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.

Generating Retinal Injury Models in Xenopus Tadpoles.

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair recently emerged as particularly appealing. A cellular source of interest for retinal repair is the Müller glial cell, which harbors stem cell potential and an extraordinary regenerative capacity in anamniotes. This potential is, however, very limited in mammals. Studying the molecular mechanisms underlying retinal regeneration in animal models with regenerative capabilities should provide insights into how to unlock the latent ability of mammalian Müller cells to regenerate the retina. This is a key step for the development of therapeutic strategies in regenerative medicine. To this aim, we developed several retinal injury paradigms in Xenopus: a mechanical retinal injury, a transgenic line allowing for nitroreductase-mediated photoreceptor conditional ablation, a retinitis pigmentosa model based on CRISPR/Cas9-mediated rhodopsin knockout, and a cytotoxic model driven by intraocular CoCl2 injections. Highlighting their advantages and disadvantages, we describe here this series of protocols that generate various degenerative conditions and allow the study of retinal regeneration in Xenopus.

TBC1D32 variants disrupt retinal ciliogenesis and cause retinitis pigmentosa.

Retinitis pigmentosa (RP) is the most common inherited retinal disease (IRD) and is characterized by photoreceptor degeneration and progressive vision loss. We report 4 patients presenting with RP from 3 unrelated families with variants in TBC1D32, which to date has never been associated with an IRD. To validate TBC1D32 as a putative RP causative gene, we combined Xenopus in vivo approaches and human induced pluripotent stem cell-derived (iPSC-derived) retinal models. Our data showed that TBC1D32 was expressed during retinal development and that it played an important role in retinal pigment epithelium (RPE) differentiation. Furthermore, we identified a role for TBC1D32 in ciliogenesis of the RPE. We demonstrated elongated ciliary defects that resulted in disrupted apical tight junctions, loss of functionality (delayed retinoid cycling and altered secretion balance), and the onset of an epithelial-mesenchymal transition-like phenotype. Last, our results suggested photoreceptor differentiation defects, including connecting cilium anomalies, that resulted in impaired trafficking to the outer segment in cones and rods in TBC1D32 iPSC-derived retinal organoids. Overall, our data highlight a critical role for TBC1D32 in the retina and demonstrate that TBC1D32 mutations lead to RP. We thus identify TBC1D32 as an IRD-causative gene.

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.

Barhl2 maintains T cell factors as repressors and thereby switches off the Wnt/ß-Catenin response driving Spemann organizer formation.

A hallmark of Wnt/ß-Catenin signaling is the extreme diversity of its transcriptional response, which varies depending on the cell and developmental context. What controls this diversity is poorly understood. In all cases, the switch from transcriptional repression to activation depends on a nuclear increase in ß-Catenin, which detaches the transcription factor T cell factor 7 like 1 (Tcf7l1) bound to Groucho (Gro) transcriptional co-repressors from its DNA-binding sites and transiently converts Tcf7/Lymphoid enhancer binding factor 1 (Lef1) into a transcriptional activator. One of the earliest and evolutionarily conserved functions of Wnt/ß-Catenin signaling is the induction of the blastopore lip organizer. Here, we demonstrate that the evolutionarily conserved BarH-like homeobox-2 (Barhl2) protein stabilizes the Tcf7l1-Gro complex and maintains the repressed expression of Tcf target genes by a mechanism that depends on histone deacetylase 1 (Hdac-1) activity. In this way, Barhl2 switches off the Wnt/ß-Catenin-dependent early transcriptional response, thereby limiting the formation of the organizer in time and/or space. This study reveals a novel nuclear inhibitory mechanism of Wnt/Tcf signaling that switches off organizer fate determination.

An atlas of Wnt activity during embryogenesis in Xenopus tropicalis.

Wnt proteins form a family of highly conserved secreted molecules that are critical mediators of cell-cell signaling during embryogenesis. Partial data on Wnt activity in different tissues and at different stages have been reported in frog embryos. Our objective here is to provide a coherent and detailed description of Wnt activity throughout embryo development. Using a transgenic Xenopus tropicalis line carrying a Wnt-responsive reporter sequence, we depict the spatial and temporal dynamics of canonical Wnt activity during embryogenesis. We provide a comprehensive series of in situ hybridization in whole-mount embryos and in cross-sections, from gastrula to tadpole stages, with special focus on neural tube, retina and neural crest cell development. This collection of patterns will thus constitute a valuable resource for developmental biologists to picture the dynamics of Wnt activity during development.

A molecular atlas of the developing ectoderm defines neural, neural crest, placode, and nonneural progenitor identity in vertebrates.

