RESUMO
Newt lens regeneration is achieved by a unique cellular regulation of transdifferentiation where the dorsal iris pigmented epithelial cells (PECs) dedifferentiate and redifferentiate into lens cells. Recent studies have shown that nuclear architecture of PECs is dynamically changed and unique epigenetic regulation in somatic nucleus is crucial in the lens transdifferentiation. Immuno-FISH, detection of protein and gene loci in nucleus, is one of the effective tools to analyze nuclear architecture of PECs. In this chapter a whole process from lentectomy to immuno-FISH is described.
Assuntos
Transdiferenciação Celular , Hibridização in Situ Fluorescente/métodos , Cristalino/citologia , Cristalino/cirurgia , Salamandridae/cirurgia , Criação de Animais Domésticos , Animais , Plasticidade Celular , DNA Ribossômico/metabolismo , Epigênese Genética , Proteínas Nucleares/metabolismoRESUMO
Hox genes encode transcription factors that regulate embryonic and post-embryonic developmental processes. The expression of Hox genes is regulated in part by the tight, spatial arrangement of conserved coding and non-coding sequences. The potential for evolutionary changes in Hox cluster structure is thought to be low among vertebrates; however, recent studies of a few non-mammalian taxa suggest greater variation than originally thought. Using next generation sequencing of large genomic fragments (>100 kb) from the red spotted newt (Notophthalamus viridescens), we found that the arrangement of Hox cluster genes was conserved relative to orthologous regions from other vertebrates, but the length of introns and intergenic regions varied. In particular, the distance between hoxd13 and hoxd11 is longer in newt than orthologous regions from vertebrate species with expanded Hox clusters and is predicted to exceed the length of the entire HoxD clusters (hoxd13-hoxd4) of humans, mice, and frogs. Many repetitive DNA sequences were identified for newt Hox clusters, including an enrichment of DNA transposon-like sequences relative to non-coding genomic fragments. Our results suggest that Hox cluster expansion and transposon accumulation are common features of non-mammalian tetrapod vertebrates.
Assuntos
DNA Intergênico/genética , Genes Homeobox/genética , Mamíferos/genética , Família Multigênica/genética , Sequências Repetitivas de Ácido Nucleico/genética , Urodelos/genética , Animais , Pareamento de Bases/genética , Cromossomos Artificiais Bacterianos/genética , Feminino , Biblioteca Gênica , Genoma/genética , Sequências Repetitivas Dispersas/genética , Íntrons/genética , Camundongos , Salamandridae/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Regeneration of the lens in newts is quite a unique process. The lens is removed in its entirety and regeneration ensues from the pigment epithelial cells of the dorsal iris via transdifferentiation. The same type of cells from the ventral iris are not capable of regenerating a lens. It is, thus, expected that differences between dorsal and ventral iris during the process of regeneration might provide important clues pertaining to the mechanism of regeneration. In this paper, we employed next generation RNA-seq to determine gene expression patterns during lens regeneration in Notophthalmus viridescens. The expression of more than 38,000 transcripts was compared between dorsal and ventral iris. Although very few genes were found to be dorsal- or ventral-specific, certain groups of genes were up-regulated specifically in the dorsal iris. These genes are involved in cell cycle, gene regulation, cytoskeleton and immune response. In addition, the expression of six highly regulated genes, TBX5, FGF10, UNC5B, VAX2, NR2F5, and NTN1, was verified using qRT-PCR. These graded gene expression patterns provide insight into the mechanism of lens regeneration, the markers that are specific to dorsal or ventral iris, and layout a map for future studies in the field.
