RESUMO
The ability of adult vertebrates to repair tissue damage is widespread and impressive; however, the ability to regenerate structurally complex organs such as the limb is limited largely to the salamanders. The fact that most of the tissues of the limb can regenerate has led investigators to question and identify the barriers to organ regeneration. From studies in the salamander, it is known that one of the earliest steps required for successful regeneration involves signaling between nerves and the wound epithelium/apical epithelial cap (AEC). In this study we confirm an earlier report that the keratinocytes of the AEC acquire their function coincident with exiting the cell cycle. We have discovered that this unique, coordinated behavior is regulated by nerve signaling and is associated with the presence of gap junctions between the basal keratinocytes of the AEC. Disruption of nerve signaling results in a loss of gap junction protein, the reentry of the cells into the cell cycle, and regenerative failure. Finally, coordinated exit from the cell cycle appears to be a conserved behavior of populations of cells that function as signaling centers during both development and regeneration.
Assuntos
Ambystoma mexicanum/fisiologia , Epitélio/fisiologia , Queratinócitos/citologia , Transdução de Sinais , Animais , Proliferação de Células , Epitélio/inervação , Queratinócitos/fisiologia , Tecido Nervoso/fisiologia , RegeneraçãoRESUMO
The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration-wound healing, cellular dedifferentiation and blastemal formation-will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.
Assuntos
Ambystoma mexicanum/fisiologia , Extremidades/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Células Germinativas/metabolismo , Regeneração/fisiologia , Ambystoma mexicanum/genética , Sequência de Aminoácidos , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Diferenciação Celular/fisiologia , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento/genética , Técnicas de Silenciamento de Genes , Dados de Sequência Molecular , Morfolinos/genética , Regeneração/genética , Cicatrização/fisiologiaRESUMO
Salamanders possess an extraordinary capacity for tissue and organ regeneration when compared to mammals. In our effort to characterize the unique transcriptional fingerprint emerging during the early phase of salamander limb regeneration, we identified transcriptional activation of some germline-specific genes within the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells into a germline-like state. In this work, we focus on one of these genes, the long interspersed nucleotide element-1 (LINE-1) retrotransposon, which is usually active in germ cells and silent in most of the somatic tissues in other organisms. LINE-1 was found to be dramatically upregulated during regeneration. In addition, higher genomic LINE-1 content was also detected in the limb regenerate when compared to that before amputation indicating that LINE-1 retrotransposition is indeed active during regeneration. Active LINE-1 retrotransposition has been suggested to have a potentially deleterious impact on genomic integrity. Silencing of activated LINE-1 by small RNAs has been reported to be part of the machinery aiming to maintain genomic integrity. Indeed, we were able to identify putative LINE-1-related piRNAs in the limb blastema. Transposable element-related piRNAs have been identified frequently in the germline in other organisms. Thus, we present here a scenario in which a unique germline-like state is established during axolotl limb regeneration, and the re-activation of LINE-1 may serve as a marker for cellular dedifferentiation in the early-stage of limb regeneration.
Assuntos
Regulação da Expressão Gênica/fisiologia , Membro Posterior , Elementos Nucleotídeos Longos e Dispersos/fisiologia , Regeneração/fisiologia , Ambystoma mexicanum , AnimaisRESUMO
The ability of animals to repair tissue damage is widespread and impressive. Among tissues, the repair and remodeling of bone occurs during growth and in response to injury; however, loss of bone above a threshold amount is not regenerated, resulting in a "critical-size defect" (CSD). The development of therapies to replace or regenerate a CSD is a major focus of research in regenerative medicine and tissue engineering. Adult urodeles (salamanders) are unique in their ability to regenerate complex tissues perfectly, yet like mammals do not regenerate a CSD. We report on an experimental model for the regeneration of a CSD in the axolotl (the Excisional Regeneration Model) that allows for the identification of signals to induce fibroblast dedifferentiation and skeletal regeneration. This regenerative response is mediated in part by BMP signaling, as is the case in mammals; however, a complete regenerative response requires the induction of a population of undifferentiated, regeneration-competent cells. These cells can be induced by signaling from limb amputation to generate blastema cells that can be grafted to the wound, as well as by signaling from a nerve and a wound epithelium to induce blastema cells from fibroblasts within the wound environment.
Assuntos
Ambystoma mexicanum/fisiologia , Desdiferenciação Celular , Extremidades/fisiopatologia , Fibroblastos/citologia , Neurônios/metabolismo , Rádio (Anatomia)/fisiopatologia , Regeneração/fisiologia , Animais , Proteínas Morfogenéticas Ósseas/metabolismo , Calo Ósseo/patologia , Linhagem da Célula , Proliferação de Células , Condrócitos/citologia , Condrócitos/transplante , Células do Tecido Conjuntivo/citologia , Extremidades/patologia , Rádio (Anatomia)/patologia , Transdução de SinaisRESUMO
Intercalation is the process whereby cells located at the boundary of a wound interact to stimulate proliferation and the restoration of the structures between the boundaries that were lost during wounding. Thus, intercalation is widely considered to be the mechanism of regeneration. When a salamander limb is amputated, the entire cascade of regeneration events is activated, and the missing limb segments and their boundaries (joints) as well as the structures within each segment are regenerated. Therefore, in an amputated limb it is not possible to distinguish between intersegmental regeneration (formation of new segments/joints) and intrasegmental regeneration (formation of structures within a given segment), and it is not possible to study the differential regulation of these two processes. We have used two models for regeneration that allow us to study these two processes independently, and report that inter- and intrasegmental regeneration are different processes regulated by different signaling pathways. New limb segments/joints can be regenerated from cells that dedifferentiate to form blastema cells in response to signaling that is mediated in part by fibroblast growth factor.
Assuntos
Ambystoma mexicanum/fisiologia , Cotos de Amputação , Regeneração , Ambystoma mexicanum/crescimento & desenvolvimento , Cotos de Amputação/anatomia & histologia , Animais , Transdução de SinaisRESUMO
BACKGROUND: Microarray analysis and 454 cDNA sequencing were used to investigate a centuries-old problem in regenerative biology: the basis of nerve-dependent limb regeneration in salamanders. Innervated (NR) and denervated (DL) forelimbs of Mexican axolotls were amputated and transcripts were sampled after 0, 5, and 14 days of regeneration. RESULTS: Considerable similarity was observed between NR and DL transcriptional programs at 5 and 14 days post amputation (dpa). Genes with extracellular functions that are critical to wound healing were upregulated while muscle-specific genes were downregulated. Thus, many processes that are regulated during early limb regeneration do not depend upon nerve-derived factors. The majority of the transcriptional differences between NR and DL limbs were correlated with blastema formation; cell numbers increased in NR limbs after 5 dpa and this yielded distinct transcriptional signatures of cell proliferation in NR limbs at 14 dpa. These transcriptional signatures were not observed in DL limbs. Instead, gene expression changes within DL limbs suggest more diverse and protracted wound-healing responses. 454 cDNA sequencing complemented the microarray analysis by providing deeper sampling of transcriptional programs and associated biological processes. Assembly of new 454 cDNA sequences with existing expressed sequence tag (EST) contigs from the Ambystoma EST database more than doubled (3935 to 9411) the number of non-redundant human-A. mexicanum orthologous sequences. CONCLUSION: Many new candidate gene sequences were discovered for the first time and these will greatly enable future studies of wound healing, epigenetics, genome stability, and nerve-dependent blastema formation and outgrowth using the axolotl model.
Assuntos
DNA Complementar/metabolismo , Extremidades/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Regeneração/genética , Análise de Sequência de DNA/métodos , Transcrição Gênica , Ambystoma , Animais , Proliferação de Células , Análise por Conglomerados , Colágeno/química , Etiquetas de Sequências Expressas , Regulação da Expressão Gênica , Fatores de Tempo , UrodelosRESUMO
BACKGROUND: The basis of genome size variation remains an outstanding question because DNA sequence data are lacking for organisms with large genomes. Sixteen BAC clones from the Mexican axolotl (Ambystoma mexicanum: c-value = 32 x 10(9) bp) were isolated and sequenced to characterize the structure of genic regions. RESULTS: Annotation of genes within BACs showed that axolotl introns are on average 10x longer than orthologous vertebrate introns and they are predicted to contain more functional elements, including miRNAs and snoRNAs. Loci were discovered within BACs for two novel EST transcripts that are differentially expressed during spinal cord regeneration and skin metamorphosis. Unexpectedly, a third novel gene was also discovered while manually annotating BACs. Analysis of human-axolotl protein-coding sequences suggests there are 2% more lineage specific genes in the axolotl genome than the human genome, but the great majority (86%) of genes between axolotl and human are predicted to be 1:1 orthologs. Considering that axolotl genes are on average 5x larger than human genes, the genic component of the salamander genome is estimated to be incredibly large, approximately 2.8 gigabases! CONCLUSION: This study shows that a large salamander genome has a correspondingly large genic component, primarily because genes have incredibly long introns. These intronic sequences may harbor novel coding and non-coding sequences that regulate biological processes that are unique to salamanders.
Assuntos
Genoma , Íntrons/genética , Urodelos/genética , Animais , Sequência de Bases , Cromossomos Artificiais Bacterianos , Biologia Computacional , Bases de Dados de Ácidos Nucleicos , Éxons , Etiquetas de Sequências Expressas , Humanos , MicroRNAs/química , Conformação de Ácido NucleicoRESUMO
OBJECTIVES: Laser reshaping of cartilage is an emerging technology aimed at replacing conventional techniques for aesthetic and reconstructive surgery. Little is known about the mechanisms of wound healing following the photothermal heating during laser reshaping and, ultimately, how collagen remodels in the irradiated tissue. Healthy hyaline and elastic cartilage as found in the ear, nose, larynx, and trachea does not express collagen type I which is characteristic of fibro-cartilage and scar tissue. The aim of the study was to determine if collagen I and II gene expression occurs within laser irradiated rabbit septal cartilage. METHODS: Nasal septum harvested from freshly euthanized New Zealand White rabbits were irradiated with an Nd:YAG laser. After 2 weeks in culture, the laser spot and surrounding non-irradiated regions were imaged using immunofluorescence staining and evaluated using reverse transcription polymerase chain reaction (RT-PCR) to determine the presence of collagen I and II, and ascertain collagen I and II gene expression, respectively. RESULTS: All laser irradiated specimens showed a cessation in collagen II gene expression within the center of the laser spot. Collagen II was expressed in the surrounding region encircling the laser spot and within the non-irradiated periphery in all specimens. Immunohistochemistry identified only type II collagen. Neither collagen I gene expression nor immunoreactivity were identified in any specimens regardless or irradiation parameters. CONCLUSIONS: Laser irradiation of rabbit septal cartilage using dosimetry parameters similar to those used in laser reshaping does not result in the detection of either collagen I gene expression or immunoreactivity. Only collagen type II was noted after laser exposure in vitro following cell culture, which suggests that the cellular response to laser irradiation is distinct from that observed in conventional wound healing. Laser irradiation of cartilage can leave an intact collagen matrix which likely allows chondrocyte recovery on an intact scaffold.
Assuntos
Colágeno/genética , Terapia com Luz de Baixa Intensidade/métodos , Cartilagens Nasais/efeitos da radiação , Septo Nasal/efeitos da radiação , Animais , Condrócitos/efeitos da radiação , Colágeno/efeitos da radiação , Modelos Animais de Doenças , Relação Dose-Resposta à Radiação , Matriz Extracelular/genética , Matriz Extracelular/efeitos da radiação , Regulação da Expressão Gênica , Imuno-Histoquímica , Cartilagens Nasais/patologia , Septo Nasal/patologia , Coelhos , Doses de Radiação , Regeneração/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sensibilidade e EspecificidadeRESUMO
Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The recently developed Accessory Limb Model (ALM) in the axolotl provides an opportunity to identify and characterize the essential signaling events that control the early steps in limb regeneration. The ALM demonstrates that limb regeneration progresses in a stepwise fashion that is dependent on signals from the wound epidermis, nerves and dermal fibroblasts from opposite sides of the limb. When all the signals are present, a limb is formed de novo. The ALM thus provides an opportunity to identify and characterize the signaling pathways that control blastema morphogenesis and limb regeneration. Our previous study provided data on cell contribution, cell migration and nerve dependency indicating that an ectopic blastema is equivalent to an amputation-induced blastema. In the present study, we have determined that formation of both ectopic blastemas and amputation-induced blastemas is regulated by the same molecular mechanisms, and that both types of blastema cells exhibit the same functions in controlling growth and pattern formation. We have identified and validated five marker genes for the early stages of wound healing, dedifferentiation and blastema formation, and have discovered that the expression of each of these markers is the same for both ectopic and amputation-induced blastemas. In addition, ectopic blastema cells interact coordinately with amputation-induced blastema cells to form a regenerated limb. Therefore, the ALM is appropriate for identifying the signaling pathways regulating the early events of tetrapod limb regeneration.
Assuntos
Ambystoma/embriologia , Amputação Cirúrgica , Coristoma , Extremidades/embriologia , Tecido Nervoso/citologia , Tecido Nervoso/embriologia , Ambystoma/genética , Sequência de Aminoácidos , Animais , Diferenciação Celular , DNA Complementar/isolamento & purificação , Derme/transplante , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Marcadores Genéticos , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Dados de Sequência Molecular , Regeneração , Reprodutibilidade dos Testes , Proteínas com Domínio T/química , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismoRESUMO
Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The Accessory Limb Model (ALM) in the axolotl allows for the identification of signals from the wound epidermis, nerves and dermal fibroblasts that interact to regenerate a limb. In the present study, we have used the ALM to identity the axolotl (Ambystoma mexicanum) orthologue of Twist (AmTwist), a basic helix-loop-helix transcription factor that is involved in the regeneration of the dermis during limb regeneration. AmTwist is expressed during the blastema stages in regeneration, but is inhibited by signals from the nerve during the early stages when dermal fibroblasts dedifferentiate to form blastema cells. As the dermis regenerates, AmTwist is expressed in association with the synthesis of type I collagen in the proximal region of the blastema. Exogenous bone morphogenetic protein-2 leads to an increase in AmTwist expression, and therefore may function as an endogenous regulator of AmTwist expression and dermis regeneration. The nerve appears to have a dual function in regeneration by coordinately regulating dedifferentiation and redifferentiation of dermal fibroblasts.
Assuntos
Ambystoma/fisiologia , Extremidades/fisiologia , Fibroblastos/citologia , Regeneração , Cicatrização , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Animais , Desdiferenciação Celular , Diferenciação Celular , Derme/citologia , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismoRESUMO
Urodele amphibians are unique adult vertebrates because they are able to regenerate body parts after amputation. Studies of urodele limb regeneration, the key model system for vertebrate regeneration, have led to an understanding of the origin of blastema cells and the importance of positional interactions between blastema cells in the control of growth and pattern formation. Progress is now being made in the identification of the signaling pathways that regulate dedifferentiation, blastema morphogenesis, growth and pattern formation. Members of the Wnt family of secreted proteins are expressed in developing and regenerating limbs, and have the potential to control growth, pattern formation and differentiation. We have studied the expression of two non-canonical Wnt genes, Wnt-5a and Wnt-5b. We report that they are expressed in equivalent patterns during limb development and limb regeneration in the axolotl (Ambystoma mexicanum), and during limb development in other tetrapods, implying conservation of function. Our analysis of the effects of ectopic Wnt-5a expression is consistent with the hypothesis that canonical Wnt signaling functions during the early stages of regeneration to control the dedifferentiation of stump cells giving rise to the regeneration-competent cells of the blastema.
Assuntos
Ambystoma mexicanum/fisiologia , Extremidades/fisiologia , