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Panagiotis A. Tsonis - One of the best experts on this subject based on the ideXlab platform.
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Video Article A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt
2016Co-Authors: Rital B. Bhavsar, Kenta Nakamura, Panagiotis A. TsonisAbstract:Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its Lens Regeneration capability. Upon Lens removal, IPE cells of the dorsal iris transdifferentiate to Lens cells and eventually form a new Lens in about a month 2,3. This property of Regeneration is never exhibited by the ventral iris cells. The Regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its Lens forming capacity whereas the ventral aggregate does not form a Lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining Regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in Lens Regeneration. Video Link The video component of this article can be found a
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a robust transcriptional program in newts undergoing multiple events of Lens Regeneration throughout their lifespan
2015Co-Authors: Konstantinos Sousounis, Panagiotis A. Tsonis, Goro Eguchi, Manisha C Yadav, Jose Luis Millan, Fubito Toyama, Chikafumi Chiba, Yukiko EguchiAbstract:Newts have the ability to repeatedly regenerate their Lens even during ageing. However, it is unclear whether this Regeneration reflects an undisturbed genetic activity. To answer this question, we compared the transcriptomes of Lenses, irises and tails from aged newts that had undergone Lens Regeneration 19 times with the equivalent tissues from young newts that had never experienced Lens Regeneration. Our analysis indicates that repeatedly regenerated Lenses showed a robust transcriptional program comparable to young never-regenerated Lenses. In contrast, the tail, which was never regenerated, showed gene expression signatures of ageing. Our analysis strongly suggests that, with respect to gene expression, the regenerated Lenses have not deviated from a robust transcriptional program even after multiple events of Regeneration throughout the life of the newt. In addition, our study provides a new paradigm in biology, and establishes the newt as a key model for the study of Regeneration in relation to ageing.
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molecular signatures that correlate with induction of Lens Regeneration in newts lessons from proteomic analysis
2014Co-Authors: Konstantinos Sousounis, Mario Looso, Thomas Braun, Rital B. Bhavsar, Marcus Kruger, Jessica L Beebe, Panagiotis A. TsonisAbstract:Amphibians have the remarkable ability to regenerate missing body parts. After complete removal of the eye Lens, the dorsal but not the ventral iris will transdifferentiate to regenerate an exact replica of the lost Lens. We used reverse-phase nano-liquid chromatography followed by mass spectrometry to detect protein concentrations in dorsal and ventral iris 0, 4, and 8 days post-lentectomy. We performed gene expression comparisons between Regeneration and intact timepoints as well as between dorsal and ventral iris. Our analysis revealed gene expression patterns associated with the ability of the dorsal iris for transdifferentiation and Lens Regeneration. Proteins regulating gene expression and various metabolic processes were enriched in Regeneration timepoints. Proteins involved in extracellular matrix, gene expression, and DNA-associated functions like DNA repair formed a Regeneration-related protein network and were all up-regulated in the dorsal iris. In addition, we investigated protein concentrations in cultured dorsal (transdifferentiation-competent) and ventral (transdifferentiation-incompetent) iris pigmented epithelial (IPE) cells. Our comparative analysis revealed that the ability of dorsal IPE cells to keep memory of their tissue of origin and transdifferentiation is associated with the expression of proteins that specify the dorso-ventral axis of the eye as well as with proteins found highly expressed in Regeneration timepoints, especially 8 days post-lentectomy. The study deepens our understanding in the mechanism of Regeneration by providing protein networks and pathways that participate in the process.
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exogenous oct 4 inhibits Lens transdifferentiation in the newt notophthalmus viridescens
2014Co-Authors: Rital B. Bhavsar, Panagiotis A. TsonisAbstract:From the cocktail of four factors that were able to induce pluripotent stem cells from differentiated cells, Oct-4, c-Myc, Sox-2 and Klf4, only Oct-4 was not expressed during Regeneration in newts. To explore the possible action of this stemness factor we developed an assay where we introduced exogenous Oct-4 protein to an in vitro system for Lens Regeneration in newts. We found that exogenous Oct-4 inhibits differentiation of iris pigmented epithelial cells into Lens cells and also regulates Sox-2 and Pax-6, both important players during Lens development. Thus, presence of Oct-4 hinders transdifferentiation of iris cells.
