RESUMO
Corneal epithelial homeostasis and regeneration are sustained by limbal stem cells (LSCs), and LSC deficiency is a major cause of blindness worldwide. Transplantation is often the only therapeutic option available to patients with LSC deficiency. However, while transplant success depends foremost on LSC frequency within grafts, a gene allowing for prospective LSC enrichment has not been identified so far. Here we show that ATP-binding cassette, sub-family B, member 5 (ABCB5) marks LSCs and is required for LSC maintenance, corneal development and repair. Furthermore, we demonstrate that prospectively isolated human or murine ABCB5-positive LSCs possess the exclusive capacity to fully restore the cornea upon grafting to LSC-deficient mice in xenogeneic or syngeneic transplantation models. ABCB5 is preferentially expressed on label-retaining LSCs in mice and p63α-positive LSCs in humans. Consistent with these findings, ABCB5-positive LSC frequency is reduced in LSC-deficient patients. Abcb5 loss of function in Abcb5 knockout mice causes depletion of quiescent LSCs due to enhanced proliferation and apoptosis, and results in defective corneal differentiation and wound healing. Our results from gene knockout studies, LSC tracing and transplantation models, as well as phenotypic and functional analyses of human biopsy specimens, provide converging lines of evidence that ABCB5 identifies mammalian LSCs. Identification and prospective isolation of molecularly defined LSCs with essential functions in corneal development and repair has important implications for the treatment of corneal disease, particularly corneal blindness due to LSC deficiency.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Transportadores de Cassetes de Ligação de ATP/metabolismo , Limbo da Córnea/citologia , Limbo da Córnea/fisiologia , Regeneração , Células-Tronco/metabolismo , Cicatrização , Subfamília B de Transportador de Cassetes de Ligação de ATP , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/deficiência , Animais , Apoptose , Biomarcadores/metabolismo , Diferenciação Celular , Proliferação de Células , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Transplante de Células-Tronco , Células-Tronco/citologia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/metabolismoRESUMO
An important aspect of wound healing is the recruitment of neutrophils to the site of infection or tissue injury. Lumican, an extracellular matrix component belonging to the small leucine rich proteoglycan (SLRP) family, is one of the major keratan sulfate proteoglycans (KSPGs) within the corneal stroma. Increasing evidence indicates that lumican can serve as a regulatory molecule for several cellular processes, including cell proliferation and migration. In the present study, we addressed the role of lumican in the process of extravasation of polymorphonuclear leukocytes (PMNs) during the early inflammatory phase present in the healing of the corneal epithelium following debridement. We used Lum(-/-) mice and a novel transgenic mouse, Lum(-/-),Kera-Lum, which expresses lumican only in the corneal stroma, to assess the role of lumican in PMN extravasation into injured corneas. Our results showed that PMNs did not readily invade injured corneas of Lum(-/-) mice and this defect was rescued by the expression of lumican in the corneas of Lum(-/-),Kera-Lum mice. The presence of lumican in situ facilitates PMN infiltration into the peritoneal cavity in casein-induced inflammation. Our findings are consistent with the notion that in addition to regulating the collagen fibril architecture, lumican acts to aid neutrophil recruitment and invasion following corneal damage and inflammation.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/imunologia , Doenças da Córnea/imunologia , Lesões da Córnea , Traumatismos Oculares/imunologia , Sulfato de Queratano/imunologia , Neutrófilos/imunologia , Animais , Córnea/imunologia , Córnea/metabolismo , Córnea/patologia , Doenças da Córnea/metabolismo , Doenças da Córnea/patologia , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Traumatismos Oculares/metabolismo , Traumatismos Oculares/patologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Lumicana , Camundongos , Lavagem Peritoneal , Cicatrização/imunologiaRESUMO
Beta-catenin signaling has been shown to play a fundamental role in embryonic development and tumorigenesis. In this study, we investigated the role of beta-catenin (Ctnnb1) in corneal homeostasis and tumorigenesis. Conditional expression of a murine Ctnnb1 gain-of-function mutation alone caused corneal neoplasia and neovascularization, resembling human ocular surface squamous neoplasia (OSSN). These corneas displayed an upregulation of cell proliferative markers (PCNA and p63), while presenting downregulation of both the Pax-6 transcription factor and the corneal differentiation marker cytokeratin 12. In addition, the expression of limbal-type keratin 15 ectopically extended to cornea, but the pattern of conjunctival keratin 4 and epidermal keratin 10 were unchanged. Moreover, epithelial E-cadherin and laminins decreased concomitantly with elevated levels of MMP-7. We also noticed a dramatic upregulation of pro-angiogenic factors (Vegf-A, Vegfr1) and angiopoietins in these corneas. Interestingly, all human OSSN specimens examined revealed nuclear beta-catenin immunoreactivity. Taken together, these results argue that beta-catenin activation is a crucial step during OSSN pathogenesis. Thus, inhibition of beta-catenin might be beneficial for treating this disease.
