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Abstract Background Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens. Results Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye. Similar studies in the zebra fish concluded that lens regeneration is not possible. Conclusions Regeneration of the lens is possible in the axolotl, but differs from both frogs and newts. Thus the axolotl iris provides a novel and more plastic strategy for lens regeneration.

During tissue regeneration, proliferation, dedifferentiation and reprogramming are necessary to restore lost structures. However, it is not fully understood how metabolism intersects with these processes. Chicken embryos can regenerate their retina through retinal pigment epithelium (RPE) reprogramming when treated with fibroblast factor 2 (FGF2). Using transcriptome profiling, we uncovered extensive regulation of gene sets pertaining to proliferation, neurogenesis and glycolysis throughout RPE-to-neural retina reprogramming. By manipulating cell media composition, we determined that glucose, glutamine or pyruvate are individually sufficient to support RPE reprogramming, identifying glycolysis as a requisite. Conversely, the activation of pyruvate dehydrogenase by inhibition of pyruvate dehydrogenase kinases, induces epithelial-to-mesenchymal transition, while simultaneously blocking the activation of neural retina fate. We also identified that epithelial-to-mesenchymal transition fate is partially driven by an oxidative environment. Our findings provide evidence that metabolism controls RPE cell fate decisions and provide insights into the metabolic state of RPE cells, which are prone to fate changes in regeneration and pathologies, such as proliferative vitreoretinopathy., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Previous studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated., Methods: Here we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery., Results: Macrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process., Conclusions: Together, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs., (© 2024. The Author(s).)

Ocular lens development entails epithelial to fiber cell differentiation, defects in which cause congenital cataracts. We report the first single-cell multiomic atlas of lens development, leveraging snRNA-seq, snATAC-seq and CUT&RUN-seq to discover previously unreported mechanisms of cell fate determination and cataract-linked regulatory networks. A comprehensive profile of cis- and trans-regulatory interactions, including for the cataract-linked transcription factor MAF, is established across a temporal trajectory of fiber cell differentiation. Furthermore, we identify an epigenetic paradigm of cellular differentiation, defined by progressive loss of the H3K27 methylation writer Polycomb repressive complex 2 (PRC2). PRC2 localizes to heterochromatin domains across master-regulator transcription factor gene bodies, suggesting it safeguards epithelial cell fate. Moreover, we demonstrate that FGF hyper-stimulation in vivo leads to MAF network activation and the emergence of novel lens cell states. Collectively, these data depict a comprehensive portrait of lens fiber cell differentiation, while defining regulatory effectors of cell identity and cataract formation., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Genomic sequences residing within introns of few genes have been shown to act as enhancers affecting expression of neighboring genes. We studied an autosomal recessive phenotypic continuum of microphthalmia, anophthalmia and ocular coloboma, with no apparent coding-region disease-causing mutation. Homozygosity mapping of several affected Jewish Iranian families, combined with whole genome sequence analysis, identified a 0.5 Mb disease-associated chromosome 2q35 locus (maximal LOD score 6.8) harboring an intronic founder variant in NHEJ1, not predicted to affect NHEJ1. The human NHEJ1 intronic variant lies within a known specifically limb-development enhancer of a neighboring gene, Indian hedgehog (Ihh), known to be involved in eye development in mice and chickens. Through mouse and chicken molecular development studies, we demonstrated that this variant is within an Ihh enhancer that drives gene expression in the developing eye and that the identified variant affects this eye-specific enhancer activity. We thus delineate an Ihh enhancer active in mammalian eye development whose variant causes human microphthalmia, anophthalmia and ocular coloboma. The findings highlight disease causation by an intronic variant affecting the expression of a neighboring gene, delineating molecular pathways of eye development., (© 2023. Springer Nature Limited and Centre of Excellence in Genomic Medicine Research, King Abdulaziz University.)

Background: Aging and regeneration are heavily linked processes. While it is generally accepted that regenerative capacity declines with age, some vertebrates, such as newts, can bypass the deleterious effects of aging and successfully regenerate a lens throughout their lifetime., Results: Here, we used Spectral-Domain Optical Coherence Tomography (SD-OCT) to monitor the lens regeneration process of larvae, juvenile, and adult newts. While all three life stages were able to regenerate a lens through transdifferentiation of the dorsal iris pigment epithelial cells (iPECs), an age-related change in the kinetics of the regeneration process was observed. Consistent with these findings, iPECs from older animals exhibited a delay in cell cycle re-entry. Furthermore, it was observed that clearance of the extracellular matrix (ECM) was delayed in older organisms., Conclusions: Collectively, our results suggest that although lens regeneration capacity does not decline throughout the lifespan of newts, the intrinsic and extrinsic cellular changes associated with aging alter the kinetics of this process. By understanding how these changes affect lens regeneration in newts, we can gain important insights for restoring the age-related regeneration decline observed in most vertebrates., Competing Interests: Declaration of competing interest None., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Salamanders have been used as research models for centuries. While they exhibit a wide range of biological features not seen in mammals, none has captivated scientists like their ability to regenerate. Interestingly, axolotl macrophages have emerged as an essential cell population for tissue regeneration. Whether the same is true in other salamanders such as newt species Notophthalmus viridescens, Cynops pyrrhogaster, or Pleurodeles waltl remains to be seen. Unfortunately, regardless of the species, molecular tools to study macrophage function in salamanders are lacking. We propose that the readily available, terminally differentiated peritoneal macrophages from newts or axolotls could be used to validate molecular reagents in the study of macrophage function during tissue regeneration in salamanders., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Lens regeneration in the adult newt illustrates a unique example of naturally occurring cell transdifferentiation. During this process, iris pigmented epithelial cells (iPECs) reprogram into a lens, a tissue that is derived from a different embryonic source. Several methodologies both in vivo and in culture have been utilized over the years to observe this phenomenon. Most recently, Optical Coherence Tomography (OCT) has been identified as an effective tool to study the lens regeneration process in continuity through noninvasive, real-time imaging of the same animal. Described in this chapter are three different methodologies that can be used to observe the newt lens regeneration process both in vivo and ex vivo., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

