The Experts below are selected from a list of 165 Experts worldwide ranked by ideXlab platform
Chikafumi Chiba - One of the best experts on this subject based on the ideXlab platform.
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Implications of a Multi-Step Trigger of Retinal Regeneration in the Adult Newt.
Biomedicines, 2017Co-Authors: Hirofumi Yasumuro, Keisuke Sakurai, Fubito Toyama, Fumiaki Maruo, Chikafumi ChibaAbstract:The Newt is an amazing four-limbed vertebrate that can regenerate various body parts including the retina. In this animal, when the neural retina (NR) is removed from the eye by surgery (retinectomy), both the NR and the retinal pigment epithelium (RPE) eventually regenerate through the process of reprogramming and proliferation of RPE cells. Thus far, we have pursued the onset mechanism of Adult Newt retinal regeneration. In this study, using an in vitro system, we found that both mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK and β-catenin were involved in cell cycle re-entry of RPE cells. MEK-ERK signaling activity in RPE cells was strengthened by retinectomy, and nuclear translocation of β-catenin in RPE cells was induced by attenuation of cell–cell contact, which was promoted by incision of the RPE or its treatment with ethylene glycol tetraacetic acid (EGTA). EGTA is a Ca2+ chelator that disrupts cadherin-mediated cell–cell adhesion. Reinforcement of MEK-ERK signaling activity was a prerequisite for nuclear translocation of β-catenin. These results suggest that retinectomy followed by attenuation of cell–cell contact may trigger cell cycle re-entry of RPE cells. This study, together with our previous findings concerning the proliferation and multipotency of Adult Newt RPE cells, provides insight into the mechanism of the multi-step trigger in which the onset of retinal regeneration in the Adult Newt is rigorously controlled.
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A Transcriptome for the Study of Early Processes of Retinal Regeneration in the Adult Newt, Cynops pyrrhogaster
2016Co-Authors: Kenta Nakamura, Hirofumi Yasumuro, Fubito Toyama, Wataru Inami, Md. Rafiqul Islam, Miyako Takayanagi, Ailidana Kunahong, Roman M. Casco-robles, Chikafumi ChibaAbstract:Retinal regeneration in the Adult Newt is a useful system to uncover essential mechanisms underlying the regeneration of body parts of this animal as well as to find clues to treat retinal disorders such as proliferative vitreoretinopathy. Here, to facilitate the study of early processes of retinal regeneration, we provide a de novo assembly transcriptome and inferred proteome of the Japanese fire bellied Newt (Cynops pyrrhogaster), which was obtained from eyeball samples of day 0–14 after surgical removal of the lens and neural retina. This transcriptome (237,120 in silico transcripts) contains most information of cDNAs/ESTs which has been reported in Newts (C. pyrrhogaster, Pleurodeles waltl and Notophthalmus viridescence) thus far. On the other hand, de novo assembly transcriptomes reported lately for N. viridescence only covered 16–31 % of this transcriptome, suggesting that most constituents of this transcriptome are specific to the regenerating eye tissues of C. pyrrhogaster. A total of 87,102 in silico transcripts of this transcriptome were functionally annotated. Coding sequence prediction in combination with functional annotation revealed that 76,968 in silico transcripts encode protein/ peptides recorded in public databases so far, whereas 17,316 might be unique. qPCR and Sanger sequencing demonstrated that this transcriptome contains much information pertaining to genes that are regulated in association with cell reprogramming, cell-cycle re-entry/proliferation, and tissue patterning in an early phase of retinal regeneration. This data also provides important insight for further investigations addressing cellular mechanisms and molecular network
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Expression of Two Classes of Pax6 Transcripts in Reprogramming Retinal Pigment Epithelium Cells of the Adult Newt
Zoological science, 2016Co-Authors: Wataru Inami, Hirofumi Yasumuro, Fubito Toyama, Fumiaki Maruo, Kenta Nakamura, Rafiqul Islam, Taro Yoshikawa, Martin Miguel Casco-robles, Chikafumi ChibaAbstract:The Adult Newt has the remarkable ability to regenerate a functional retina from retinal pigment epithelium (RPE) cells, even when the neural retina (NR) is completely lost from the eye. In this system, RPE cells are reprogrammed into a unique state of multipotent cells, named RPESCs, in an early phase of retinal regeneration. However, the signals that trigger reprogramming remain unknown. Here, to approach this issue we focused on Pax6, a transcription factor known to be expressed in RPESCs. We first identified four classes (v1, v2, v3 and v4) of Pax6 variants in the eye of Adult Newt, Cynops pyrrhogaster. These variants were expressed in most tissues of the intact eye in different combinations but not in the RPE, choroid or sclera. On the basis of this information, we investigated the expression of Pax6 in RPE cells after the NR was removed from the eye by surgery (retinectomy), and found that two classes (v1 and v2) of Pax6 variants were newly expressed in RPE cells 10 days after retinectomy, both in vivo and in vitro (RLEC system). In the RLEC system, we found that Pax6 expression is mediated through a pathway separate from the MEK-ERK pathway, which is required for cell cycle re-entry of RPE cells. These results predict the existence of a pathway that may be of fundamental importance to a better understanding of the reprogramming of RPE cells in vivo.