Assuntos
Transformação Celular Neoplásica/patologia , Córnea/metabolismo , Córnea/patologia , Proteínas Mutantes/metabolismo , beta Catenina/metabolismo , Envelhecimento/patologia , Animais , Membrana Basal/enzimologia , Membrana Basal/patologia , Caderinas/metabolismo , Diferenciação Celular , Membrana Celular/metabolismo , Membrana Celular/patologia , Núcleo Celular/metabolismo , Proliferação de Células , Transformação Celular Neoplásica/metabolismo , Regulação para Baixo , Epitélio Corneano/metabolismo , Epitélio Corneano/patologia , Neoplasias Oculares/metabolismo , Neoplasias Oculares/patologia , Humanos , Hiperplasia , Metaloproteinase 7 da Matriz/metabolismo , Camundongos , Modelos Biológicos , Neoplasias de Células Escamosas/metabolismo , Neoplasias de Células Escamosas/patologia , Transporte Proteico , Regulação para CimaRESUMO
Limbal stem cell deficiency (LSCD) leads to severe ocular surface abnormalities that can result in the loss of vision. The most successful therapy currently being used is transplantation of limbal epithelial cell sheets cultivated from a limbal biopsy obtained from the patient's healthy, contralateral eye or cadaveric tissue. In this study, we investigated the therapeutic potential of murine vibrissae hair follicle bulge-derived stem cells (HFSCs) as an autologous stem cell (SC) source for ocular surface reconstruction in patients bilaterally affected by LSCD. This study is an expansion of our previously published work showing transdifferentiation of HFSCs into cells of a corneal epithelial phenotype in an in vitro system. In this study, we used a transgenic mouse model, K12(rtTA/rtTA) /tetO-cre/ROSA(mTmG) , which allows for HFSCs to change color, from red to green, once differentiation to corneal epithelial cells occurs and Krt12, the corneal epithelial-specific differentiation marker, is expressed. HFSCs were isolated from transgenic mice, amplified by clonal expansion on a 3T3 feeder layer, and transplanted on a fibrin carrier to the eye of LSCD wild-type mice (n = 31). The HFSC transplant was able to reconstruct the ocular surface in 80% of the transplanted animals; differentiating into cells with a corneal epithelial phenotype, expressing Krt12, and repopulating the corneal SC pool while suppressing vascularization and conjunctival ingrowth. These data highlight the therapeutic properties of using HFSC to treat LSCD in a mouse model while demonstrating a strong translational potential and points to the niche as a key factor for determining stem cell differentiation.
Assuntos
Doenças da Córnea/cirurgia , Epitélio Corneano/citologia , Anormalidades do Olho/cirurgia , Folículo Piloso/transplante , Limbo da Córnea/anormalidades , Limbo da Córnea/cirurgia , Transplante de Células-Tronco , Células-Tronco/fisiologia , Animais , Transdiferenciação Celular , Células Cultivadas , Folículo Piloso/citologia , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vibrissas/citologia , Vibrissas/transplanteRESUMO
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.