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plasticity for axolotl Lens Regeneration is associated with age related changes in gene expression
2014Co-Authors: Konstantinos Sousounis, Antony Athippozhy, Randal S Voss, Panagiotis A. TsonisAbstract:Mexican axolotls lose potential for Lens Regeneration 2 weeks after hatching. We used microarrays to identify differently expressed genes before and after this critical time, using RNA isolated from iris. Over 3700 genes were identified as differentially expressed in response to lentectomy between young (7 days post-hatching) and old (3 months post-hatching) axolotl larvae. Strikingly, many of the genes were only expressed in the early or late iris. Genes that were highly expressed in young iris significantly enriched electron transport chain, transcription, metabolism, and cell cycle gene ontologies, all of which are associated with Lens Regeneration. In contrast, genes associated with cellular differentiation and tissue maturation were uniquely expressed in old iris. Many of these expression differences strongly suggest that young and old iris samples were collected before and after the spleen became developmentally competent to produce and secrete cells with humoral and innate immunity functions. Our study establishes the axolotl as a powerful model to investigate age-related cellular differentiation and immune system ontogeny within the context of tissue Regeneration.
Katia Del Riotsonis - One of the best experts on this subject based on the ideXlab platform.
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in vivo imaging of newt Lens Regeneration novel insights into the Regeneration process
2021Co-Authors: Weihao Chen, Georgios Tsissios, Anthony Sallese, Byran J Smucker, Junfan Chen, Hui Wang, Anhthu Nguyen, Katia Del RiotsonisAbstract: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.
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in vivo imaging of newt Lens Regeneration with oct
2021Co-Authors: Weihao Chen, Katia Del Riotsonis, Georgios Tsissios, Anthony Sallese, Byran J Smucker, A T Nguyen, Junfan Chen, Hui WangAbstract:Newts have the exceptional capability of regenerating the Lens through their lifetime. The transparency of the anterior chamber makes OCT an idea imaging technology to track the entire process of the Lens Regeneration in vivo without interruptions. We demonstrated, for the first time, that OCT can capture not only essential morphological changes similar to the changes observed in histology but some fine structures, like zonular fibers, which are not visible in histology. Our initial results warrant the future research of tailoring OCT for dynamically imaging the Lens Regeneration in newts.
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Lens Regeneration a historical perspective
2018Co-Authors: Natalia M Vergara, George Tsissios, Katia Del RiotsonisAbstract: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.
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Lens Regeneration in axolotl new evidence of developmental plasticity
2012Co-Authors: Nobuyasu Maki, Kenta Nakamura, Katia Del Riotsonis, Rinako Suetsugumaki, Saulius Sumanas, Jie Zhu, Panagiotis A. TsonisAbstract: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.
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the role of pax 6 in Lens Regeneration
2006Co-Authors: Mayur Madhavan, Tracy Haynes, Mindy K Call, Panagiotis A. Tsonis, Nicholas C Frisch, Craig M Minich, Katia Del RiotsonisAbstract: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.
Nobuhiko Mizuno - One of the best experts on this subject based on the ideXlab platform.
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determinative roles of fgf and wnt signals in iris derived Lens Regeneration in newt eye
2008Co-Authors: Toshinori Hayashi, Nobuhiko Mizuno, Hisato KondohAbstract:Total Regeneration of experimentally excised Lens from the dorsal part of the iris-pigmented epithelium of newts has been a key model of tissue Regeneration via cells originating from a foreign tissue. Due to the strict spatial restriction of the Lens origin in the newt iris, it has often been assumed that only the dorsal iris cells are endowed with an intrinsic potential to give rise to Lens tissues. However, our reinvestigation of the process revealed completely different mechanisms underlying Lens Regeneration and its spatial restriction, comprising the following two steps: (i) Fibroblast growth factor (FGF) 2-dependent proliferation of iris-pigmented epithelium and activation of early Lens genes (Pax6, Sox2, MafB) over the entire circumference of the iris; and (ii) dorsal iris-restricted activation of the canonical Wnt signals (involving Wnt2b and Frizzeld4) that leads to localized expression of late Lens genes (Prox1, Sox1, β-crystallin). Injection of FGF2 into normal eyes specifically elicited the second Lens development from the dorsal iris, and the administration of recombinant Wnt3a to the cultured iris-pigmented epithelium caused even ventral iris-derived Lens development. Thus, it is concluded that the regulation of FGF2 and Wnt signals is a determinative of the iris-derived Lens Regeneration in the newt eye.