Assuntos
Cristalino/metabolismo , Salamandridae/genética , Salamandridae/fisiologia , Transcriptoma/genética , Animais , Perfilação da Expressão Gênica , Iris/metabolismo , Iris/fisiologia , Cristalino/fisiologia , Notophthalmus viridescens/genética , Notophthalmus viridescens/fisiologia , Reação em Cadeia da Polimerase em Tempo Real , Regeneração/genética , Regeneração/fisiologiaRESUMO
PURPOSE: Notophthalmus viridescens, the red-spotted newt, possesses tremendous regenerative capabilities. Among the tissues and organs newts can regenerate, the lens is regenerated via transdifferentiation of the pigment epithelial cells of the dorsal iris, following complete removal (lentectomy). Under normal conditions, the same cells from the ventral iris are not capable of regenerating. This study aims to further understand the initial signals of lens regeneration. METHODS: We performed microarray analysis using RNA from a dorsal or ventral iris isolated 1, 3, and 5 days after lentectomy and compared to RNA isolated from an intact iris. This analysis was supported with quantitative real-time polymerase chain reaction (qRT-PCR) of selected genes. RESULTS: Microarrays showed 804 spots were differentially regulated 1, 3, and 5 days post-lentectomy in the dorsal and ventral iris. Functional annotation using Gene Ontology revealed interesting terms. Among them, factors related to cell cycle and DNA repair were mostly upregulated, in the microarray, 3 and 5 days post-lentectomy. qRT-PCR for rad1 and vascular endothelial growth factor receptor 1 showed upregulation for the dorsal iris 3 and 5 days post- lentectomy and for the ventral iris 5 days post-lentectomy. Rad1 was also upregulated twofold more in the dorsal iris than in the ventral iris 5 days post-lentectomy (p<0.001). Factors related to redox homeostasis were mostly upregulated in the microarray in all time points and samples. qRT-PCR for glutathione peroxidase 1 also showed upregulation in all time points for the ventral and dorsal iris. For the most part, mitochondrial enzymes were downregulated with the notable exception of cytochrome c-related oxidases that were mostly upregulated at all time points. qRT-PCR for cytochrome c oxidase subunit 2 showed upregulation especially 3 days post-lentectomy for the dorsal and ventral iris (p<0.001). Factors related to extracellular matrix and tissue remodeling showed mostly upregulation (except collagen I) for all time points and samples. qRT-PCR for stromelysin 1/2 alpha and avidin showed upregulation in all the time points for the dorsal and ventral iris. CONCLUSIONS: The results show that the dorsal iris and the ventral iris follow the same general pattern with some distinct differences especially 5 days after lentectomy. In addition, while the expression of genes involved in DNA repair, redox homeostasis, and tissue remodeling in preparation for proliferation and transdifferentiation is altered in the entire iris, the response is more prominent in the dorsal iris following lentectomy.
Assuntos
Cristalino/fisiologia , Notophthalmus viridescens/genética , Notophthalmus viridescens/fisiologia , Regeneração/genética , Animais , Transdiferenciação Celular/genética , Reparo do DNA/genética , Genes cdc , Iris/citologia , Iris/fisiologia , Cristalino/citologia , Notophthalmus viridescens/anatomia & histologia , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Epitélio Pigmentado Ocular/citologia , Reação em Cadeia da Polimerase em Tempo Real , Regeneração/fisiologia , TranscriptomaRESUMO
During newt lens regeneration a unique transdifferentiation event occurs. In this process, dorsal iris pigmented epithelial cells transdifferentiate into lens cells. This system should provide a new insight into cellular plasticity in basic and applied research. Recently, a series of approaches to study epigenetic reprogramming during transdifferentiation have been performed. In this review, we introduce the regulation of dynamic regulation of core-histone modifications and the emergence of an oocyte-type linker histone during transdifferentiation. Finally, we show supporting evidence that there are common strategies of reprogramming between newt somatic cell in transdifferentiation and oocytes after somatic cell nuclear transfer.
Assuntos
Epigenômica , Cristalino/fisiologia , Regeneração , Salamandridae/fisiologia , Sequência de Aminoácidos , Animais , Transdiferenciação Celular , Reprogramação Celular , Histonas/fisiologia , Dados de Sequência MolecularRESUMO
BACKGROUND: Among vertebrates lens regeneration is most pronounced in newts, which have the ability to regenerate the entire lens throughout their lives. Regeneration occurs from the dorsal iris by transdifferentiation of the pigment epithelial cells. Interestingly, the ventral iris never contributes to regeneration. Frogs have limited lens regeneration capacity elicited from the cornea during pre-metamorphic stages. The axolotl is another salamander which, like the newt, regenerates its limbs or its tail with the spinal cord, but up until now all reports have shown that it does not regenerate the lens. RESULTS: Here we present a detailed analysis during different stages of axolotl development, and we show that despite previous beliefs the axolotl does regenerate the lens, however, only during a limited time after hatching. We have found that starting at stage 44 (forelimb bud stage) lens regeneration is possible for nearly two weeks. Regeneration occurs from the iris but, in contrast to the newt, regeneration can be elicited from either the dorsal or the ventral iris and, occasionally, even from both in the same eye. Similar studies in the zebra fish concluded that lens regeneration is not possible. CONCLUSIONS: Regeneration of the lens is possible in the axolotl, but differs from both frogs and newts. Thus the axolotl iris provides a novel and more plastic strategy for lens regeneration.