The retinal pigment epithelium (RPE) exhibits a diverse range of plasticity across vertebrates and is a potential source of cells for the regeneration of retinal neurons. Embryonic amniotes possess a transitory ability to regenerate neural retina through the reprogramming of RPE cells in an FGF-dependent manner. Chicken RPE can regenerate neural retina at embryonic day 4 (E4), but RPE neural competence is lost by embryonic day 5 (E5). To identify mechanisms that underlie loss of regenerative competence, we performed RNA and ATAC sequencing using E4 and E5 chicken RPE, as well as at both stages following retinectomy and FGF2 treatment. We find that genes associated with neural retina fate remain FGF2-inducible in the non-regenerative E5 RPE. Coinciding with fate restriction, RPE cells stably exit the cell cycle and dampen the expression of cell cycle progression genes normally expressed during regeneration, including E2F1 . E5 RPE exhibits progressive activation of gene pathways associated with mature function independently of retinectomy or FGF2 treatment, including retinal metabolism, pigmentation synthesis, and ion transport. Moreover, the E5 RPE fails to efficiently repress OTX2 expression in response to FGF2. Predicted OTX2 binding motifs undergo robust accessibility increases in E5 RPE, many of which coincide with putative regulatory elements for genes known to facilitate RPE differentiation and maturation. Together, these results uncover widespread alterations in gene regulation that culminate in the loss of RPE neural competence and implicate OTX2 as a key determinant in solidifying the RPE fate. These results yield valuable insight to the basis of RPE lineage restriction during early development and will be of importance in understanding the varying capacities for RPE-derived retinal regeneration observed among vertebrates., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Tangeman, Pérez-Estrada, Van Zeeland, Liu, Danciutiu, Grajales-Esquivel, Smucker, Liang and Del Rio-Tsonis.)

Purpose: To establish optical coherence tomography (OCT) as an in vivo imaging modality for investigating the process of newt lens regeneration., Methods: Spectral-domain OCT was employed for in vivo imaging of the newt lens regeneration process. A total of 37 newts were lentectomized and followed by OCT imaging over the course of 60 to 80 days. Histological images were obtained at several time points to compare with the corresponding OCT images. Volume measurements were also acquired., Results: OCT can identify the key features observed in corresponding histological images based on the scattering differences from various eye tissues, such as the cornea, intact and regenerated lens, and the iris. Lens volume measurements from three-dimensional OCT images showed that the regenerating lens size increased linearly until 60 days post-lentectomy., Conclusions: Using OCT imaging, we were able to track the entire process of newt lens regeneration in vivo for the first time. Three-dimensional OCT images allowed us to volumetrically quantify and visualize the dynamic spatial relationships between tissues during the regeneration process. Our results establish OCT as an in vivo imaging modality to track/analyze the entire lens regeneration process from the same animal., Translational Relevance: Lens regeneration in newts represents a unique example of vertebrate tissue plasticity. Investigating the cellular and morphological events that govern this extraordinary process in vivo will advance our understanding and shed light on developing new therapies to treat blinding disorders in higher vertebrates.

Age-related macular degeneration (AMD) is a retinal disease that affects 196 million people and causes nearly 9% of blindness worldwide. While several pharmacological approaches slow the effects of AMD, in our opinion, cell-based strategies offer the most likely path to a cure. We describe the design and initial characterization of a kerateine (obtained by reductive extraction from keratin proteins) aerogel-electrospun polycaprolactone fiber scaffold system. The scaffolds mimic key features of the choroid and the Bruch's membrane, which is the basement membrane to which the cells of the retinal pigment epithelium (RPE) attach. The scaffolds had elastic moduli of 2-7.2 MPa, a similar range as native choroid and Bruch's membrane. ARPE-19 cells attached to the polycaprolactone fibers, remained viable for one week, and proliferated to form a monolayer reminiscent of that needed for retinal repair. These constructs could serve as a model system for testing cell and/or drug treatment strategies or directing ex vivo retinal tissue formation in the treatment of AMD., (© 2021. Biomedical Engineering Society.)

The plasticity of human retinal pigment epithelium (RPE) has been observed during proliferative vitreoretinopathy, a defective repair process during which injured RPE gives rise to fibrosis. In contrast, following injury, the RPE of the embryonic chicken can be reprogrammed to regenerate neural retina in a fibroblast growth factor 2 (FGF2)-dependent manner. To better explore the mechanisms underlying embryonic RPE reprogramming, we used laser capture microdissection to isolate RNA from (1) intact RPE, (2) transiently reprogrammed RPE (t-rRPE) 6 h post-retinectomy, and (3) reprogrammed RPE (rRPE) 6 h post-retinectomy with FGF2 treatment. Using RNA-seq, we observed the acute repression of genes related to cell cycle progression in the injured t-rRPE, as well as up-regulation of genes associated with injury. In contrast, the rRPE was strongly enriched for mitogen-activated protein kinase (MAPK)-responsive genes and retina development factors, confirming that FGF2 and the downstream MAPK cascade are the main drivers of embryonic RPE reprogramming. Clustering and pathway enrichment analysis was used to create an integrated network of the core processes associated with RPE reprogramming, including key terms pertaining to injury response, migration, actin dynamics, and cell cycle progression. Finally, we employed gene set enrichment analysis to suggest a previously uncovered role for epithelial-mesenchymal transition (EMT) machinery in the initiation of embryonic chick RPE reprogramming. The EMT program is accompanied by extensive, coordinated regulation of extracellular matrix (ECM) associated factors, and these observations together suggest an early role for ECM and EMT-like dynamics during reprogramming. Our study provides for the first time an in-depth transcriptomic analysis of embryonic RPE reprogramming and will prove useful in guiding future efforts to understand proliferative disorders of the RPE and to promote retinal regeneration.