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Determinative role of Wnt signals in dorsal iris-derived Lens Regeneration in newt eye
2006Co-Authors: Toshinori Hayashi, Ritsuko Takada, Shinji Takada, Nobuhiko Mizuno, Hisato KondohAbstract:Abstract We have previously shown that Lens Regeneration from the pigmented epithelium of the dorsal iris in the adult newt eye proceeds in two steps after Lens removal or intraocular FGF2 injection. The FGF2-dependent proliferation of iris pigmented epithelium and activation of early Lens genes that occur over the entire circumference of the iris comprise the first step, while subsequent dorsally confined Lens development marks the second step. Here, we investigated the expression of Wnt and Wnt receptor Frizzled genes in Lens-regenerating iris tissues. Wnt2b and Frizzled4 were activated only in the dorsal half of the iris in synchrony with the occurrence of the second step, whereas Wnt5a and Frizzled2 were activated in both halves throughout the period of the first and second steps. Cultured explants of the iris-derived pigmented epithelium in the presence of FGF2 underwent dorsal-specific Lens development fully recapitulating the in vivo Lens Regeneration process. Under these conditions, Wnt inhibitors Dkk1, which specifically inhibits the canonical signal pathway, and/or sFRP1 repressed the Lens development, while exogenous Wnt3a, which generally activates the canonical pathway like Wnt2b, stimulated Lens development from the dorsal iris epithelium and even caused Lens development from the ventral iris epithelium, albeit at a reduced rate. Wnt5a did not elicit Lens development from the ventral epithelium. These observations indicate that dorsal-specific activation of Wnt2b determines the dorsally limited development of Lens from the iris pigmented epithelium.
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requirement for betab1 crystallin promoter of xenopus laevis in embryonic Lens development and Lens Regeneration
2005Co-Authors: Nobuhiko Mizuno, Yoko Ueda, Hisato KondohAbstract:Regulation of the Lens-specific βB1-crystallin promoter in Xenopus laevis was investigated using transgenic larvae and tadpoles. Comparison of the promoter sequence with that of chicken βB1-crystallin gene indicates significant sequence similarity over a span of several hundred base pairs starting from the transcriptional start site. Remarkably, PL-1 and PL-2 sequences identified in the chicken promoter as essential binding sites of MAF, Pax6 and Prox1 transcription factors were conserved. Mutations of X (Xenopus) PL-1 and XPL-2 sequences eliminated the promoter activity, indicating a conserved mechanism regulating βB1-crystallin promoter among vertebrate species. A stepwise deletion of the promoter sequence starting from 2800 bp indicated that the proximal 260 bp directly upstream of the transcription initiation site is sufficient for eliciting Lens-specific expression, but the 150 bp promoter sequence is inactive despite it containing the XPL-1 and XPL-2 sequences, suggesting the presence of an additional and essential regulatory sequence located between −150 and −260 bp. Activity of the βB1-crystallin promoter during Lens Regeneration from cornea was examined using transgenic tadpoles and found to have the same dependence on promoter regions as in embryonic Lens development, indicating that gene regulation is largely shared by the two Lens-generating processes.
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Generation of transgenic newt Cynops pyrrhogaster for Regeneration study.