Assuntos
Ambystoma mexicanum/crescimento & desenvolvimento , Ambystoma mexicanum/fisiologia , Cristalino/crescimento & desenvolvimento , Cristalino/fisiologia , Regeneração/fisiologia , Animais , Proliferação de Células , Embrião não Mamífero/citologia , Cabeça/crescimento & desenvolvimento , Larva/citologia , Cristalino/citologia , Cristalino/cirurgiaRESUMO
PURPOSE: To evaluate the effects of complement employing a mouse model for secondary cataract. METHODS: The role of complement receptor C5a (CD88) was evaluated after cataract surgery in mice. An antagonist specific to C5a receptor was administered intraperitoneally to mice. Epithelial to mesenchymal transition (EMT) was evaluated by alpha-smooth muscle actin (α-SMA) staining and proliferation by bromodeoxyuridine (5-bromo-2'-deoxyuridine, BrdU) incorporation. Gene expression patterns was examined by microarray analysis and quantitative polymerase chain reaction (QPCR). RESULTS: We found that administration of a C5aR antagonist in C57BL/6J mice decreases EMT, as evidenced by α-SMA expression, and cell proliferation. Gene expression by microarray analysis reveals discreet steps of gene regulation in the two major stages that of EMT and lens fiber differentiation in vivo. A hallmark of the microarray analysis is that the antagonist seems to be a novel stage-specific regulator of crystallin genes. At week two, which is marked by lens fiber differentiation genes encoding 12 crystallins and 3 lens-specific structural proteins were severely down-regulated. CONCLUSIONS: These results suggest a possible therapeutic role of an antagonist to C5aR in preventing secondary cataracts after surgery. Also these results suggest that crystallin gene expression can be regulated by pro-inflammatory events in the eye.
Assuntos
Catarata/metabolismo , Cristalinas/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Peptídeos Cíclicos/administração & dosagem , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Actinas/análise , Animais , Bromodesoxiuridina/análise , Catarata/tratamento farmacológico , Catarata/genética , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Complemento C5a/metabolismo , Cristalinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica , Injeções Intraperitoneais , Cristalino/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Procedimentos Cirúrgicos Oftalmológicos/efeitos adversos , Peptídeos Cíclicos/uso terapêutico , Reação em Cadeia da Polimerase , Receptor da Anafilatoxina C5a/metabolismoRESUMO
Here we describe a protocol for gene loss of function during regeneration in newts, specifically applied to lens regeneration. Knockdown with the use of morpholinos can be achieved both in vitro and in vivo, depending on the experimental design. These methods achieve desirable levels of gene knockdown, and thus can be compared with methods developed for use in other animals, such as zebrafish. The technology has been applied to study molecular mechanisms during the process of lens regeneration by knocking down genes at specific stages and examining their effects on other genes and lens differentiation. The protocol can take a few days or up to 20 d to complete, depending on the duration of the experiment.
Assuntos
Técnicas de Silenciamento de Genes , Cristalino/fisiologia , Salamandridae/genética , Animais , Modelos Animais , RegeneraçãoRESUMO
The great regenerative abilities of newts provide the impetus for studies at the molecular level. However, efficient methods for gene regulation have historically been quite limited. Here we describe a protocol for transgenically expressing exogenous genes in the newt Cynops pyrrhogaster. This method is simple: a reaction mixture of I-SceI meganuclease and a plasmid DNA carrying a transgene cassette flanked by the enzyme recognition sites is directly injected into fertilized eggs. The protocol achieves a high efficiency of transgenesis, comparable to protocols used in other animal systems, and it provides a practical number of transgenic newts (â¼20% of injected embryos) that survive beyond metamorphosis and that can be applied to regenerative studies. The entire protocol for obtaining transgenic adult newts takes 4-5 months.
Assuntos
Técnicas de Transferência de Genes , Salamandridae/genética , Animais , Animais Geneticamente Modificados/genética , Feminino , MasculinoRESUMO
PURPOSE: Reprogramming of pigmented epithelial cells (PECs) is a decisive process in newt lens regeneration. After lens removal PECs in dorsal iris dedifferentiate and revert to stem cell-like cells, and transdifferentiate into lens cells. Our purpose is to know how global histone modifications are regulated in the reprogramming of PECs. METHODS: Iris sections were stained using various histone modification-specific antibodies. The intensity of stained signal in nucleus of PECs was measured and changes in histone modification during dedifferentiation were evaluated. RESULTS: During dedifferentiation of PECs histone modifications related to gene activation were differentially regulated. Although tri-methylated histone H3 lysine 4 (TriMeH3K4) and acetylated histone H4 (AcH4) were increased, acetylated histone H3 lysine 9 (AcH3K9) was decreased during dedifferentiation. Among all gene repression-related modifications analyzed only tri-methylated histone H3 lysine 27 (TriMeH3K27) showed a significant change. Although in the dorsal iris TriMeH3K27 was kept at same levels after lentectomy, in ventral iris it was increased. CONCLUSIONS: Histone modifications are dynamically changed during dedifferentiation of PECs. A coordination of gene activation-related modifications, increasing of TriMeH3K4 and AcH4 and decreasing of AcH3K9, as well as regulation of TriMeH3K27, could be a hallmark of chromatin regulation during newt dedifferentiation.