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A transcriptome for the study of early processes of retinal regeneration in the Adult Newt, Cynops pyrrhogaster.
PloS one, 2014Co-Authors: Kenta Nakamura, Hirofumi Yasumuro, Fubito Toyama, Wataru Inami, Rafiqul Islam, Miyako Takayanagi, Ailidana Kunahong, Roman M. Casco-robles, Chikafumi ChibaAbstract:Retinal regeneration in the Adult Newt is a useful system to uncover essential mechanisms underlying the regeneration of body parts of this animal as well as to find clues to treat retinal disorders such as proliferative vitreoretinopathy. Here, to facilitate the study of early processes of retinal regeneration, we provide a de novo assembly transcriptome and inferred proteome of the Japanese fire bellied Newt (Cynops pyrrhogaster), which was obtained from eyeball samples of day 0–14 after surgical removal of the lens and neural retina. This transcriptome (237,120 in silico transcripts) contains most information of cDNAs/ESTs which has been reported in Newts (C. pyrrhogaster, Pleurodeles waltl and Notophthalmus viridescence) thus far. On the other hand, de novo assembly transcriptomes reported lately for N. viridescence only covered 16–31% of this transcriptome, suggesting that most constituents of this transcriptome are specific to the regenerating eye tissues of C. pyrrhogaster. A total of 87,102 in silico transcripts of this transcriptome were functionally annotated. Coding sequence prediction in combination with functional annotation revealed that 76,968 in silico transcripts encode protein/peptides recorded in public databases so far, whereas 17,316 might be unique. qPCR and Sanger sequencing demonstrated that this transcriptome contains much information pertaining to genes that are regulated in association with cell reprogramming, cell-cycle re-entry/proliferation, and tissue patterning in an early phase of retinal regeneration. This data also provides important insight for further investigations addressing cellular mechanisms and molecular networks underlying retinal regeneration as well as differences between retinal regeneration and disorders. This transcriptome can be applied to ensuing comprehensive gene screening steps, providing candidate genes, regardless of whether annotated or unique, to uncover essential mechanisms underlying early processes of retinal regeneration.
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mek erk signaling in Adult Newt retinal pigment epithelium cells is strengthened immediately after surgical induction of retinal regeneration
Neuroscience Letters, 2012Co-Authors: Aki Mizuno, Wataru Inami, Hirofumi Yasumuro, Taro Yoshikawa, Chikafumi ChibaAbstract:Adult Newt retinal pigment epithelium (RPE) cells are mitotically quiescent in the physiological condition, but upon a traumatic injury of the neural retina (NR) they re-enter the cell-cycle and eventually regenerate the missing NR. Here, to understand the mechanism underlying the cell-cycle re-entry of RPE cells following NR injury, we first investigated changes in MEK-ERK signaling activity in RPE cells upon removal of the NR (retinectomy) from the eye of living animals, and found that ERK-mediated signaling activity is elevated quickly (in 30 min) upon retinectomy. In addition, we found, in in vitro analyses, that immediate early activation of MEK-ERK signaling may occur in RPE cells upon NR injury, intensifying the MEK-ERK signaling itself through up-regulation of the expression of constituent molecules in the pathway, and that 1-h blockade of such early MEK-ERK signaling interferes with the cell-cycle re-entry, which occurs 5-10 days later. Together, these results provide us with insight that elevation of MEK-ERK signaling activity upon NR injury may be a key process for mitotically quiescent RPE cells to re-enter the cell-cycle, leading to retinal regeneration.