2005Co-Authors: Yoko Ueda, Hisato Kondoh, Nobuhiko MizunoAbstract:To take advantage of the ample potential for tissue Regeneration by the newt, a technique to create transgenic newt was developed. The technique was based on a procedure for producing transgenic Xenopus, but modified to adapt to the different sperm morphology and to overcome the refractoriness of newt eggs to activation by normal cleavage. Sperm was collected from mature testes early in winter, permeabilized with digitonin, but without treatment of egg extract. Efficient egg activation was achieved by coinjection of inositol 1,4,5-trisphosphate (IP3) with DNA-sperm nucleus complex. Transgenic Cynops for EGFP/DsRed2 genes under the control of cytomegalovirus (CMV) enhancer/promoter showed nonmosaic widespread expression of reporter genes in embryos, swimming larvae, and adults after metamorphosis. Transgenic newt carrying EGFP gene under regulation of betaB1-crystallin promoter expressed the transgene uniquely in the Lens. During Lens Regeneration after Lens removal, EGFP expression occurred, reflecting the Lens Regeneration process. The newt transgenesis technique described here is likely to be of wide use in monitoring and manipulating gene expression in the study of molecular mechanisms underlying tissue Regeneration.
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fgf2 triggers iris derived Lens Regeneration in newt eye
2004Co-Authors: Toshinori Hayashi, Nobuhiko Mizuno, Mitsumasa Okamoto, Yoko Ueda, Hisato KondohAbstract:Lens Regeneration in newts occurs exclusively from the dorsal aspect of the iris pigment epithelium. Although the phenomenon has been a paradigm of experimental tissue Regeneration, little is understood about how it is initiated and restricted to the dorsal iris. Here we show among various growth factors injected in an intact eye, a single injection of FGF2 specifically caused morphological changes of the iris characteristic of Lens Regeneration, induced expression of transcription factor genes Pax6, Sox2 and MafB, as well as endogenous Fgf2 in both dorsal and ventral halves, and provoked second Lens development only from the dorsal iris. FGF2 protein accumulated in the iris tissue after the Lens was removed, and injection of a soluble form of FGF receptor titrating FGF2 inhibited all reactions observed after the Lens removal or after administration of FGF2. These results indicate that FGF2 and/or related molecules trigger Lens Regeneration from the dorsal iris in the newt. The observations also indicate that the absence of Lens Regeneration from the ventral iris is due to a block in a later phase of Lens developmental pathway.
Mitsumasa Okamoto - One of the best experts on this subject based on the ideXlab platform.
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bmp inhibition driven regulation of six 3 underlies induction of newt Lens Regeneration
2005Co-Authors: Matthew W Grogg, Mindy K Call, Mitsumasa Okamoto, Katia Del Riotsonis, Natalia M Vergara, Panagiotis A. TsonisAbstract:Lens Regeneration in adult newts is a classic example of how cells can faithfully regenerate a complete organ through the process of transdifferentiation1,2,3,4,5,6. 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 embryogenesis7,8. 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 embryos9, 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.
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fgf2 triggers iris derived Lens Regeneration in newt eye
2004Co-Authors: Toshinori Hayashi, Nobuhiko Mizuno, Mitsumasa Okamoto, Yoko Ueda, Hisato KondohAbstract:Lens Regeneration in newts occurs exclusively from the dorsal aspect of the iris pigment epithelium. Although the phenomenon has been a paradigm of experimental tissue Regeneration, little is understood about how it is initiated and restricted to the dorsal iris. Here we show among various growth factors injected in an intact eye, a single injection of FGF2 specifically caused morphological changes of the iris characteristic of Lens Regeneration, induced expression of transcription factor genes Pax6, Sox2 and MafB, as well as endogenous Fgf2 in both dorsal and ventral halves, and provoked second Lens development only from the dorsal iris. FGF2 protein accumulated in the iris tissue after the Lens was removed, and injection of a soluble form of FGF receptor titrating FGF2 inhibited all reactions observed after the Lens removal or after administration of FGF2. These results indicate that FGF2 and/or related molecules trigger Lens Regeneration from the dorsal iris in the newt. The observations also indicate that the absence of Lens Regeneration from the ventral iris is due to a block in a later phase of Lens developmental pathway.