Background: Mathematical approaches have been for decades used to probe the structure of DNA sequences. This has led to the development of Bioinformatics. In this exploratory work, a novel mathematical method is applied to probe the DNA structure of two related viral families: those of coronaviruses and those of influenza viruses. The coronaviruses are SARS-CoV-2, SARS-CoV-1, and MERS. The influenza viruses include H1N1-1918, H1N1-2009, H2N2-1957, and H3N2-1968., Methods: The mathematical method used is the slow feature analysis (SFA), a rather new but promising method to delineate complex structure in DNA sequences., Results: The analysis indicates that the DNA sequences exhibit an elaborate and convoluted structure akin to complex networks. We define a measure of complexity and show that each DNA sequence exhibits a certain degree of complexity within itself, while at the same time there exists complex inter-relationships between the sequences within a family and between the two families. From these relationships, we find evidence, especially for the coronavirus family, that increasing complexity in a sequence is associated with higher transmission rate but with lower mortality., Conclusions: The complexity measure defined here may hold a promise and could become a useful tool in the prediction of transmission and mortality rates in future new viral strains.

Cellular reprogramming resets the epigenetic landscape to drive shifts in transcriptional programmes and cell identity. The embryonic chick can regenerate a complete neural retina, after retinectomy, via retinal pigment epithelium (RPE) reprogramming in the presence of FGF2. In this study, we systematically analysed the reprogramming competent chick RPE prior to injury, and during different stages of reprogramming. In addition to changes in the expression of genes associated with epigenetic modifications during RPE reprogramming, we observed dynamic changes in histone marks associated with bivalent chromatin (H3K27me3/H3K4me3) and intermediates of the process of DNA demethylation including 5hmC and 5caC. Comprehensive analysis of the methylome by whole-genome bisulphite sequencing (WGBS) confirmed extensive rearrangements of DNA methylation patterns including differentially methylated regions (DMRs) found at promoters of genes associated with chromatin organization and fibroblast growth factor production. We also identified Tet methylcytosine dioxygenase 3 (TET3) as an important factor for DNA demethylation and retina regeneration, capable of reprogramming RPE in the absence of exogenous FGF2. In conclusion, we demonstrate that injury early in RPE reprogramming triggers genome-wide dynamic changes in chromatin, including bivalent chromatin and DNA methylation. In the presence of FGF2, these dynamic modifications are further sustained in the commitment to form a new retina. Our findings reveal active DNA demethylation as an important process that may be applied to remove the epigenetic barriers in order to regenerate retina in mammals., Abbreviations: bp: Base pair; DMR: Differentially methylated region; DMC: Differentially methylated cytosines; GFP: Green fluorescent protein; PCR: Polymerase chain reaction. TET: Ten-eleven translocation; RPE: retinal pigment epithelium.

Purpose: Early in mammalian eye development, VSX2 , BRN3b , and RCVRN expression marks neural retinal progenitors (NRPs), retinal ganglion cells (RGCs), and photoreceptors (PRs), respectively. The ability to create retinal organoids from human induced pluripotent stem cells (hiPSC) holds great potential for modeling both human retinal development and retinal disease. However, no methods allowing the simultaneous, real-time monitoring of multiple specific retinal cell types during development currently exist., Methods: CRISPR/Cas9-mediated homology-directed repair (HDR) in hiPSCs facilitated the replacement of the VSX2 (Progenitor), BRN3b (Ganglion), and RCVRN (Photoreceptor) stop codons with sequences encoding a viral P2A peptide fused to Cerulean, green fluorescent protein, and mCherry reporter genes, respectively, to generate a triple transgenic reporter hiPSC line called PGP1. This was accomplished by co-electroporating HDR templates and sgRNA/Cas9 vectors into hiPSCs followed by antibiotic selection. Functional validation of the PGP1 hiPSC line included the ability to generate retinal organoids, with all major retinal cell types, displaying the expression of the three fluorescent reporters consistent with the onset of target gene expression. Disaggregated organoids were also analyzed by fluorescence-activated cell sorting and fluorescent populations were tested for the expression of the targeted gene., Results: Retinal organoids formed from the PGP1 line expressed appropriate fluorescent proteins consistent with the differentiation of NRPs, RGCs, and PRs. Organoids produced from the PGP1 line expressed transcripts consistent with the development of all major retinal cell types., Conclusions and Translational Relevance: The PGP1 line offers a powerful new tool to study retinal development, retinal reprogramming, and therapeutic drug screening., Competing Interests: Disclosure: P.T. Lam, PGP1 line (P); C. Gutierrez, None; K. Del Rio-Tsonis, PGP1 line (P); M.L. Robinson, PGP1 line (P), (Copyright 2020 The Authors.)

The use of antioxidants in tissue regeneration has been studied, but their mechanism of action is not well understood. Here, we analyze the role of the antioxidant N-acetylcysteine (NAC) in retina regeneration. Embryonic chicks are able to regenerate their retina after its complete removal from retinal stem/progenitor cells present in the ciliary margin (CM) of the eye only if a source of exogenous factors, such as FGF2, is present. This study shows that NAC modifies the redox status of the CM, initiates self-renewal of the stem/progenitor cells, and induces regeneration in the absence of FGF2. NAC works as an antioxidant by scavenging free radicals either independently or through the synthesis of glutathione (GSH), and/or by reducing oxidized proteins through a thiol disulfide exchange activity. We dissected the mechanism used by NAC to induce regeneration through the use of inhibitors of GSH synthesis and the use of other antioxidants with different biochemical structures and modes of action, and found that NAC induces regeneration through its thiol disulfide exchange activity. Thus, our results provide, for the first time, a biochemical basis for induction of retina regeneration. Furthermore, NAC induction was independent of FGF receptor signaling, but dependent on the MAPK (pErk1/2) pathway., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

The idea of regenerating injured body parts has captivated human imagination for centuries, and the topic still remains an area of extensive scientific research. This review focuses on the process of lens regeneration: its history, our current knowledge, and the questions that remain unanswered. By highlighting some of the milestones that have shaped our understanding of this phenomenon and the contributions of scientists who have dedicated their lives to investigating these questions, we explore how regeneration enquiry evolved into the science it is today, and how technological advances accelerated our understanding of these remarkable processes.