Panagiotis A. Tsonis - One of the best experts on this subject based on the ideXlab platform.
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Regeneration of retinotectal projections after optic tectum removal in Adult Newts.
Molecular vision, 2007Co-Authors: Mitsumasa Okamoto, Hatsuki Ohsawa, Toshinori Hayashi, Katsushi Owaribe, Panagiotis A. TsonisAbstract:PURPOSE When injured, the Adult Newt possesses the remarkable capability to regenerate tissues and organs with return of function and physiology. One example is the Newt eye, in which regeneration can restore normal vision if the retina or lens has been removed. We wanted to examine how the retinotectal projections regenerate after removal of the brain's optic tectum and establish this animal as a model for retinal projection as well as a central nervous system regeneration model. METHODS A major portion of the left optic tectum was removed in several Adult Newts, and the animals were monitored postoperatively for eight months to observe regeneration and innervation. Cell proliferation was examined by histological methods and by BrdU incorporation. RESULTS We observed that Adult Newts have the capability to the excised optic tectum. As indicated by horseradish peroxidase staining, 80% of the retinotectal projection area was regenerated eight months after the operation, even though the wound closed much earlier. Our study provides the first quantitation of regeneration of the retinotectal projections. The ependymal cells that line the ventricle were the most likely source of the regenerated tectum. After removal, cell proliferation was detected only in the ependymal cells layer. Double staining of proliferating cells and neurons was limited, indicating that direct transition of ependymal cells is a possibility. CONCLUSIONS The retinotectal projections after removal of the Adult Newt optic tectum can be readily re-established. Thus, this model can become indispensable for the study of vision restoration and neurogenesis.
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Identification of MicroRNAs and Other Small RNAs from the Adult Newt Eye
Molecular vision, 2006Co-Authors: Evgeny Makarev, Jason R. Spence, Katia Del Rio-tsonis, Panagiotis A. TsonisAbstract: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 to30 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.
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A spontaneous melanoma-like tumor in the Adult Newt Triturus cristatus.
Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine, 1991Co-Authors: Nicolaos P. Zilakos, Panagiotis A. TsonisAbstract:We present here a case of a spontaneous melanoma-like tumor in the Adult Newt Triturus cristatus, lying above the scapula. The melanized cellular mass had infiltrated the subcutaneous connective tissu
Jean-marie Cabelguen - One of the best experts on this subject based on the ideXlab platform.
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EPAXIAL AND LIMB MUSCLE ACTIVITY DURING SWIMMING AND TERRESTRIAL STEPPING IN THE Adult Newt, PLEURODELES WALTL
Journal of Neurophysiology, 1997Co-Authors: Isabelle Delvolvé, Jean-marie CabelguenAbstract:Delvolve, Isabelle, Tiaza Bem, and Jean-Marie Cabelguen. Epaxial and limb muscle activity during swimming and terrestrial stepping in the Adult Newt, Pleurodeles waltl. J. Neurophysiol. 78: 638–650, 1997. We have investigated the patterns of activation of epaxial musculature during both swimming and overground stepping in an Adult Newt ( Pleurodeles waltl ) with the use of electromyographic (EMG) recordings from different sites of the myomeric muscle dorsalis trunci along the body axis. The locomotor patterns of some limb muscles have also been investigated. During swimming, the epaxial myomeres are rhythmically active, with a strict alternation between opposite myomeres located at the same longitudinal site. The pattern of intersegmental coordination consists of three successively initiated waves of EMG activity passing posteriorly along the anterior trunk, the midtrunk, and the posterior trunk, respectively. Swimming is also characterized by a tonic activation of forelimb (dorsalis scapulae and extensor ulnae) and hindlimb (puboischiotibialis and puboischiofemoralis internus) muscles and a rhythmic activation of muscles (latissimus dorsi and caudofemoralis) acting both on limb and body axis. The latter matched the activation pattern of epaxial myomeres at the similar vertebral level. During overground stepping, the midtrunk myomeres express single synchronous bursts whereas the myomeres of the anterior trunk and those of the posterior trunk display a double bursting pattern in the form of two waves of EMG activity propagating in opposite directions. During overground stepping, the limb muscles and muscles acting on both limb and body axis were found to be rhythmically active and usually displayed a double bursting pattern. The main conclusion of this investigation is that the patterns of intersegmental coordination during both swimming and overground stepping in the Adult Newt are related to the presence of limbs and that they can be considered as hybrid lampreylike patterns. Thus it is hypothesized that, in Newt, a chain of coupled segmental oscillatory networks, similar to that which constitutes the central pattern generator (CPG) for swimming in the lamprey, can account for both trunk motor patterns if it is influenced by limb CPGs in a way depending on the locomotor mode. During swimming, the segmental networks located close to the girdles receive extra tonic excitation coming from the limb CPGs, whereas during stepping, the axial CPGs are entrained to some extent by the limb oscillators.
