activated in the postembryonic CMZ. To further investigate the functional implications of this, we generated transgenic, hormone-inducible canonical Wnt pathway activating and repressing systems, which are directed to specifically intersect at the nuclear endpoint of transcriptional Wnt target gene activation. We found that postembryonic induction of the canonical Wnt pathway in transgenic retinas resulted in increased proliferation in the CMZ compartment. This is most likely due to delayed cell cycle exit, as inferred from a pulse-chase experiment on 5-bromo-2'-deoxyuridine-labeled retinal precursors. Conversely, repression of the canonical Wnt pathway inhibited proliferation of CMZ cells. Neither activation nor repression of the Wnt pathway affected the differentiated cells in the central retina. We conclude that even at postembryonic stages, the canonical Wnt signaling pathway continues to have a major function in promoting proliferation and maintaining retinal stem cells. These findings may contribute to the eventual design of vertebrate, stem cell-based retinal therapies. Disclosure of potential conflicts of interest is found at the end of this article.

Ptf1a triggers GABAergic neuronal cell fates in the retina.

BACKGROUND: In recent years, considerable knowledge has been gained on the molecular mechanisms underlying retinal cell fate specification. However, hitherto studies focused primarily on the six major retinal cell classes (five types of neurons of one type of glial cell), and paid little attention to the specification of different neuronal subtypes within the same cell class. In particular, the molecular machinery governing the specification of the two most abundant neurotransmitter phenotypes in the retina, GABAergic and glutamatergic, is largely unknown. In the spinal cord and cerebellum, the transcription factor Ptf1a is essential for GABAergic neuron production. In the mouse retina, Ptf1a has been shown to be involved in horizontal and most amacrine neurons differentiation. RESULTS: In this study, we examined the distribution of neurotransmitter subtypes following Ptf1a gain and loss of function in the Xenopus retina. We found cell-autonomous dramatic switches between GABAergic and glutamatergic neuron production, concomitant with profound defects in the genesis of amacrine and horizontal cells, which are mainly GABAergic. Therefore, we investigated whether Ptf1a promotes the fate of these two cell types or acts directly as a GABAergic subtype determination factor. In ectodermal explant assays, Ptf1a was found to be a potent inducer of the GABAergic subtype. Moreover, clonal analysis in the retina revealed that Ptf1a overexpression leads to an increased ratio of GABAergic subtypes among the whole amacrine and horizontal cell population, highlighting its instructive capacity to promote this specific subtype of inhibitory neurons. Finally, we also found that within bipolar cells, which are typically glutamatergic interneurons, Ptf1a is able to trigger a GABAergic fate. CONCLUSION: Altogether, our results reveal for the first time in the retina a major player in the GABAergic versus glutamatergic cell specification genetic pathway.

A general role of hedgehog in the regulation of proliferation.

The Hedgehog (Hh) pathway regulates proliferation in a variety of tissues, however its specific effects on the cell cycle are unclear. During retinal proliferation in particular, the role of Hh has been controversial, with studies variably suggesting a stimulatory or an inhibitory effect on proliferation. Our recent data provide an underlying mechanism, which reconciles these different views. We showed that Hh signaling in the retina accelerates the G(1) and G(2) phases of the cell cycle and then pushes these rapidly dividing cells out of the cell cycle prematurely. From this and other evidence, we propose that Hh converts quiescent retinal stem cells into fast-cycling transient amplifying progenitors that are closer to cell cycle exit and differentiation. This is, we suggest, likely to be a general role of Hh in the nervous system and other tissues. This function of Hh in cell cycle kinetics and cell cycle exit may have implications for tumorigenesis and brain evolution.