Assuntos
Proteínas Morfogenéticas Ósseas/antagonistas & inibidores , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Cristalino/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Regeneração/fisiologia , Salamandridae/fisiologia , Ambystoma , Animais , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo I/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Iris/citologia , Iris/efeitos dos fármacos , Iris/crescimento & desenvolvimento , Iris/fisiologia , Cristalino/citologia , Cristalino/efeitos dos fármacos , Cristalino/crescimento & desenvolvimento , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Epitélio Pigmentado Ocular/metabolismo , Regeneração/efeitos dos fármacos , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Salamandridae/genética , Tretinoína/farmacologia , Proteína Homeobox SIX3RESUMO
PURPOSES: To determine the relationship between immunohistochemical reactivity to osteopontin, vimentin, keratin 8/18, LZTS1, and beta-catenin and clinical and histopathological prognostic factors for metastasis and death in archival specimens of primary uveal melanomas, and the prognostic value of the evaluated study variables for death from metastasis. METHODS: Retrospective analysis of clinical records and formalin-fixed, paraffin-embedded slides of primary uveal melanomas treated by enucleation during May 1 1999, through June 30 2009. Immunofluorescent staining of each tumor was assessed on newly prepared histologic slides after the application of antibodies directed against five biomarkers associated with unfavorable prognosis in uveal melanoma. RESULTS: After exclusions, our study group consisted of 82 cases. Immunofluorescence was observed in 40.2% of specimens evaluated for keratin, 50.0% evaluated for osteopontin, 26.8% evaluated for ß-catenin, 65.9% evaluated for vimentin, and 70.7% evaluated for LZTS1. Through available follow-up, 27 patients (32.9%) were dead of confirmed or suspected metastatic uveal melanoma. None of the patients whose tumor exhibited strong immunoreactivity to ß-catenin died of metastasis. In contrast, patients whose tumor exhibited immunoreactivity of any intensity to LZTS1 were more likely to develop metastasis. In multivariate Cox proportional hazards modeling, a composite variable that took into account the immunostaining for both ß-catenin and LZTS1 had a statistically significant relationship with patient's survival time. CONCLUSIONS: Our study suggests that conventional clinical and histopathological prognostic factors, and immunoexpression of ß-catenin and LZTS1 combined may allow better prognostication of metastasis than clinical and histomorphological factors alone.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Coroide/mortalidade , Neoplasias da Coroide/patologia , Corpo Ciliar/patologia , Melanoma/mortalidade , Melanoma/secundário , Proteínas de Neoplasias/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Coroide/metabolismo , Corpo Ciliar/metabolismo , Proteínas de Ligação a DNA/metabolismo , Enucleação Ocular , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Queratina-18/metabolismo , Queratina-8/metabolismo , Masculino , Melanoma/metabolismo , Pessoa de Meia-Idade , Osteopontina/metabolismo , Prognóstico , Estudos Retrospectivos , Proteínas Supressoras de Tumor/metabolismo , Vimentina/metabolismo , beta Catenina/metabolismoRESUMO
Lens regeneration in adult newts is possible by transdifferentiation of the pigment epithelial cells (PECs) of the dorsal iris. The same cells in the ventral iris are not capable of such a process. To understand this difference in regenerative competency, we examined gene expression of 373 genes in the intact dorsal and ventral irises as well as in irises during the process of lens regeneration. We found similar signatures of gene expression in dorsal and ventral with several cases of even higher levels in the ventral iris. Such transcriptional activity in the regeneration-incompetent ventral iris was unexpected and calls for a revision of our views about mechanisms of lens regeneration induction.
Assuntos
Iris/metabolismo , Cristalino/fisiologia , Regeneração/genética , Salamandridae/genética , Salamandridae/fisiologia , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Cristalino/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da PolimeraseRESUMO
PURPOSE: It has been shown that after extracapsular lens removal by anterior capsulotomy in the mouse, the lens can be regenerated. However, as the capsular bag is filled with fibers, epithelial to mesenchymal transition (EMT), an event which is common after cataract surgery as well, takes place during early stages. This study, using a unique mouse model, was undertaken to identify novel regulators and networks in order to more clearly understand secondary cataracts at the molecular level. METHODS: We examined global gene expression via microarray analysis of mouse lens regeneration after extracapsular surgery. Gene expression at different times after surgery was correlated with the processes of EMT, which is seen in the initial stages of regeneration, and lens fiber differentiation, which occurs later. RESULTS: Several notable patterns were observed from the gene clustering data. It was obvious from the analysis that initially there is a response to injury, extensive matrix remodeling, and severe downregulation of genes encoding lens structural proteins. The patterns returned gradually to normal three weeks after surgery. New genes were identified from the clustering results that might be potential regulators of EMT and lens differentiation. CONCLUSIONS: With this approach, we demonstrated the utility of a mouse model to study secondary cataracts at the molecular level. Extension of these studies in mice with known mutations affecting EMT or lens differentiation should allow the identification of the crucial molecular players that could lead to better treatments of secondary cataracts.