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EPAXIAL AND LIMB MUSCLE ACTIVITY DURING SWIMMING AND TERRESTRIAL STEPPING IN THE Adult Newt, PLEURODELES WALTL
Journal of neurophysiology, 1997Co-Authors: Isabelle Delvolvé, Tiaza Bem, Jean-marie CabelguenAbstract:Delvolve, Isabelle, Tiaza Bem, and Jean-Marie Cabelguen. Epaxial and limb muscle activity during swimming and terrestrial stepping in the Adult Newt, Pleurodeles waltl. J. Neurophysiol. 78: 638–650...
Roy A Tassava - One of the best experts on this subject based on the ideXlab platform.
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timing the commitment to a wound healing response of denervated limb stumps in the Adult Newt notophthalmus viridescens
Wound Repair and Regeneration, 2006Co-Authors: Judith D Salleyguydon, Roy A TassavaAbstract:Adult Newt limbs that are denervated 1 day after amputation undergo a wound-healing response and, although they become reinnervated, will not regenerate unless reinjured. Experiments were designed to determine when denervated limb stumps of Adult Newts become committed to a wound-healing response. In Experiment I, limbs were amputated and denervated 1 day after amputation. On days 7, 14, 21, 28, and 35 days after the initial amputation and denervation, stumps were reamputated to remove the distal tip. This design varied the time the distal stump was devoid of nerves before reamputation. None of the limbs reamputated at 7 days regenerated. About half of the limbs reamputated at 14 days regenerated and almost all of those reamputated at days 21, 28, and 35 regenerated. In Experiment II, limbs were denervated and then amputated on day 7 or 14, at two different levels. Limbs with a short stump became innervated earlier and regeneration occurred more frequently at both levels of amputation. The results of these experiments show that denervated limb stumps become committed to a wound-healing response between days 7 and 14 after amputation/denervation. If sufficient nerves arrive before day 7, a regeneration response is initiated. If the stump is denervated for 14 days or longer, commitment to wound healing occurs and ingrowing nerves cannot initiate a regeneration response.
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Tail regeneration and ependymal outgrowth in the Adult Newt, Notophthalmus viridescens, are adversely affected by experimentally produced ischemia.
Journal of experimental zoology. Part A Comparative experimental biology, 2005Co-Authors: Roy A Tassava, Yan HuangAbstract:Spinal axons of the Adult Newt will regenerate when the spinal cord is severed or when the tail is amputated. Ischemia and associated hypoxia have been correlated with poor central nervous system regeneration in mammals. To test the effects of ischemia on Newt spinal cord regeneration, the spinal cord and major blood vessels of the Newt tail were severed 2 cm caudal to the cloaca as a primary injury. This primary injury severely reduced circulation in the caudal direction for 7 days; by day 8, circulation was largely restored. After various periods of time after primary injury, tails were amputated 1 cm caudal to the primary injury (in the area of ischemia) and tested for regeneration. If the tail was amputated within 5 days of the primary injury, regeneration did not occur. If amputation was 7 days or longer after the primary injury, a regenerative response occurred. Histology showed that in the non-regenerating tails the spinal cord and associated ependyma, known to be important to tail regeneration, had degenerated in the rostral direction. Such degeneration was prevented when tails were first amputated and allowed to form blastemas before the primary injury. The data indicate that the first 5-7 days of blastema formation are particularly sensitive to compromised blood flow (ischemia/hypoxia). It follows that mechanisms must be present in the Adult Newt to reduce ischemia to a minimum and thus allow ependymal outgrowth and tail regeneration.