Complement components have been implicated in a wide variety of functions including neurogenesis, proliferation, cell migration, differentiation, cancer, and more recently early development and regeneration. Following our initial observations indicating that C3a/C3aR signaling induces chick retina regeneration, we analyzed its role in chick eye morphogenesis. During eye development, the optic vesicle (OV) invaginates to generate a bilayer optic cup (OC) that gives rise to the retinal pigmented epithelium (RPE) and neural retina. We show by immunofluorescence staining that C3 and the receptor for C3a (the cleaved and active form of C3), C3aR, are present in chick embryos during eye morphogenesis in the OV and OC. Interestingly, C3aR is mainly localized in the nuclear compartment at the OC stage. Loss of function studies at the OV stage using morpholinos or a blocking antibody targeting the C3aR (anti-C3aR Ab), causes eye defects such as microphthalmia and defects in the ventral portion of the eye that result in coloboma. Such defects were not observed when C3aR was disrupted at the OC stage. Histological analysis demonstrated that microphthalmic eyes were unable to generate a normal optic stalk or a closed OC. The dorsal/ventral patterning defects were accompanied by an expansion of the ventral markers Pax2, cVax and retinoic acid synthesizing enzyme raldh-3 (aldh1a3) domains, an absence of the dorsal expression of Tbx5 and raldh-1 (aldh1a1) and a re-specification of the ventral RPE to neuroepithelium. In addition, the eyes showed overall decreased expression of Gli1 and a change in distribution of nuclear β-catenin, suggesting that Shh and Wnt pathways have been affected. Finally, we observed prominent cell death along with a decrease in proliferating cells, indicating that both processes contribute to the microphthalmic phenotype. Together our results show that C3aR is necessary for the proper morphogenesis of the OC. This is the first report implicating C3aR in eye development, revealing an unsuspected hitherto regulator for proper chick eye morphogenesis., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

Being involved in many important biological processes, miRNAs can regulate gene expression by targeting mRNAs to facilitate their degradation or translational inhibition. Many miRNA sequencing studies reveal that miRNA variations such as isomiRs and "arm switching" are biologically relevant. However, existing standalone tools usually do not provide comprehensive, detailed information on miRNA variations. To deepen our understanding of miRNA variability, we developed a new standalone tool called "mirPRo" to quantify known miRNAs and predict novel miRNAs. Compared with the most widely used standalone program, miRDeep2, mirPRo offers several new functions including read cataloging based on genome annotation, optional seed region check, miRNA family expression quantification, isomiR identification and categorization, and "arm switching" detection. Our comparative data analyses using three datasets from mouse, human and chicken demonstrate that mirPRo is more accurate than miRDeep2 by avoiding over-counting of sequence reads and by implementing different approaches in adapter trimming, mapping and quantification. mirPRo is an open-source standalone program (https://sourceforge.net/projects/mirpro/).

The chick embryo has prevailed as one of the major models to study developmental biology, cell biology and regeneration. From all the anatomical features of the chick embryo, the eye is one of the most studied. In the chick embryo, the eye develops between 26 and 33 h after incubation (Stages 8-9, Hamburger and Hamilton, 1951). It originates from the posterior region of the forebrain, called the diencephalon. However, the vertebrate eye includes tissues from different origins including surface ectoderm (lens and cornea), anterior neural plate (retina, iris, ciliary body and retinal pigmented epithelium) and neural crest/head mesoderm (stroma of the iris and of the ciliary body as well as choroid, sclera and part of the cornea). After gastrulation, a single eye field originates from the anterior neural plate and is characterized by the expression of eye field transcriptional factors (EFTFs) that orchestrate the program for eye development. Later in development, the eye field separates in two and the optic vesicles form. After several inductive interactions with the lens placode, the optic cup forms. At Stages 14-15, the outer layer of the optic cup becomes the retinal pigmented epithelium (RPE) while the inner layer forms the neuroepithelium that eventually differentiates into the retina. One main advantage of the chick embryo, is the possibility to perform experiments to over-express or to down-regulate gene expression in a place and time specific manner to explore gene function and regulation. The aim of this protocol is to describe the electroporation techniques at Stages 8-12 (anterior neural fold and optic vesicle stages) and Stages 19-26 (eye cup, RPE and neuroepithelium). We provide a full description of the equipment, materials and electrode set up as well as a detailed description of the highly reproducible protocol including some representative results. This protocol has been adapted from our previous publications Luz-Madrigal et al. (2014) and Zhu et al. (2014).

In the present study we explored the role of β-catenin in mediating chick retina regeneration. The chick can regenerate its retina by activating stem/progenitor cells present in the ciliary margin (CM) of the eye or via transdifferentiation of the retinal pigmented epithelium (RPE). Both modes require fibroblast growth factor 2 (FGF2). We observed, by immunohistochemistry, dynamic changes of nuclear β-catenin in the CM and RPE after injury (retinectomy). β-Catenin nuclear accumulation was transiently lost in cells of the CM in response to injury alone, while the loss of nuclear β-catenin was maintained as long as FGF2 was present. However, nuclear β-catenin positive cells remained in the RPE in response to injury and were BrdU-/p27+, suggesting that nuclear β-catenin prevents those cells from entering the cell cycle. If FGF2 is present, the RPE undergoes dedifferentiation and proliferation concomitant with loss of nuclear β-catenin. Moreover, retinectomy followed by disruption of active β-catenin by using a signaling inhibitor (XAV939) or over-expressing a dominant negative form of Lef-1 induces regeneration from both the CM and RPE in the absence of FGF2. Our results imply that β-catenin protects cells of the CM and RPE from entering the cell cycle in the developing eye, and specifically for the RPE during injury. Thus inactivation of β-catenin is a pre-requisite for chick retina regeneration.