Assuntos
Desdiferenciação Celular , Histonas/metabolismo , Cristalino/citologia , Cristalino/fisiologia , Processamento de Proteína Pós-Traducional , Regeneração/fisiologia , Salamandridae/fisiologia , Animais , Desdiferenciação Celular/genética , Regeneração/genética , Ativação TranscricionalRESUMO
BACKGROUND: Lens regeneration in adult newts occurs via transdifferentiation of the pigment epithelial cells (PECs) of the dorsal iris. The same source of cells from the ventral iris is not able to undergo this process. In an attempt to understand this restriction we have studied in the past expression patterns of miRNAs. Among several miRNAs we have found that mir-148 shows an up-regulation in the ventral iris, while members of the let-7 family showed down-regulation in dorsal iris during dedifferentiation. METHODOLOGY/PRINCIPAL FINDINGS: We have performed gain- and loss-of-function experiments of mir-148 and let-7b in an attempt to delineate their function. We find that up-regulation of mir-148 caused significant decrease in the proliferation rates of ventral PECs only, while up-regulation of let-7b affected proliferation of both dorsal and ventral PECs. Neither miRNA was able to affect lens morphogenesis or induction. To further understand how this effect of miRNA up-regulation is mediated we examined global expression of miRNAs after up-regulation of mir148 and let-7b. Interestingly, we identified a novel level of mirRNA regulation, which might indicate that miRNAs are regulated as a network. CONCLUSION/SIGNIFICANCE: The major conclusion is that different miRNAs can control proliferation in the dorsal or ventral iris possibly by a different mechanism. Of interest is that down-regulation of the let-7 family members has also been documented in other systems undergoing reprogramming, such as in stem cells or oocytes. This might indicate that reprogramming during newt regeneration shares common molecular signatures with reprogramming in stem or germ cells. On the other hand that miRNAs can regulate the levels of other miRNAs is a novel level of regulation, which might provide new insights on their function.
Assuntos
Cristalino/citologia , Cristalino/fisiologia , MicroRNAs/genética , MicroRNAs/metabolismo , Regeneração , Salamandridae/genética , Salamandridae/fisiologia , Animais , Diferenciação Celular , Proliferação de Células , Regulação da Expressão Gênica , Iris/citologia , Iris/metabolismo , Cristalino/metabolismo , Luciferases/metabolismo , Epitélio Pigmentado Ocular/citologia , TransfecçãoRESUMO
The ability to reprogram in vivo a somatic cell after differentiation is quite limited. One of the most impressive examples of such a process is transdifferentiation of pigmented epithelial cells (PECs) to lens cells during lens regeneration in newts. However, very little is known of the molecular events that allow newt cells to transdifferentiate. Histone B4 is an oocyte-type linker histone that replaces the somatic-type linker histone H1 during reprogramming mediated by somatic cell nuclear transfer (SCNT). We found that B4 is expressed and required during transdifferentiation of PECs. Knocking down of B4 decreased proliferation and increased apoptosis, which resulted in considerable smaller lens. Furthermore, B4 knockdown altered gene expression of key genes of lens differentiation and nearly abolished expression of gamma-crystallin. These data are the first to show expression of oocyte-type linker histone in somatic cells and its requirement in newt lens transdifferentiation and suggest that transdifferentiation in newts might share common strategies with reprogramming after SCNT.
Assuntos
Histonas/metabolismo , Cristalino/metabolismo , Salamandridae/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Proliferação de Células , Transdiferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Feminino , Histonas/genética , Imuno-Histoquímica , Cristalino/citologia , Dados de Sequência Molecular , Técnicas de Transferência Nuclear , Regeneração/genética , Regeneração/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Salamandridae/genética , gama-Cristalinas/genética , gama-Cristalinas/metabolismoRESUMO
PURPOSE: The adult newt can regenerate lens from pigmented epithelial cells (PECs) of the dorsal iris via dedifferentiation. The purpose of this research is to obtain sequence resources for a newt lens regeneration study and to obtain insights of dedifferentiation at the molecular level. METHODS: mRNA was purified from iris during dedifferentiation and its cDNA library was constructed. From the cDNA library 10,449 clones were sequenced and analyzed. RESULTS: From 10,449 reads, 780 contigs and 1,666 singlets were annotated. The presence of several cancer- and apoptosis-related genes during newt dedifferentiation was revealed. Moreover, several candidate genes, which might participate in reprogramming during dedifferentiation, were also found. CONCLUSIONS: The expression of cancer- and apoptosis-related genes could be hallmarks during dedifferentiation. The expression sequence tag (EST) resource is useful for the future study of newt dedifferentiation, and the sequence information is available in GenBank (accession numbers; FS290155-FS300559).