Assuntos
Expressão Gênica , Cristalino/fisiopatologia , Regeneração/genética , Animais , Catarata/etiologia , Sistemas Computacionais , Cristalinas/genética , Cristalinas/metabolismo , Epitélio/fisiopatologia , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica , Cristalino/metabolismo , Mesoderma , Camundongos , Camundongos Endogâmicos C57BL , Família Multigênica , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
Lens regeneration in newts is a remarkable process, whereby a lost tissue is replaced by transdifferentiation of adult tissues that only a few organisms possess. In this review, we will touch on the approaches being used to study this phenomenon, recent advances in the field of lens regeneration, similarities and differences between development and regeneration, as well as the potential role stem cells may play in understanding this process.
Assuntos
Cristalino/fisiologia , Regeneração/fisiologia , Animais , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Cristalino/citologia , Cristalino/crescimento & desenvolvimento , Salamandridae/fisiologia , Células-Tronco/fisiologiaRESUMO
Based on the role of retinoblastoma (Rb) in lens development and in the cell cycle reentry of muscle cells during limb regeneration, we have analyzed expression or Rb patterns in intact and lens regeneration-undergoing newt eyes. We find that in intact newt eye Rb is expressed in the retina as a gradient with higher levels in the photoreceptor layer and virtually no expression in the ganglion layer. In addition, a second gradient was detected within the photoreceptor layer with expression diminishing at the dorsal and ventral regions. In the intact lens, Rb is expressed in the lens epithelium and in the differentiating lens fibers at the bow region. During lens regeneration, Rb is expressed very strongly in the differentiating lens fibers, but not in the lens epithelium. Using an antibody specific to the hyperphosphorylated form of Rb, we detected the inactive protein only in the pigment epithelial cells of the iris. These distinct patterns might be related to the regenerative potential of the lens in the newt.
Assuntos
Olho/metabolismo , Cristalino/fisiologia , Notophthalmus viridescens/genética , Regeneração/fisiologia , Retinoblastoma/genética , Retinoblastoma/metabolismo , Animais , Expressão Gênica , Cristalino/metabolismo , Retina/citologia , Retina/metabolismoRESUMO
PURPOSE: Dexamethasone (DEX) regulates aqueous humor outflow by inducing a reorganization of the cytoskeleton to form cross-linked actin networks (CLANs) in trabecular meshwork (TM) cells. Rho-associated protein kinase (ROCK) has been demonstrated to have an important role in this process, but the upstream components leading to its activation remain elusive. The purpose of the study is to demonstrate that noncanonical Wnt signaling mediates the DEX-induced CLAN formation in TM cells. METHODS: The TM cells were treated with 100 nM DEX in low serum medium for over 7 days. The medium was changed every 3 days. The cells were harvested and subjected to molecular analysis for the expression of Wnt ligands. Stress fiber structures were revealed by Phalloidin staining. Lentivirus-based shRNA against noncanonical Wnt receptor (Ror2) was used to determine the role of noncanonical Wnt signaling in DEX-induced CLAN formation. RESULTS: The DEX induced stress fiber rearrangement in TM cells. A noncanonical Wnt ligand (Wnt5a) was upregulated by DEX as demonstrated by Wnt ligand degenerate PCR, real-time quantitative PCR (qRT-PCR), and Western blotting. Knocking-down Ror2, the receptor of noncanonical Wnt signaling, abolished the effects of DEX on the TM cells. CONCLUSIONS: Our data suggest that DEX induces the upregulation of noncanonical Wnt ligand Wnt5a. Recombinant WNT5a protein induces CLAN formation through the noncanonical Wnt receptor ROR2/RhoA/ROCK signaling axis. Given the similarities between DEX-induced ocular hypertension and primary open-angle glaucoma, our results provide a mechanism of action for applying ROCK inhibitor to treat primary open-angle glaucoma.
Assuntos
Actinas/metabolismo , DNA/genética , Dexametasona/farmacologia , Glaucoma de Ângulo Aberto/genética , Proteínas Proto-Oncogênicas/genética , Malha Trabecular/metabolismo , Regulação para Cima , Proteínas Wnt/genética , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/patologia , Células Cultivadas , Glaucoma de Ângulo Aberto/tratamento farmacológico , Glaucoma de Ângulo Aberto/metabolismo , Glucocorticoides/farmacologia , Humanos , Proteínas Proto-Oncogênicas/biossíntese , Reação em Cadeia da Polimerase em Tempo Real , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/biossíntese , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Malha Trabecular/citologia , Malha Trabecular/efeitos dos fármacos , Proteínas Wnt/biossíntese , Proteína Wnt-5aRESUMO
Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-ß receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.