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Vasculature in pre-blastema and nerve-dependent blastema stages of regenerating forelimbs of the Adult Newt, Notophthalmus viridescens.
The Journal of experimental zoology, 2002Co-Authors: Mona A.e. Rageh, Luciara Mendenhall, Essam El-din A. Moussad, Sarah E. Abbey, Anthony L. Mescher, Roy A TassavaAbstract:Immunocytochemistry utilizing a monoclonal antibody (BV1; blood vessel 1) highly reactive to the vasculature of the Adult Newt showed that a developing vasculature was present during early, pre-blastema, and early-bud blastema stages of forelimb regeneration in this species. Infusion of Prussian Blue and DiI into the brachial artery further delineated the intactness of this early vasculature. Finally, macroscopic observations of vascular flow underneath the apical epithelial cap (AEC) and microsurgical removal of the AEC and observation of subsequent bleeding buttressed the conclusion that an intact vasculature exists during early nerve-dependent stages of Newt forelimb regeneration. The results suggest that this process of neovascular formation is angiogenesis, i.e., the formation of new vessels from pre-existing vessels in the stump. Furthermore, angiogenesis is an ongoing process initiated early after amputation. Blastema cells and the AEC are likely sources of factors that stimulate neovascularization. J. Exp. Zool. 292:255–266, 2002. © 2002 Wiley-Liss, Inc.
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retinoic acid enhances monoclonal antibody we3 reactivity in the regenerate epithelium of the Adult Newt
Journal of Morphology, 1992Co-Authors: Roy A TassavaAbstract:Monoclonal antibody (mAb) WE3 recognizes an antigen that is developmentally expressed in the wound epithelium during Adult Newt limb regeneration. Experiments were designed to determine whether retinoic acid (RA), dissolved in dimethyl sulfoxide (DMSO) and administered by intraperitoneal injection, would enhance the temporal appearance of the WE3 antigen. RA given on days 1 or 4 after amputation, when the WE3 antigen is not yet detectable, resulted in moderate reactivity to mAb 2 days after injection and strong reactivity throughout the wound epithelium 4 days after injection. DMSO alone had no enhancing effect. RA also caused limb skin epidermis to exhibit reactivity to mAb WE3, initially near the amputation level, but then also more proximally. By 4 and 6 days after RA injection, epidermis of the flank, eye lid, and unamputated hind limbs also became strongly reactive to mAb WE3. Outer layers of skin epidermis were shed, resulting in an epidermis only one or two cells thick. Epidermis of Newts given DMSO alone remained non-reactive to mAb WE3. When RA was given on days 7 and 10 after amputation, when a low level of mAb WE3 reactivity is already present in the wound epithelium, a considerable enhancement of mAb WE3 reactivity occurred through the next few days. No such enhancement was seen with DMSO alone. RA also greatly increased mAb WE3 reactivity in the wound epithelium of denervated limbs, in which case the wound epithelial reactivity to mAb WE3 is normally low. Retinol palmitate also increased mAb WE3 reactivity. The results raise the possibility that the WE3 antigen is a component of most if not all retinoid target tissues in Newts. © 1992 Wiley-Liss, Inc.
Kenta Nakamura - One of the best experts on this subject based on the ideXlab platform.