During vertebrate neurulation, the embryonic ectoderm is patterned into lineage progenitors for neural plate, neural crest, placodes and epidermis. Here, we use Xenopus laevis embryos to analyze the spatial and temporal transcriptome of distinct ectodermal domains in the course of neurulation, during the establishment of cell lineages. In order to define the transcriptome of small groups of cells from a single germ layer and to retain spatial information, dorsal and ventral ectoderm was subdivided along the anterior-posterior and medial-lateral axes by microdissections. Principal component analysis on the transcriptomes of these ectoderm fragments primarily identifies embryonic axes and temporal dynamics. This provides a genetic code to define positional information of any ectoderm sample along the anterior-posterior and dorsal-ventral axes directly from its transcriptome. In parallel, we use nonnegative matrix factorization to predict enhanced gene expression maps onto early and mid-neurula embryos, and specific signatures for each ectoderm area. The clustering of spatial and temporal datasets allowed detection of multiple biologically relevant groups (e.g., Wnt signaling, neural crest development, sensory placode specification, ciliogenesis, germ layer specification). We provide an interactive network interface, EctoMap, for exploring synexpression relationships among genes expressed in the neurula, and suggest several strategies to use this comprehensive dataset to address questions in developmental biology as well as stem cell or cancer research.

PFKFB4 control of AKT signaling is essential for premigratory and migratory neural crest formation.

Neural crest (NC) specification comprises an early phase, initiating immature NC progenitors formation at neural plate stage, and a later phase at neural fold stage, resulting in a functional premigratory NC that is able to delaminate and migrate. We found that the NC gene regulatory network triggers upregulation of pfkfb4 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 4) during this late specification phase. As shown in previous studies, PFKFB4 controls AKT signaling in gastrulas and glycolysis rate in adult cells. Here, we focus on PFKFB4 function in NC during and after neurulation, using time-controlled or hypomorph depletions in vivo We find that PFKFB4 is essential both for specification of functional premigratory NC and for its migration. PFKFB4-depleted embryos fail to activate n-cadherin and late NC specifiers, and exhibit severe migration defects resulting in craniofacial defects. AKT signaling mediates PFKFB4 function in NC late specification, whereas both AKT signaling and glycolysis regulate migration. These findings highlight novel and essential roles of PFKFB4 activity in later stages of NC development that are wired into the NC gene regulatory network.

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.

Expression and localization of aromatase during fetal mouse testis development.

BACKGROUND: Both androgens and estrogens are necessary to ensure proper testis development and function. Studies on endocrine disruptors have highlighted the importance of maintaining the balance between androgens and estrogens during fetal development, when testis is highly sensitive to environmental disturbances. This balance is regulated mainly through an enzymatic cascade that converts irreversibly androgens into estrogens. The most important and regulated component of this cascade is its terminal enzyme: the cytochrome p450 19A1 (aromatase hereafter). This study was conducted to improve our knowledge about its expression during mouse testis development. FINDINGS: By RT-PCR and western blotting, we show that full-length aromatase is expressed as early as 12.5 day post-coitum (dpc) with maximal expression at 17.5 dpc. Two additional truncated transcripts were also detected by RT-PCR. Immunostaining of fetal testis sections and of gonocyte-enriched cell cultures revealed that aromatase is strongly expressed in fetal Leydig cells and at variable levels in gonocytes. Conversely, it was not detected in Sertoli cells. CONCLUSIONS: This study shows for the first time that i) aromatase is expressed from the early stages of fetal testis development, ii) it is expressed in mouse gonocytes suggesting that fetal germ cells exert an endocrine function in this species and that the ratio between estrogens and androgens may be higher inside gonocytes than in the interstitial fluid. Furthermore, we emphasized a species-specific cell localization. Indeed, previous works found that in the rat aromatase is expressed both in Sertoli and Leydig cells. We propose to take into account this species difference as a new concept to better understand the changes in susceptibility to Endocrine Disruptors from one species to another.