Assuntos
Proteoglicanas de Sulfatos de Condroitina/metabolismo , Sulfato de Queratano/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Adulto , Idoso , Animais , Western Blotting , Linhagem Celular , Células Cultivadas , Proteoglicanas de Sulfatos de Condroitina/genética , Feminino , Imunofluorescência , Humanos , Sulfato de Queratano/genética , Lumicana , Masculino , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Simulação de Dinâmica Molecular , Proteínas Serina-Treonina Quinases/genética , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptor do Fator de Crescimento Transformador beta Tipo I , Receptores de Fatores de Crescimento Transformadores beta/genética , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
PURPOSE: The aim of this study was to elucidate the mechanisms governing epithelial cell migration and proliferation during wound healing. METHODS: The authors used wound healing of mouse corneal epithelium to examine the role TGF-ß signaling plays during the healing process. To achieve this goal, they used transgenic mice in which the TGF-ß receptor type II (Tbr2) was conditionally ablated from the corneal epithelium. Epithelium debridement wounds were made, followed by the assessment of cell migration, proliferation, and immunostaining of various signaling pathway components. RESULTS: The authors showed that in the absence of TGF-ß signaling corneal epithelial wound healing is delayed by 48 hours; this corresponds to a delay in p38MAPK activation. Despite the delayed p38MAPK activation, ATF2, a substrate of p38MAPK, is still phosphorylated, leading to the suppression of cell proliferation at the leading edge of the wound. These data provide evidence that in the absence of TGF-ß signaling, the suppression of cell proliferation during the early stages of wound healing is maintained through the JNK activation of ATF2. CONCLUSIONS; Together the data presented here demonstrate the importance of the TGF-ß and MAPK signaling pathways in corneal epithelial wound healing.
Assuntos
Comunicação Celular/fisiologia , Epitélio Corneano/fisiologia , Traumatismos Oculares/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Cicatrização/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Fator 2 Ativador da Transcrição/metabolismo , Animais , Movimento Celular , Proliferação de Células , Desbridamento , Epitélio Corneano/lesões , Traumatismos Oculares/patologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Masculino , Camundongos , Camundongos Transgênicos , Fosforilação , Ferimentos não Penetrantes/metabolismo , Ferimentos não Penetrantes/patologiaRESUMO
PURPOSE: The purpose of this study was to characterize a Krt12-Cre knock-in mouse line for corneal epithelium-specific gene ablation and to analyze the allelic selection of the keratin 12 (Krt12) gene during corneal type-epithelium differentiation. METHODS: Knock-in mice were generated by gene targeting. The authors examined the expression patterns of several reporter genes in the corneas of bitransgenic Krt12cre/+/ROSA(EGFP), Krt12Cre/+/ZEG, and Krt12Cre/+/ZAP mouse lines. Krt12 and cre recombinase (Cre) immunostaining was performed. Corneal epithelial cells from bitransgenic Krt12Cre/+/ROSA(EGFP) mice were examined by fluorescence-activated cell sorter. RESULTS: Mosaic and spiral expression patterns of EGFP were observed in young and adult bitransgenic Krt12Cre/+/ZEG mice, respectively. Immunostaining revealed that Cre- cells were also Krt12 negative in the corneal epithelia of Krt12Cre/-/ZAP mice. Using FACS analysis, 60% to 70% of the corneal epithelial cells from Krt12Cre/+/ROSAEGFP mice were EGFP positive, whereas 20% to 30% were negative. RT-PCR revealed that EGFP+ cells express both Krt12Cre and Krt12+ alleles, whereas EGFP- cells express only Krt12+. In the Krt12Cre/- cornea, the number of epithelial cells expressing Cre is the same as that found in Krt12Cre/Cre, which can be explained by the fragility of corneal epithelial cells that did not produce Krt12 because the Krt12Cre allele was not transcribed. CONCLUSIONS: These observations are consistent with the notion that clonal limbal stem cells randomly activate Krt12 alleles in the process of terminal differentiation. The authors suggest that this selection is advantageous for retaining epithelial cells expressing the Krt12+ allele and that it allows tolerance to structural mutations of Krt12.