Background: One of the promises in regenerative medicine is to regenerate or replace damaged tissues. The embryonic chick can regenerate its retina by transdifferentiation of the retinal pigmented epithelium (RPE) and by activation of stem/progenitor cells present in the ciliary margin. These two ways of regeneration occur concomitantly when an external source of fibroblast growth factor 2 (FGF2) is present after injury (retinectomy). During the process of transdifferentiation, the RPE loses its pigmentation and is reprogrammed to become neuroepithelium, which differentiates to reconstitute the different cell types of the neural retina. Somatic mammalian cells can be reprogrammed to become induced pluripotent stem cells by ectopic expression of pluripotency-inducing factors such as Oct4, Sox2, Klf4, c-Myc and in some cases Nanog and Lin-28. However, there is limited information concerning the expression of these factors during natural regenerative processes. Organisms that are able to regenerate their organs could share similar mechanisms and factors with the reprogramming process of somatic cells. Herein, we investigate the expression of pluripotency-inducing factors in the RPE after retinectomy (injury) and during transdifferentiation in the presence of FGF2., Results: We present evidence that upon injury, the quiescent (p27(Kip1)+/BrdU-) RPE cells transiently dedifferentiate and express sox2, c-myc and klf4 along with eye field transcriptional factors and display a differential up-regulation of alternative splice variants of pax6. However, this transient process of dedifferentiation is not sustained unless FGF2 is present. We have identified lin-28 as a downstream target of FGF2 during the process of retina regeneration. Moreover, we show that overexpression of lin-28 after retinectomy was sufficient to induce transdifferentiation of the RPE in the absence of FGF2., Conclusion: These findings delineate in detail the molecular changes that take place in the RPE during the process of transdifferentiation in the embryonic chick, and specifically identify Lin-28 as an important factor in this process. We propose a novel model in which injury signals initiate RPE dedifferentiation, while FGF2 up-regulates Lin-28, allowing for RPE transdifferentiation to proceed.

Patients with congenital disorder of glycosylation (CDG), type Ib (MPI-CDG or CDG-Ib) have mutations in phosphomannose isomerase (MPI) that impair glycosylation and lead to stunted growth, liver dysfunction, coagulopathy, hypoglycemia, and intestinal abnormalities. Mannose supplements correct hypoglycosylation and most symptoms by providing mannose-6-P (Man-6-P) via hexokinase. We generated viable Mpi hypomorphic mice with residual enzymatic activity comparable to that of patients, but surprisingly, these mice appeared completely normal except for modest (~15%) embryonic lethality. To overcome this lethality, pregnant dams were provided 1-2% mannose in their drinking water. However, mannose further reduced litter size and survival to weaning by 40 and 66%, respectively. Moreover, ~50% of survivors developed eye defects beginning around midgestation. Mannose started at birth also led to eye defects but had no effect when started after eye development was complete. Man-6-P and related metabolites accumulated in the affected adult eye and in developing embryos and placentas. Our results demonstrate that disturbing mannose metabolic flux in mice, especially during embryonic development, induces a highly specific, unanticipated pathological state. It is unknown whether mannose is harmful to human fetuses during gestation; however, mothers who are at risk for having MPI-CDG children and who consume mannose during pregnancy hoping to benefit an affected fetus in utero should be cautious.

Identifying the initiation signals for tissue regeneration in vertebrates is one of the major challenges in regenerative biology. Much of the research thus far has indicated that certain growth factors have key roles. Here we show that complement fragment C3a is sufficient to induce complete regeneration of the embryonic chick retina from stem/progenitor cells present in the eye, independent of fibroblast growth factor receptor signaling. Instead, C3a induces retina regeneration via STAT3 activation, which in turn activates the injury- and inflammation-responsive factors, IL-6, IL-8 and TNF-α. This activation sets forth regulation of Wnt2b, Six3 and Sox2, genes associated with retina stem and progenitor cells. Thus, our results establish a mechanism for retina regeneration based on injury and inflammation signals. Furthermore, our results indicate a unique function for complement anaphylatoxins that implicate these molecules in the induction and complete regeneration of the retina, opening new avenues of experimentation in the field.

Comparative studies of lens and retina regeneration have been conducted within a wide variety of animals over the last 100 years. Although amphibians, fish, birds and mammals have all been noted to possess lens- or retina-regenerative properties at specific developmental stages, lens or retina regeneration in adult animals is limited to lower vertebrates. The present review covers the newest perspectives on lens and retina regeneration from these different model organisms with a focus on future trends in regeneration research.

The high recurrence rate of secondary cataract (SC) is caused by the intrinsic differentiation activity of residual lens epithelial cells after extra-capsular lens removal. The objective of this study was to identify changes in the microRNA (miRNA) expression profile during mouse SC formation and to selectively manipulate miRNA expression for potential therapeutic intervention. To model SC, mouse cataract surgery was performed and temporal changes in the miRNA expression pattern were determined by microarray analysis. To study the potential SC counterregulative effect of miRNAs, a lens capsular bag in vitro model was used. Within the first 3 wks after cataract surgery, microarray analysis demonstrated SC-associated expression pattern changes of 55 miRNAs. Of the identified miRNAs, miR-184 and miR-204 were chosen for further investigations. Manipulation of miRNA expression by the miR-184 inhibitor (anti-miR-184) and the precursor miRNA for miR-204 (pre-miR-204) attenuated SC-associated expansion and migration of lens epithelial cells and signs of epithelial to mesenchymal transition such as α-smooth muscle actin expression. In addition, pre-miR-204 attenuated SC-associated expression of the transcription factor Meis homeobox 2 (MEIS2). Examination of miRNA target binding sites for miR-184 and miR-204 revealed an extensive range of predicted target mRNA sequences that were also a target to a complex network of other SC-associated miRNAs with possible opposing functions. The identification of the SC-specific miRNA expression pattern together with the observed in vitro attenuation of SC by anti-miR-184 and pre-miR-204 suggest that miR-184 and miR-204 play a significant role in the control of SC formation in mice that is most likely regulated by a complex competitive RNA network.

