Caroline Borday

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.

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/β-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.

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.

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. STEM CELLS 2012;30:2784–2795

Neural tube patterning by Krox20 and emergence of a respiratory control.

Recent data begin to bridge the gap between developmental events controlling hindbrain neural tube regional patterning and the emergence of breathing behaviour in the fetus and its vital adaptive function after birth. In vertebrates, Hox paralogs and Hox-regulating genes orchestrate, in a conserved manner, the transient formation of developmental compartments in the hindbrain, the rhombomeres, in which rhythmic neuronal networks of the brainstem develop. Genetic inactivation of some of these genes in mice leads to pathological breathing at birth pointing to the vital importance of rhombomere 3 and 4 derived territories for maintenance of the breathing frequency. In chick embryo at E7, we investigated neuronal activities generated in neural tube islands deriving from combinations of rhombomeres isolated at embryonic day E1.5. Using a gain of function approach, we reveal a role of the transcription factor Krox20, specifying rhombomeres 3 and 5, in inducing a rhythm generator at the parafacial level of the hindbrain. The developmental genes selecting and regionally coordinating the fate of CNS progenitors may hold further clues to conserved aspects of neuronal network formation and function. However, the most immediate concern is to take advantage of early generated rhythmic activities in the hindbrain to pursue their downstream cellular and molecular targets, for it seems likely that it will be here that rhythmogenic properties will eventually take on a vital role at birth.

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.

Ontogeny of central rhythm generation in chicks and rodents

Recent studies help in understanding how the basic organization of brainstem neuronal circuits along the anterior-posterior (AP) axis is set by the Hox-dependent segmentation of the neural tube in vertebrate embryos. Neonatal respiratory abnormalities in Krox20(-/-), Hoxa1(-/-) and kreisler mutant mice indicate the vital role of a para-facial (Krox20-dependent, rhombomere 4-derived) respiratory group, that is distinct from the more caudal rhythm generator called Pre-Bötzinger complex. Embryological studies in the chick suggest homology and conservation of this Krox20-dependent induction of parafacial rhythms in birds and mammals. Calcium imaging in embryo indicate that rhythm generators may derive from different cell lineages within rhombomeres. In mice, the Pre-Bötzinger complex is found to be distinct from oscillators producing the earliest neuronal activity, a primordial low-frequency rhythm. In contrast, in chicks, maturation of the parafacial generator is tightly linked to the evolution of this primordial rhythm. It seems therefore that ontogeny of brainstem rhythm generation involves conserved processes specifying distinct AP domains in the neural tube, followed by diverse, lineage-specific regulations allowing the emergence of organized rhythm generators at a given AP level.

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. DOI: http://dx.doi.org/10.7554/eLife.08488.001

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.

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.