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regulated Lens Regeneration from isolated pigmented epithelial cells of newt iris in culture in response to fgf2 4
2002Co-Authors: Toshinori Hayashi, Nobuhiko Mizuno, Atsushi Kuroiwa, Katsushi Owaribe, Mitsumasa OkamotoAbstract:When a Lens is removed from the newt eye, a new Lens is regenerated from the pigmented epithelial cells of the dorsal iris, whereas the ventral iris never shows such an ability. It is important to clarify the nature of signaling molecules which act directly on the iris cells to accomplish Lens Regeneration from the iris and also to gain insight into the mechanism of dorso-ventral difference of the Regeneration potential. To examine the effects of exogenous factors, we established an in vitro culture of reaggregates made from dissociated pigmented epithelial cells of dorsal or ventral halves of newt iris. Foci of depigmented cells appeared within the cell reaggregates, regardless of their origins, when the cell reaggregates were cultured with FGF2 or FGF4. In contrast, only the depigmented cells in the dorsal iris cell reaggregates underwent extensive proliferation and developed a Lens with the synthesis of Lens-specific crystallins, recapitulating the normal Lens Regeneration. On the other hand, neither FGF8, FGF10, EGF, VEGF, nor IGF promoted Lens development from iris cell reaggregates. Consistent with the FGF-specific action, FGFR-specific inhibitor SU5402 suppressed the Lens development from the cultured cell reaggregates. These results demonstrated that FGF2 or FGF4 is essential for the in vitro Lens Regeneration from the pigmented cells of the dorsal iris. In addition, these findings indicated that unequal competence in the dorsal and ventral iris to FGF2/4 contributes to the difference in Lens forming ability between them.
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highly efficient transfection system for functional gene analysis in adult amphibian Lens Regeneration
2001Co-Authors: Toshinori Hayashi, Hisato Kondoh, Nobuhiko Mizuno, Atsushi Yamagishi, Atsushi Kuroiwa, Mitsumasa OkamotoAbstract:The analysis of newt Lens Regeneration has been an important subject in developmental biology. Recently, it has been reported that the genes involved in the normal eye development are also expressed in the regenerative process of Lens Regeneration in the adult newt. However, functional analysis of these genes has not been possible, because there is no system to introduce genes efficiently into the cells involved in the Regeneration. In the present study, lipofection was used as the method for gene transfer in cultured pigmented iris cells that can transdifferentiate into Lens cells in newt Lens Regeneration. Positive expression of a reporter gene was obtained in more than 70% of cells. In addition, the aggregate derived from gene-transfected cells maintained its expression at a high level for a long time within the host tissue. To verify the effectiveness of this model system with a reporter gene in Lens Regeneration, Pax6, which is suggested to be involved in normal eye development and Lens Regeneration, was transfected. Ectopic expression of Lens-specific crystallins was obtained in cells that show no such activity in normal Lens Regeneration. These results made it possible for the first time to analyze the molecular mechanism of Lens Regeneration in the adult newt.
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Lens formation by pigmented epithelial cell reaggregate from dorsal iris implanted into limb blastema in the adult newt
1999Co-Authors: Mayumi Ito, Toshinori Hayashi, Atsushi Kuroiwa, Mitsumasa OkamotoAbstract:In newt Lens Regeneration, the dorsal iris has Lens forming ability and the ventral iris has no such capability, whereas there is no difference in the morphological criteria. To investigate the real aspects of this characteristic Lens Regeneration in the newt at the cellular level, a useful model system was constructed by transplanting the dorsal and ventral reaggregate derived from singly dissociated pigmented epithelial cells of the iris into the blastema of the forelimb in the newt. The Lens was formed from the dorsal reaggregate with high efficiency, but not from the ventral one. No Lens formation was observed in the implantation of the reaggregate into the tissue of the intact limbs. In detailed examination of the process of Lens formation from the reaggregate, it was shown that tubular formation was the first step in the rearrangement of cells within the reaggregate. This was followed by depigmentation, vesicle formation with active cell growth, and the final step was Lens fiber formation by transdifferentiation of epithelial cells composing the Lens vesicle. The process was almost the same as in situ Lens Regeneration except the reconstitution of the two-layered epithelial structure was embodied as flattened tubular formation in the first step. The present study made it possible for the first time to examine Lens forming ability in the reaggregate mixed with dorsal and ventral cells, because the formation of a reaggregate was started from singly dissociated cells of the dorsal and ventral cells of the iris. Mixed reaggregate experiments indicated that the existence of the dorsal cells in a cluster within the reaggregate is important in Lens formation, and ventral cells showed an inhibitory effect on the formation. The present study demonstrated that the limb system thus constructed was effective for the analysis of Lens formation at the cellular level and made it possible to examine the role of dorsal and ventral cells in Lens Regeneration.