The onset mechanism of proliferation in mitotically quiescent retinal pigment epithelium (RPE) cells is still obscure in humans and newts, although it can be a clinical target for manipulating both retinal diseases and regeneration. To address this issue, we investigated factors or signaling pathways involved in the first cell-cycle entry of RPE cells upon retinal injury using a newt retina-less eye-cup culture system in which the cells around the wound edge of the RPE exclusively enter the cell cycle. We found that MEK-ERK signaling is necessary for their cell-cycle entry, and signaling pathways whose activities can be modulated by heparin, such as Wnt-, Shh-, and thrombin-mediated pathways, are capable of regulating the cell-cycle entry. Furthermore, we found that the cells inside the RPE have low proliferation competence even in the presence of serum, suggesting inversely that a loss of cell-to-cell contact would allow the cells to enter the cell cycle., (© 2011 John Wiley & Sons A/S.)

Here we describe a protocol for gene loss of function during regeneration in newts, specifically applied to lens regeneration. Knockdown with the use of morpholinos can be achieved both in vitro and in vivo, depending on the experimental design. These methods achieve desirable levels of gene knockdown, and thus can be compared with methods developed for use in other animals, such as zebrafish. The technology has been applied to study molecular mechanisms during the process of lens regeneration by knocking down genes at specific stages and examining their effects on other genes and lens differentiation. The protocol can take a few days or up to 20 d to complete, depending on the duration of the experiment.

The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (∼20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 4-5 months.

The ability to reprogram in vivo a somatic cell after differentiation is quite limited. One of the most impressive examples of such a process is transdifferentiation of pigmented epithelial cells (PECs) to lens cells during lens regeneration in newts. However, very little is known of the molecular events that allow newt cells to transdifferentiate. Histone B4 is an oocyte-type linker histone that replaces the somatic-type linker histone H1 during reprogramming mediated by somatic cell nuclear transfer (SCNT). We found that B4 is expressed and required during transdifferentiation of PECs. Knocking down of B4 decreased proliferation and increased apoptosis, which resulted in considerable smaller lens. Furthermore, B4 knockdown altered gene expression of key genes of lens differentiation and nearly abolished expression of gamma-crystallin. These data are the first to show expression of oocyte-type linker histone in somatic cells and its requirement in newt lens transdifferentiation and suggest that transdifferentiation in newts might share common strategies with reprogramming after SCNT.

In this study, we present data indicating that mammalian stem cell pluripotency-inducing factors are expressed during lens and limb regeneration in newts. The apparent expression even in intact tissues and the ensued regulation during regeneration raises the possibility that these factors might regulate tissue-specific reprogramming and regeneration. Furthermore, these factors should enable us to understand the similarities and differences between animal regeneration in the newt and stem cell strategies in mammals. Developmental Dynamics 238:1613-1616, 2009. (c) 2009 Wiley-Liss, Inc.

Purpose: Retinal regeneration research holds potential for providing new avenues for the treatment of degenerative diseases of the retina. Various animal models have been used to study retinal regeneration over the years, providing insights into different aspects of this process. However the mechanisms that drive this important phenomenon remain to be fully elucidated. In the present study, we introduce and characterize a new model system for retinal regeneration research that uses the tadpole of the African clawed frog, Xenopus laevis., Methods: The neural retina was surgically removed from Xenopus laevis tadpoles at stages 51-54, and a heparin-coated bead soaked in fibroblast growth factor 2 (FGF-2) was introduced in the eyes to induce regeneration. Histological and immunohistochemical analyses as well as DiI tracing were performed to characterize the regenerate. A similar surgical approach but with concomitant removal of the anterior portion of the eye was used to assess the capacity of the retinal pigmented epithelium (RPE) to regenerate a retina. Immunohistochemistry for FGF receptors 1 and 2 and phosphorylated extracellular signal-regulated protein kinase (pERK) was performed to start elucidating the intracellular mechanisms involved in this process. The role of the mitogen activated protein kinase (MAPK) pathway was confirmed through a pharmacological approach using the MAPK kinase (MEK) inhibitor U0126., Results: We observed that Xenopus laevis tadpoles were able to regenerate a neural retina upon induction with FGF-2 in vivo. The regenerated tissue has the characteristics of a differentiated retina, as assessed by the presence and distribution of different retinal cell markers, and DiI tracing indicated that it is able to form an optic nerve. We also showed that retinal regeneration in this system could take place independently of the presence of the anterior eye tissues. Finally, we demonstrated that FGF-2 treatment induces ERK phosphorylation in the pigmented epithelia 10 days after retinectomy, and that inhibition of the MAPK pathway significantly decreases the amount of retina regenerated at 30 days post-operation., Conclusions: Regeneration of a complete neural retina can be achieved in larval Xenopus laevis through activation of the MAPK signaling pathway by administering exogenous FGF-2. This mechanism is conserved in other animal models, which can regenerate their retina via pigmented epithelium transdifferentiation. Our results provide an alternative approach to retinal regeneration studies, capitalizing on the advantages of the Xenopus laevis tadpole as a model system.

Adult newts are able to regenerate their retina and lens after injury or complete removal through transdifferentiation of the pigmented epithelial tissues of the eye. This process needs to be tightly controlled, and several different mechanisms are likely to be recruited for this function. The Na(+)/K(+) ATPase is a transmembrane protein that establishes electrochemical gradients through the transport of Na(+) and K(+) and has been implicated in the modulation of key cellular processes such as cell division, migration and adhesion. Even though it is expressed in all cells, its isoform composition varies with cell type and is tightly controlled during development and regeneration. In the present study we characterize the expression pattern of Na(+)/K(+) ATPase alpha1 in the adult newt eye and during the process of lens and retina regeneration. We show that this isoform is up-regulated in undifferentiated cells during transdifferentiation. Such change in composition could be one of the mechanisms that newt cells utilize to modulate this process.