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A Transcriptome for the Study of Early Processes of Retinal Regeneration in the Adult Newt, Cynops pyrrhogaster
2016Co-Authors: Kenta Nakamura, Hirofumi Yasumuro, Fubito Toyama, Wataru Inami, Md. Rafiqul Islam, Miyako Takayanagi, Ailidana Kunahong, Roman M. Casco-robles, Chikafumi ChibaAbstract:Retinal regeneration in the Adult Newt is a useful system to uncover essential mechanisms underlying the regeneration of body parts of this animal as well as to find clues to treat retinal disorders such as proliferative vitreoretinopathy. Here, to facilitate the study of early processes of retinal regeneration, we provide a de novo assembly transcriptome and inferred proteome of the Japanese fire bellied Newt (Cynops pyrrhogaster), which was obtained from eyeball samples of day 0–14 after surgical removal of the lens and neural retina. This transcriptome (237,120 in silico transcripts) contains most information of cDNAs/ESTs which has been reported in Newts (C. pyrrhogaster, Pleurodeles waltl and Notophthalmus viridescence) thus far. On the other hand, de novo assembly transcriptomes reported lately for N. viridescence only covered 16–31 % of this transcriptome, suggesting that most constituents of this transcriptome are specific to the regenerating eye tissues of C. pyrrhogaster. A total of 87,102 in silico transcripts of this transcriptome were functionally annotated. Coding sequence prediction in combination with functional annotation revealed that 76,968 in silico transcripts encode protein/ peptides recorded in public databases so far, whereas 17,316 might be unique. qPCR and Sanger sequencing demonstrated that this transcriptome contains much information pertaining to genes that are regulated in association with cell reprogramming, cell-cycle re-entry/proliferation, and tissue patterning in an early phase of retinal regeneration. This data also provides important insight for further investigations addressing cellular mechanisms and molecular network
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Expression of Two Classes of Pax6 Transcripts in Reprogramming Retinal Pigment Epithelium Cells of the Adult Newt
Zoological science, 2016Co-Authors: Wataru Inami, Hirofumi Yasumuro, Fubito Toyama, Fumiaki Maruo, Kenta Nakamura, Rafiqul Islam, Taro Yoshikawa, Martin Miguel Casco-robles, Chikafumi ChibaAbstract:The Adult Newt has the remarkable ability to regenerate a functional retina from retinal pigment epithelium (RPE) cells, even when the neural retina (NR) is completely lost from the eye. In this system, RPE cells are reprogrammed into a unique state of multipotent cells, named RPESCs, in an early phase of retinal regeneration. However, the signals that trigger reprogramming remain unknown. Here, to approach this issue we focused on Pax6, a transcription factor known to be expressed in RPESCs. We first identified four classes (v1, v2, v3 and v4) of Pax6 variants in the eye of Adult Newt, Cynops pyrrhogaster. These variants were expressed in most tissues of the intact eye in different combinations but not in the RPE, choroid or sclera. On the basis of this information, we investigated the expression of Pax6 in RPE cells after the NR was removed from the eye by surgery (retinectomy), and found that two classes (v1 and v2) of Pax6 variants were newly expressed in RPE cells 10 days after retinectomy, both in vivo and in vitro (RLEC system). In the RLEC system, we found that Pax6 expression is mediated through a pathway separate from the MEK-ERK pathway, which is required for cell cycle re-entry of RPE cells. These results predict the existence of a pathway that may be of fundamental importance to a better understanding of the reprogramming of RPE cells in vivo.
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A transcriptome for the study of early processes of retinal regeneration in the Adult Newt, Cynops pyrrhogaster.