Les androgènes et les oestrogènes sont indispensables au développement et aux fonctions du testicule. Le testicule est particulièrement sensible aux perturbateurs endocriniens pendant le développement fœtal et beaucoup de perturbateurs endocriniens agissent en modifiant la balance oestrogènes/androgènes. Physiologiquement, cette balance est régulée par une cascade enzymatique qui convertit irréversiblement les androgènes en oestrogènes. Le composant principal de cette cascade est le cytochrome p450 19A1 (appelé couramment aromatase). Le but de ce travail a été d'étudier l'expression de l'aromatase testiculaire au cours du développement fœtal chez la souris.En utilisant une approche par RT-PCR et par western blot, nous avons montré que l'aromatase est exprimée dès 12,5 jours post-conception (jpc) et que l'expression est maximum à 17,5 jpc. Deux transcripts tronqués ont également été détectés par RT-PCR. La localisation cellulaire de l'aromatase a été étudiée par immunohistologie et par immunomarquage après séparation des cellules testiculaires. Cette enzyme est très fortement exprimée dans les cellules de Leydig fœtales. Elle est également exprimée dans les gonocytes mais plus faiblement et à un niveau variable selon les cellules. En revanche, elle est indétectable dans les cellules de Sertoli.En conclusion, cette étude montre pour la première fois chez la souris que 1) l'aromatase est exprimée dès le début de l'ontogenèse testiculaire, 2) elle est exprimée dans les gonocytes suggérant que ces cellules interviennent dans l'endocrinologie testiculaire et que le rapport oestrogènes/androgènes est plus important dans les gonocytes que dans le liquide interstitiel. En outre, on sait que, chez le fœtus de rat l'aromatase est essentiellement exprimée par les cellules de Sertoli. Nous proposons de prendre en compte cette différence inter-espèces comme un nouveau concept pour comprendre les différences de sensibilité aux perturbateurs endocriniens d'une espèce à l'autre.

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.

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.

Induction of a parafacial rhythm generator by rhombomere 3 in the chick embryo.

Observations of knock-out mice suggest that breathing at birth requires correct development of a specific hindbrain territory corresponding to rhombomeres (r) 3 and 4. Focusing on this territory, we examined the development of a neuronal rhythm generator in the chick embryo. We show that rhythmic activity in r4 is inducible after developmental stage 10 through interaction with r3. Although the nature of this interaction remains obscure, we find that the expression of Krox20, a segmentation gene responsible for specifying r3 and r5, is sufficient to endow other rhombomeres with the capacity to induce rhythmic activity in r4. Induction is robust, because it can be reproduced with r2 and r6 instead of r4 and with any hindbrain territory that normally expresses Krox20 (r3, r5) or can be forced to do so (r1, r4). Interestingly, the interaction between r4 and r3/r5 that results in rhythm production can only take place through the anterior border of r4, revealing a heretofore unsuspected polarity in individual rhombomeres. The r4 rhythm generator appears to be homologous to a murine respiratory parafacial neuronal system developing in r4 under the control of Krox20 and Hoxa1. These results identify a late role for Krox20 at the onset of neurogenesis.

Developmental gene control of brainstem function: views from the embryo.

The respiratory rhythm is generated within the hindbrain reticular formation, rostrally in the vicinity of the facial nucleus and caudally within the vagal/glossopharyngeal domain. This is probably one of the best models to understand how genes have been selected and conserved to control adaptive behaviour in vertebrates. The para-facial region is well understood with respect to the transcription factors that underlie antero-posterior specification of neural progenitors in the embryo. Hox paralogs and Hox-regulating genes kreisler and Krox-20 govern transient formation of developmental compartments, the rhombomeres, in which rhythmic neuronal networks develop. Hox are master genes selecting and coordinating the developmental fate of reticular and motor neurons thereby specifying patterns of motor activities operating throughout life. Neuronal function and development are also tightly linked in the vagal/glossopharyngeal domain. At this level, bdnf acts as a neurotrophin of peripheral chemoafferent neural populations and as a neuromodulator of the central rhythmogenic respiratory circuits. A general view is now emerging on the role of developmental transcription and trophic factors allowing the coordinated integration of different neuronal types to produce, and eventually refine, respiratory rhythmic pattern in a use-dependent manner.

Developmental molecular switches regulating breathing patterns in CNS.

The present paper presents some of the molecular switches that may operate at early embryonic stages to make development of the brainstem respiratory rhythm generator a robust and irreversible process. We concentrate on the role of transient Hox-related gene expression patterns in register with the regionalisation of the rhombencephalic neural tube along the antero-posterior axis. Using different recording and isolation procedures in chick embryos, we show that the hindbrain is subdivided at E1 into developmental units (rhombomeres) intrinsically able to produce rhythm generating neuronal circuits active at E5. At E6, intrinsic cues also allow a progressive maturation of episodic rhythm generators that persists after isolation of the hindbrain in vitro and requires odd/even rhombomeric interactions at E1. From these results and from respiratory pathologies observed in transgenic mice, we are beginning to understand that, despite diversity of breathing patterns and adaptations, there are links between developmental control genes and adult respiration.