Jonathan J Henry - One of the best experts on this subject based on the ideXlab platform.
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the role of sensory innervation in cornea Lens Regeneration
2019Co-Authors: Kimberly J. Perry, Paul W. Hamilton, Surabhi Sonam, Ratnakar Singh, Jonathan J HenryAbstract:BACKGROUND Numerous sensory nerves in the cornea contribute to normal tissue homeostasis. Interestingly, cells within the basal corneal epithelium can regenerate new Lenses in the frog, Xenopus. In this study, we investigated whether cornea sensory nerves or their neuropeptides are important for supporting cornea-Lens Regeneration. RESULTS Attempts to sever the trigeminal nerve trunk, which provides sensory nerve branches to the cornea, did not inhibit Lens Regeneration. However, using this approach we found that it was not possible to completely disrupt sensory innervation, as these nerves are able to quickly regenerate back to the cornea. On the other hand, attenuation of neuropeptide levels with capsaicin was found to significantly inhibit Lens Regeneration, as visualized by a reduction of Substance P. These treatments also led to a reduction of cornea sensory innervation. Interestingly, inhibition of the Substance P-preferred receptor NK-1 with Spantide II did not affect Lens-Regeneration rates. CONCLUSIONS This study provides evidence that cornea nerves support cornea-Lens Regeneration, which could occur through the release of various neurotrophic factors. Substance P, however, does not appear to be the critical component of this signaling pathway. Further studies are needed to investigate what role other known neurotrophic factors may play in this process.
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understanding the basis of cyp26 mediated regulation of Lens Regeneration using ex vivo eye cultures and 4 oxo ra
2019Co-Authors: Alvin G Thomas, Mohd Tayyab Adil, Jonathan J HenryAbstract:Abstract PURPOSE Xenopus has the remarkable ability to regenerate a Lens from the basal cornea epithelial cells in response to signals from the retina. Previous work demonstrated that the Retinoic Acid (RA) metabolizing enzyme CYP26 is expressed in the cornea, and that its activity is required for Lens Regeneration. Gaps remain in our knowledge as to whether CYP26 is needed only to attenuate RA signaling via RA elimination, or whether it also acts to generate retinoid metabolites, such as 4-oxo-RA, to act as signaling ligands. Other key questions are why CYP26 antagonism, but not exogenous retinoids, can reduce cell division in the cornea, and when during Regeneration CYP26 is important. MATERIALS AND METHODS Ex vivo cultures supplemented with RA, 4-oxo-RA, or the CYP26 inhibitor Liarozole were used to assay the effects of these compounds on Lens Regeneration. Similarly, corneas were explanted, cultured in the presence of these compounds, and assayed for mitotic changes by counting anti-Histone H3 positive nuclei. qPCRs validated responsiveness to these compounds. RESULTS Ex vivo cultures showed that when the media was supplemented with the RA metabolite 4-oxo-RA in addition to Liarozole, Lens Regeneration was still inhibited. 4-oxo-RA also does not rescue the loss of cell division in the cornea that is observed upon CYP26 antagonism. Liarozole inhibited Regeneration when added 12 hours after lentectomy, but not when added 48 hours after. CONCLUSIONS These data show that the necessity of CYP26 is not explained as a generator of 4-oxo-RA for Regeneration. Moreover, Liarozole-induced mitotic reduction is not explained by 4-oxo-RA deficiency. These results support a model of RA-independent mitotic regulation by CYP26, though other retinoid metabolites may be active. Finally, CYP26 activity is only needed between 12 and 48 hours post-surgery, showing that its action is required only during the earliest stages of Lens Regeneration. Financial interests The authors declare no competing financial interests.
