generating-retinal-injury-models-in-xenopus-tadpoles

Generating Retinal Injury Models in <em>Xenopus</em> Tadpoles

Retinal neurodegenerative diseases are the leading causes of blindness. Among numerous therapeutic strategies being explored, stimulating self-repair ...

Université Paris-Saclay

Thyroid hormones (TH) have been mainly associated with post-embryonic development and adult homeostasis but few studies report direct experimental evidence for TH function at very early phases of embryogenesis. We assessed the outcome of altered TH signaling on early embryogenesis using the amphibian Xenopus as a model system. Precocious exposure to the TH antagonist NH-3 or impaired thyroid receptor beta function led to severe malformations related to neurocristopathies. These include pathologies with a broad spectrum of organ dysplasias arising from defects in embryonic neural crest cell (NCC) development. We identified a specific temporal window of sensitivity that encompasses the emergence of NCCs. Although the initial steps in NCC ontogenesis appeared unaffected, their migration properties were severely compromised both in vivo and in vitro. Our data describe a role for TH signaling in NCCs migration ability and suggest severe consequences of altered TH signaling during early phases of embryonic development.

Implication of thyroid hormone signaling in neural crest cells migration: Evidence from thyroid hormone receptor beta knockdown and NH3 antagonist studies.

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.

Müller glial cell reactivation in Xenopus models of retinal degeneration.

In anamniotes, somite compartimentalization in the lateral somitic domain leads simultaneously to myotome and dermomyotome formation. In the myotome, Xenopus Sox5 is co-expressed with Myod1 in the course of myogenic differentiation. Here, we studied the function of Sox5 using a Myod1-induced myogenic transcription assay in pluripotent cells of animal caps. We found that Sox5 enhances myogenic transcription of muscle markers Des, Actc1, Ckm and MyhE3. The use of chimeric transactivating or transrepressive Sox5 proteins indicates that Sox5 acts as a transrepressor and indirectly stimulates myogenic transcription except for the slow muscle-specific genes Myh7L, Myh7S, Myl2 and Tnnc1. We showed that this role is shared by Sox6, which is structurally similar to Sox5, both belonging to the SoxD subfamily of transcription factors. Moreover, Sox5 can antagonize the inhibitory function of Meox2 on myogenic differentiation. Meox2 which is a dermomyotome marker, represses myogenic transcription in Myod-induced myogenic transcription assay and in Nodal5-induced mesoderm from animal cap assay. The inhibitory function of Meox2 and the pro-myogenic function of Sox5 were confirmed during Xenopus normal development by the use of translation-blocking oligomorpholinos and dexamethasone inducible chimeric Sox5 and Meox2 proteins. We have therefore identified a new function for SoxD proteins in muscle cells, which can indirectly enhance myogenic transcription through transrepression, in addition to the previously identified function as a direct repressor of slow muscle-specific genes.

Xenopus SOX5 enhances myogenic transcription indirectly through transrepression.

Xenopus is an attractive model system for regeneration studies, as it exhibits an extraordinary regenerative capacity compared to mammals. It is commonly used to study body part regeneration following amputation, for instance of the limb, the tail, or the retina. Models with more subtle injuries are also needed for human degenerative disease modeling, allowing for the study of stem cell recruitment for the regeneration of a given cellular subtype. We present here a model to ablate photoreceptor cells in the Xenopus retina. This method is based on the nitroreductase/metronidazole (NTR/MTZ) system, a combination of chemical and genetic tools, allowing for the conditional ablation of targeted cells. This type of approach establishes Xenopus as a powerful model to study cellular regeneration and stem cell regulation.

Conditional Chemogenetic Ablation of Photoreceptor Cells in Xenopus Retina.

A hallmark of Wnt/β-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 β-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/β-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/β-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.

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

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.

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

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.  is the most frequently mutated gene in this disease. We here developed  gene editing-based models of retinitis pigmentosa in two  species,  and , by using CRISPR/Cas9 technology. In both of them, loss of  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  and . While a significant proportion of Müller cells re-enter into the cell cycle in , their proliferation remains extremely limited in . 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.

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

In multicellular eukaryotic organisms, the initiation of DNA replication occurs asynchronously throughout S-phase according to a regulated replication timing program. Here, using  egg extracts, we showed that Yap (Yes-associated protein 1), a downstream effector of the Hippo signalling pathway, is required for the control of DNA replication dynamics. We found that Yap is recruited to chromatin at the start of DNA replication and identified Rif1, a major regulator of the DNA replication timing program, as a novel Yap binding protein. Furthermore, we show that either Yap or Rif1 depletion accelerates DNA replication dynamics by increasing the number of activated replication origins. In  embryos, using a Trim-Away approach during cleavage stages devoid of transcription, we found that either Yap or Rif1 depletion triggers an acceleration of cell divisions, suggesting a shorter S-phase by alterations of the replication program. Finally, our data show that Rif1 knockdown leads to defects in the partitioning of early versus late replication foci in retinal stem cells, as we previously showed for Yap. Altogether, our findings unveil a non-transcriptional role for Yap in regulating replication dynamics. We propose that Yap and Rif1 function as brakes to control the DNA replication program in early embryos and post-embryonic stem cells.

Article	Year
Implication of thyroid hormone signaling in neural crest cells migration: Evidence from thyroid hormone receptor beta knockdown and NH3 antagonist studies. Molecular and cellular endocrinology\| PubMed ID: 27619407	2017
Müller glial cell reactivation in Xenopus models of retinal degeneration. Glia\| PubMed ID: 28548249	2017
Xenopus SOX5 enhances myogenic transcription indirectly through transrepression. Developmental biology\| PubMed ID: 30071218	2018
Conditional Chemogenetic Ablation of Photoreceptor Cells in Xenopus Retina. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 30151764	2018
Barhl2 maintains T cell factors as repressors and thereby switches off the Wnt/β-Catenin response driving Spemann organizer formation. Development (Cambridge, England)\| PubMed ID: 31036545	2019
Linking YAP to Müller Glia Quiescence Exit in the Degenerative Retina. Cell reports\| PubMed ID: 31067458	2019
CRISPR/Cas9-Mediated Models of Retinitis Pigmentosa Reveal Differential Proliferative Response of Müller Cells between and . Cells\| PubMed ID: 35269429	2022
A non-transcriptional function of Yap regulates the DNA replication program in . eLife\| PubMed ID: 35838349	2022

Albert Chesneau

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