Assuntos
Células Epiteliais/citologia , Queratina-12/genética , Limbo da Córnea/citologia , Limbo da Córnea/fisiologia , Células-Tronco/citologia , Alelos , Animais , Diferenciação Celular/fisiologia , Células Epiteliais/fisiologia , Epitélio Corneano/citologia , Epitélio Corneano/fisiologia , Expressão Gênica/fisiologia , Técnicas de Introdução de Genes , Genes Reporter , Proteínas de Fluorescência Verde/genética , Óperon Lac , Camundongos , Camundongos Transgênicos , Mosaicismo , Células-Tronco/fisiologiaRESUMO
MicroRNAs are known to regulate the expression of many mRNAs by binding to complementary target sequences at the 3'UTRs. Because of such properties, miRNAs may regulate tissue-specific mRNAs as a cell undergoes transdifferentiation during regeneration. We have tested this hypothesis during lens and hair cell regeneration in newts using microarray analysis. We found that distinct sets of miRNAs are associated with lens and hair cell regeneration. Members of the let-7 family are expressed in both events and they are regulated in a similar fashion. All the let-7 members are down regulated during the initiation of regeneration, which is characterized by dedifferentiation of terminally differentiated cells. This is the first report to correlate expression of miRNAs as novel regulators of vertebrate regeneration, alluding to a novel mechanism whereby transdifferentiation occurs.
Assuntos
Células Ciliadas Auditivas Internas/citologia , Células Ciliadas Auditivas Internas/metabolismo , Cristalino/citologia , Cristalino/metabolismo , Proteoma/metabolismo , Regeneração/fisiologia , Salamandridae/metabolismo , Animais , Diferenciação Celular/fisiologia , Regulação da Expressão Gênica/fisiologia , Cristalino/crescimento & desenvolvimento , MicroRNAs/genética , Morfogênese/fisiologia , Salamandridae/anatomia & histologia , Salamandridae/crescimento & desenvolvimentoRESUMO
The newt is one of the few organisms that is able to undergo lens regeneration as an adult. This review will examine the signaling pathways that are involved in this amazing phenomenon. In addition to outlining the current research involved in elucidating the key signaling molecules in lens regeneration, we will also highlight some of the similarities and differences between lens regeneration and development.
Assuntos
Cristalino/fisiologia , Regeneração/fisiologia , Transdução de Sinais/fisiologia , Animais , HumanosRESUMO
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.
Assuntos
Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Cristalino/fisiologia , Fatores de Transcrição Box Pareados/metabolismo , Regeneração , Proteínas Repressoras/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Células Epiteliais/citologia , Iris/citologia , Cristalino/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/deficiência , Epitélio Pigmentado Ocular/citologia , Epitélio Pigmentado Ocular/efeitos dos fármacos , Regeneração/efeitos dos fármacos , SalamandridaeRESUMO
In this chapter, we have touched upon some of the key processes of vertebrate limb regeneration from the formation of the wound epithelium to pattern formation, to provide a picture of the many complex and intricate facets of this system. Our synthesis incorporates recent advances in molecular biology, which has revealed some important factors related to the initiation, induction and patterning in limb regeneration.
Assuntos
Padronização Corporal/fisiologia , Extremidades/crescimento & desenvolvimento , Extremidades/lesões , Substâncias de Crescimento/metabolismo , Modelos Biológicos , Regeneração/fisiologia , Animais , Humanos , Vertebrados , Cicatrização/fisiologiaRESUMO
Lens regeneration in adult mice is possible when the lens capsule is left behind after lentectomy. The lens is regenerated by the remaining adherent lens epithelial cells, which differentiate to form lens fibres within days, showing normal morphology and bow regions. Epithelial to mesenchymal cell transformation is also seen during the early stages. The mouse, therefore, can become an indispensable animal model for cataract research, surgery and therapy.
Assuntos
Catarata/fisiopatologia , Cristalino/fisiologia , Regeneração , Animais , Extração de Catarata , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos EndogâmicosRESUMO
Lens regeneration in adult newts is always initiated from the dorsal iris by transdifferentiation of the pigment epithelial cells. One of the most important early events should be the ability of pigment epithelial cells to dedifferentiate and re-enter the cell cycle. As a first step in an attempt to study this event, we have decided to examine the effects of a cyclin-dependent kinase-2 inhibitor on lens regeneration. At the appropriate concentration, this inhibitor completely abolished the ability of pigment epithelial cells to form a new lens, but it did not stop them from dedifferentiating and forming a small lens vesicle. The effects of this inhibitor seem to be mediated by its opposite effects on cell proliferation and apoptosis. The inhibitor significantly reduced cell proliferation and enhanced apoptosis of pigment epithelial cells both in vitro and in vivo and of the regenerating lens in vivo.