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methods for examining Lens Regeneration in xenopus
2019Co-Authors: Jonathan J Henry, Kimberly J. Perry, Paul W. HamiltonAbstract:Some vertebrates are able to regenerate the Lens following its removal. This includes species in the genus Xenopus (i.e., X. laevis, X. tropicalis, and X. borealis), the only anurans known to undergo Lens Regeneration. In Xenopus the regenerated Lens is derived de novo from cells located within the basal-most layer of the larval corneal epithelium, and is triggered by factors provided by the neural retina. In larval frogs the corneal epithelium is underlain by an endothelium separated from the corneal epithelium except for a small central attachment (i.e., the "stromal-attracting center"). This connection grows larger as the stroma forms and the frogs approach metamorphosis. Here we provide instructions for performing lentectomies (removal of the original Lens) to study Lens Regeneration.
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diverse evolutionary origins and mechanisms of Lens Regeneration
2018Co-Authors: Jonathan J Henry, Paul W. HamiltonAbstract:In this review, we compare and contrast the three different forms of vertebrate Lens Regeneration: Wolffian Lens Regeneration, cornea-Lens Regeneration, and Lens Regeneration from Lens epithelial cells. An examination of the diverse cellular origins of these Lenses, their unique phylogenetic distribution, and the underlying molecular mechanisms, suggests that these different forms of Lens Regeneration evolved independently and utilize neither conserved nor convergent mechanisms to regulate these processes.
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Lens Regeneration from the cornea requires suppression of wnt β catenin signaling
2016Co-Authors: Paul W. Hamilton, Yu Sun, Jonathan J HenryAbstract:The frog, Xenopus laevis, possesses a high capacity to regenerate various larval tissues, including the Lens, which is capable of complete Regeneration from the cornea epithelium. However, the molecular signaling mechanisms of cornea-Lens Regeneration are not fully understood. Previous work has implicated the involvement of the Wnt signaling pathway, but molecular studies have been very limited. Iris-derived Lens Regeneration in the newt (Wolffian Lens Regeneration) has shown a necessity for active Wnt signaling in order to regenerate a new Lens. Here we provide evidence that the Wnt signaling pathway plays a different role in the context of cornea-Lens Regeneration in Xenopus. We examined the expression of frizzled receptors and wnt ligands in the frog cornea epithelium. Numerous frizzled receptors (fzd1, fzd2, fzd3, fzd4, fzd6, fzd7, fzd8, and fzd10) and wnt ligands (wnt2b.a, wnt3a, wnt4, wnt5a, wnt5b, wnt6, wnt7b, wnt10a, wnt11, and wnt11b) are expressed in the cornea epithelium, demonstrating that this tissue is transcribing many of the ligands and receptors of the Wnt signaling pathway. When compared to flank epithelium, which is Lens Regeneration incompetent, only wnt11 and wnt11b are different (present only in the cornea epithelium), identifying them as potential regulators of cornea-Lens Regeneration. To detect changes in canonical Wnt/β-catenin signaling occurring within the cornea epithelium, axin2 expression was measured over the course of Regeneration. axin2 is a well-established reporter of active Wnt/β-catenin signaling, and its expression shows a significant decrease at 24 h post-lentectomy. This decrease recovers to normal endogenous levels by 48 h. To test whether this signaling decrease was necessary for Lens Regeneration to occur, regenerating eyes were treated with either 6-bromoindirubin-3'-oxime (BIO) or 1-azakenpaullone - both activators of Wnt signaling - resulting in a significant reduction in the percentage of cases with successful Regeneration. In contrast, inhibition of Wnt signaling using either the small molecule IWR-1, treatment with recombinant human Dickkopf-1 (rhDKK1) protein, or transgenic expression of Xenopus DKK1, did not significantly affect the percentage of successful Regeneration. Together, these results suggest a model where Wnt/β-catenin signaling is active in the cornea epithelium and needs to be suppressed during early Lens Regeneration in order for these cornea cells to give rise to a new lentoid. While this finding differs from what has been described in the newt, it closely resembles the role of Wnt signaling during the initial formation of the Lens placode from the surface ectoderm during early embryogenesis.