To examine underlying mechanisms of urodele lens regeneration we have employed a proteomic analysis of 650 proteins involved in several signaling pathways. We compared expression of these proteins between the regeneration-competent dorsal iris and the regeneration-incompetent ventral iris in the newt. After a series of screenings we selected several proteins to evaluate their expression quantitatively on immunoblots. We then used these selected proteins to compare their expression between the dorsal iris of the newt and the iris of the axolotl, another urodele, which does not regenerate the lens. In the newt we find that most proteins are expressed in both dorsal and ventral iris, even though there is differential regulation. Moreover, several of these proteins are expressed in the axolotl iris as well and for some of them their expression is consistent with the regeneration potential.

We identified a mechanism whereby retina regeneration in the embryonic chick can be induced by the contribution of stem/progenitor cells. We show that bone morphogenetic protein (BMP) signaling is sufficient and necessary to induce retina regeneration and that its action can be divided into two phases. By 3 days after postretinectomy (d PR), the BMP pathway directs proliferation and regeneration through the activation of Smad (canonical BMP pathway) and the up-regulation of FGF signaling by the MAPK pathway. By 7d PR, it induces apoptosis by activating p38 (a noncanonical BMP pathway) and down-regulating FGF signaling (by both MAPK and AKT pathways). Apoptosis at this later stage can be prevented, and BMP-induced regeneration can be further induced by inhibition of p38. These results unravel a mechanism for stem/progenitor cell-mediated retina regeneration, where BMP activation establishes a cross-talk with the FGF pathway and selectively activates the canonical and noncanonical BMP pathways. Retina stem/progenitor cells exist in other species, including humans. Thus, our findings provide insights on how retinal stem cells can be activated for possible regenerative therapies.

The embryonic chick is able to regenerate the retina after it has been removed. We have previously shown that proliferating stem/progenitor cells present in the ciliary body/ciliary marginal zone (CB/CMZ) of the chick eye are responsible for regeneration, which can be induced by ectopic fibroblast growth factor-2 (FGF2) or Sonic hedgehog (Shh). Here, we reveal the mechanisms showing how FGF2 and Shh signaling are interdependent during retina regeneration. If the FGF pathway is inhibited, regeneration stimulated by Shh is inhibited. Likewise, if the Hedgehog pathway is inhibited, regeneration stimulated by FGF2 is inhibited. We examined early signaling events in the CB/CMZ and found that FGF2 or Shh induced a robust Erk phosphorylation during the early stages of retina regeneration. Shh also up-regulated the expression of several members of the FGF signaling pathway. We show that ectopic FGF2 or Shh overexpression increased the number of phosphohistone 3 (PH3)-positive cells in the CB/CMZ and inhibition of either pathway decreased the number of PH3-positive cells. Additionally, both FGF and Hh signaling are required for cell survival in the CB/CMZ, whereas Hh and not FGF signaling plays a role in maintaining the identity of the retinal progenitor population in this region. Combined, our results support a model where the FGF and Hedgehog pathways work together to stimulate retina regeneration., ((c) 2007 Wiley-Liss, Inc.)

Purpose: To elucidate the early cellular events that take place during induction of retina regeneration in the embryonic chick, focusing on the relationship between fibroblast growth factor (FGF) signaling and the regulation of Pax6 and Mitf., Methods: The retina of embryonic day 4 (E4) chicks was removed and a heparin coated bead soaked in fibroblast growth factor 2 (FGF2) was placed into the optic cup. The pharmacological inhibitor PD173074 was used to inhibit FGF receptors, PD98059 was used to inhibit MAP kinase-kinase/extracellular signal-regulated kinase (MEK/Erk) signaling. Retroviral constructs for paired box 6 (Pax6), MEK, and microphthalmia (Mitf) were also used in overexpression studies. Immunohistochemistry was used to examine pErk, Pax6, Mitf, and melanosomal matrix protein 115 (MMP115) immunoreactivity and bromodeoxyuridine (BrdU) incorporation at different time points after removing the retina., Results: The embryonic chick has the ability to regenerate a new retina by the process of transdifferentiation of the retinal pigment epithelium (RPE). We observed that during the induction of transdifferentiation, downregulation of Mitf was not sufficient to induce transdifferentiation at E4 and that FGF2 was required to drive Pax6 protein expression and cell proliferation, both of which are necessary for transdifferentiation. Furthermore, we show that FGF2 works through the FGFR/MEK/Erk signaling cascade to increase Pax6 expression and proliferation. Ectopic Mitf expression was able to inhibit transdifferentiation by acting downstream of FGFR/MEK/Erk signaling, likely by inhibiting the increase in Pax6 protein in the RPE., Conclusions: FGF2 stimulates Pax6 expression during induction of transdifferentiation of the RPE through FGFR/MEK/Erk signaling cascade. This Pax6 expression is accompanied by an increase in BrdU incorporation. In addition, we show that Mitf is spontaneously downregulated after removal of the retina even in the absence of FGF2. This Mitf downregulation is not accompanied by Pax6 upregulation, demonstrating that FGF2 stimulated Pax6 upregulation is required for transdifferentiation of the RPE. Furthermore, we show that ectopic Mitf expression is able to protect the RPE from FGF2 induced transdifferentiation by inhibiting Pax6 upregulation.