PloS one, 2014Co-Authors: Kenta Nakamura, Hirofumi Yasumuro, Fubito Toyama, Wataru Inami, Rafiqul Islam, Miyako Takayanagi, Ailidana Kunahong, Roman M. Casco-robles, Chikafumi ChibaAbstract:Retinal regeneration in the Adult Newt is a useful system to uncover essential mechanisms underlying the regeneration of body parts of this animal as well as to find clues to treat retinal disorders such as proliferative vitreoretinopathy. Here, to facilitate the study of early processes of retinal regeneration, we provide a de novo assembly transcriptome and inferred proteome of the Japanese fire bellied Newt (Cynops pyrrhogaster), which was obtained from eyeball samples of day 0–14 after surgical removal of the lens and neural retina. This transcriptome (237,120 in silico transcripts) contains most information of cDNAs/ESTs which has been reported in Newts (C. pyrrhogaster, Pleurodeles waltl and Notophthalmus viridescence) thus far. On the other hand, de novo assembly transcriptomes reported lately for N. viridescence only covered 16–31% of this transcriptome, suggesting that most constituents of this transcriptome are specific to the regenerating eye tissues of C. pyrrhogaster. A total of 87,102 in silico transcripts of this transcriptome were functionally annotated. Coding sequence prediction in combination with functional annotation revealed that 76,968 in silico transcripts encode protein/peptides recorded in public databases so far, whereas 17,316 might be unique. qPCR and Sanger sequencing demonstrated that this transcriptome contains much information pertaining to genes that are regulated in association with cell reprogramming, cell-cycle re-entry/proliferation, and tissue patterning in an early phase of retinal regeneration. This data also provides important insight for further investigations addressing cellular mechanisms and molecular networks underlying retinal regeneration as well as differences between retinal regeneration and disorders. This transcriptome can be applied to ensuing comprehensive gene screening steps, providing candidate genes, regardless of whether annotated or unique, to uncover essential mechanisms underlying early processes of retinal regeneration.
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Evidence for Notch signaling involvement in retinal regeneration of Adult Newt.
Brain research, 2006Co-Authors: Kenta Nakamura, Chikafumi ChibaAbstract:Abstract Involvement of Notch signaling in retinal regeneration by transdifferentiation of pigment epithelium cells was investigated using the Adult Newt Cynops pyrrhogaster. During retinal regeneration, cells expressing Notch-1 first appeared in the regenerating retina one to two cells thick (stage E-3) originated from the retinal pigment epithelium (RPE) cells, and increased in number as the regenerating retina increased in thickness. Notch-1 expression was decreased in the central retina in association with cell differentiation and became restricted to the peripheral retina. Administration of a Notch signaling blocker DAPT resulted in the appearance of a cluster of neurons, earlier than in normal regeneration, along the regenerating retina 1–3 cells thick (stage E-3 to I-1). Immunoblot analysis suggested that DAPT could perturb the processing of Notch-1. Similar results were obtained in the Newt embryonic retinal development. These results suggest that the Notch-1 signaling system may be reset to regulate neurogenesis during retinal regeneration. However, PCR analysis revealed that the Adult Newt RPE cells express Hes-1, neurogenin1 and sometimes Delta-1 Hes-1, neurogenin1 and sometimes Delta-1 all of which are differently regulated in association with retinal regeneration, implying that Notch signaling might also be involved early in the process of transdifferentiation.
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Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of Adult Newt
The Journal of comparative neurology, 2006Co-Authors: Chikafumi Chiba, Kanako Susaki, Yuko Kaneko, Fumiaki Maruo, Kenta Nakamura, Akika Hoshino, Yuka Yamano, Osamu Kuwata, Takehiko SaitoAbstract:Adult Newts can regenerate their entire retina through transdifferentiation of the retinal pigment epithelium (RPE). The objective of this study was to redescribe the retina regeneration process by means of modern biological techniques. We report two different antibodies (RPE-No.112 and MAB5428) that recognize the Newt homolog of RPE65, which is involved in the visual cycle and exclusively label the RPE cell-layer in the Adult Newt eye. We analyzed the process of retinal regeneration by immunohistochemistry and immunoblotting and propose that this process should be divided into nine stages. We found that the RPE65 protein is present in the RPE-derived new retinal rudiment at 14 days postoperative (po) and in the regenerating retinas at the 3-4 cell stage (19 days po). These observations suggest that certain characteristics of RPE cells overlap with those of retinal stem/progenitor cells during the period of transdifferentiation. However, RPE65 protein was not detected in either retinal stem/progenitor cells in the ciliary marginal zone (CMZ) of Adult eyes or in neuroepithelium present during retina development, where it was first detected in differentiated RPE. Moreover, the gene expression of RPE65 was drastically downregulated in the early phase of transdifferentiation (by 10 days po), while those of Connexin43 and Pax-6, both expressed in regenerating retinas, were differently upregulated. These observations suggest that the RPE65 protein in the RPE-derived retinal rudiment may represent the remainder after protein degradation or discharge rather than newly synthesized protein.