Assuntos
Desdiferenciação Celular/genética , Etiquetas de Sequências Expressas/metabolismo , Perfilação da Expressão Gênica , Cristalino/citologia , Cristalino/fisiologia , Regeneração/genética , Salamandridae/genética , Animais , Apoptose/genética , Reprogramação Celular/genética , Regulação da Expressão Gênica , Iris/citologia , Iris/metabolismo , Neoplasias/genética , Análise de Sequência de DNARESUMO
In this study, we present data indicating that mammalian stem cell pluripotency-inducing factors are expressed during lens and limb regeneration in newts. The apparent expression even in intact tissues and the ensued regulation during regeneration raises the possibility that these factors might regulate tissue-specific reprogramming and regeneration. Furthermore, these factors should enable us to understand the similarities and differences between animal regeneration in the newt and stem cell strategies in mammals. Developmental Dynamics 238:1613-1616, 2009. (c) 2009 Wiley-Liss, Inc.
Assuntos
Células-Tronco Pluripotentes/fisiologia , Regeneração/fisiologia , Salamandridae , Animais , Extremidades/anatomia & histologia , Extremidades/fisiologia , Proteínas de Homeodomínio/classificação , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Cristalino/anatomia & histologia , Cristalino/fisiologia , Fator 3 de Transcrição de Octâmero/classificação , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Filogenia , Salamandridae/anatomia & histologia , Salamandridae/fisiologiaRESUMO
Urodele amphibians (newts and salamanders) are important animal models for understanding regeneration mechanisms and genome evolution. We constructed ideograms of BrdU/dT- and C-banded karyotypes in the Japanese fire-belly newt, Cynops pyrrhogaster, which is useful as a model animal with extremely high ability of regeneration. We also established a high-resolution FISH mapping system for newts, and localized satellite DNA sequences, 18S rDNAs, telomeric (TTAGGG)n repeats and seven functional genes, including genes associated with lens regeneration, tyrosinase and two types of gamma crystallins, to chromosomes of the newt. The 18S rDNAs were localized to three chromosomal pairs in males, whereas the chromosomal locations were highly variable in females. No hybridization signals were detected for the telomeric (TTAGGG)n sequence. All three lens regeneration-related genes were mapped on the short arm of chromosome 7, suggesting that the location of the genes in the same linkage group may be correlated with the regulation of gene expression associated with chromatin dynamics in interphase nuclei during regeneration. The chromosomal distribution and nucleotide sequences of pericentric satellite DNA sequences were well conserved between C. pyrrhogaster and European newts; in contrast, there was species specificity of nucleotide sequences for centromere-specific satellite DNAs.
Assuntos
Cromossomos/genética , Hibridização in Situ Fluorescente/métodos , Salamandridae/genética , Animais , Mapeamento Cromossômico/métodos , DNA/genética , DNA Ribossômico/genética , Feminino , Cariotipagem , Masculino , Regeneração/genética , Sequências Repetitivas de Ácido Nucleico , Salamandridae/fisiologia , Telômero/genéticaRESUMO
In newt regeneration, differentiated cells can revert to stem cell-like cells in which the proliferative ability and multipotentiality are restored after dedifferentiation. However, the molecular events that occur during the dedifferentiation still remain obscure. Nucleostemin has been identified in mammals as a nucleolar protein specific to stem cells and cancer cells. In this study, a newt nucleostemin homologue was cloned and its regulation was analyzed. During lens regeneration, the expression of nucleostemin was activated and nucleostemin rapidly accumulated in the nucleoli of dedifferentiating pigmented epithelial cells 2 days before cell cycle reentry. During limb regeneration, nucleostemin also accumulated in the nucleoli of degenerating multinucleate muscle fibers before blastema formation. These findings suggest that nucleostemin plays a role in the dedifferentiation of newt cells and can provide crucial clues for addressing the molecular events at early steps of cellular dedifferentiation in newts.