Purpose: MicroRNAs (miRNAs) are capable of controlling gene expression by targeting complimentary sequences in many mRNAs. Thus, a small number of miRNAs are capable of regulating expression of many different genes. miRNAs have been found in all animals from Drosophila to human and they are highly conserved. This work was undertaken in order to identify such RNAs in the newt eye., Methods: Cloning of these RNAs was attempted after isolating and fractionating total RNA from the adult newt eye. A gel slice ranging from about 15 to 30 nucleotides in length was cut and the extracted RNA was cloned after several processes involving reverse transcription and linker addition. For expression analysis and verification during the process of lens regeneration we used as a probe mir-124a., Results: Several microRNAs, piRNAs and other small RNAS were identified. Some of them have eye specific gene targets in other species, but for many a function in the eye remains to be attributed. Expression of miR-124a showed an interesting regulation in the lens regeneration-competent dorsal iris., Conclusions: The cloned miRNAs and other small RNAs are the first to be reported for this animal and might bear significance in regulating processes that are unique to the newt eye, i.e., regeneration of the lens and retina.

Pax-6 is a master regulator of eye development and is expressed in the dorsal and ventral iris during newt lens regeneration. We show that expression of Pax-6 during newt lens regeneration coincides with cell proliferation. By knocking down expression of Pax-6 via treatment with morpholinos, we found that proliferation of iris pigment epithelial cells was dramatically reduced both in vitro and in vivo, and, as a result, lens regeneration was significantly retarded. However, induction of dedifferentiation in the dorsal iris was not inhibited. Pax-6 knockdown early in lens regeneration resulted in inhibition of crystallin expression and retardation of lens fiber induction. Once crystallin expression and differentiation of lens fibers has ensued, however, loss of function of Pax-6 did not affect crystallin expression and lens fiber maintenance, even though the effects on proliferation persisted. These results conclusively show that Pax-6 is associated with distinct early events during lens regeneration, namely control of cell proliferation and subsequent lens fiber differentiation.

Lens regeneration in adult newts is a classic example of how cells can faithfully regenerate a complete organ through the process of transdifferentiation. After lens removal, the pigment epithelial cells of the dorsal, but not the ventral, iris dedifferentiate and then differentiate to form a new lens. Understanding how this process is regulated might provide clues about why lens regeneration does not occur in higher vertebrates. The genes six-3 and pax-6 are known to induce ectopic lenses during embryogenesis. Here we tested these genes, as well as members of the bone morphogenetic protein (BMP) pathway that regulate establishment of the dorsal-ventral axis in embryos, for their ability to induce lens regeneration. We show that the lens can be regenerated from the ventral iris when the BMP pathway is inhibited and when the iris is transfected with six-3 and treated with retinoic acid. In intact irises, six-3 is expressed at higher levels in the ventral than in the dorsal iris. During regeneration, however, only expression in the dorsal iris is significantly increased. Such an increase is seen in ventral irises only when they are induced to transdifferentiate by six-3 and retinoic acid or by BMP inhibitors. These data suggest that lens regeneration can be achieved in noncompetent adult tissues and that this regeneration occurs through a gene regulatory mechanism that is more complex than the dorsal expression of lens regeneration-specific genes.

Tissue regeneration will soon become an avenue for repair of damaged or diseased tissues as stem cell niches have been found in almost every organ of the vertebrate body including the CNS. In addition, different animals display an array of regenerative capabilities that are currently being researched to dissect the molecular mechanisms involved. This review concentrates on the different ways in which CNS tissues such as brain, spinal cord and retina can regenerate or display neurogenic potential and how these abilities are modulated by morphogens., ((c) 2005 Wiley Periodicals, Inc.)

This study identifies and characterizes retinal stem cells (RSCs) in early postnatal to seventh-decade human eyes. Different subregions of human eyes were dissociated and cultured by using a clonal sphere-forming assay. The stem cells were derived only from the pars plicata and pars plana of the retinal ciliary margin, at a frequency of approximately 1:500. To test for long-term self-renewal, both the sphere assay and monolayer passaging were used. By using the single sphere passaging assay, primary spheres were dissociated and replated, and individual spheres demonstrated 100% self-renewal, with single spheres giving rise to one or more new spheres in each subsequent passage. The clonal retinal spheres were plated under differentiation conditions to assay the differentiation potential of their progeny. The spheres were produced all of the different retinal cell types, demonstrating multipotentiality. Therefore, the human eye contains a small population of cells (approximately equal to 10,000 cells per eye) that have retinal stem-cell characteristics (proliferation, self-renewal, and multipotentiality). To test the in vivo potential of the stem cells and their progeny, we transplanted dissociated human retinal sphere cells, containing both stem cells and progenitors, into the eyes of postnatal day 1 NOD/SCID mice and embryonic chick eyes. The progeny of the RSCs were able to survive, migrate, integrate, and differentiate into the neural retina, especially as photoreceptors. Their facile isolation, integration, and differentiation suggest that human RSCs eventually may be valuable in treating human retinal diseases.

The embryonic chick has the ability to regenerate its retina after it has been completely removed. Here, we provide a detailed characterization of retina regeneration in the embryonic chick at the cellular level. Retina regeneration can occur in two distinct manners. The first is via transdifferentiation, which is induced by members of the Fibroblast growth factor (Fgf) family. The second type of retinal regeneration occurs from the anterior margin of the eye, near the ciliary body (CB) and ciliary marginal zone (CMZ). We show that regeneration from the CB/CMZ is the result of proliferating stem/progenitor cells. This type of regeneration is also stimulated by Fgf2, but we show that it can be activated by Sonic hedgehog (Shh) overexpression when no ectopic Fgf2 is present. Shh-stimulated activation of CB/CMZ regeneration is inhibited by the Fgf receptor (Fgfr) antagonist, PD173074. This indicates that Shh-induced regeneration acts through the Fgf signaling pathway. In addition, we show that the hedgehog (Hh) pathway plays a role in maintenance of the retina pigmented epithelium (RPE), as ectopic Shh expression inhibits transdifferentiation and Hh inhibition increases the transdifferentiation domain. Ectopic Shh expression in the regenerating retina also results in a decrease in the number of ganglion cells present and an increase in apoptosis mostly in the presumptive ganglion cell layer (GCL). However, Hh inhibition increases the number of ganglion cells but does not have an effect on cell death. Taken together, our results suggest that the hedgehog pathway is an important modulator of retina regeneration.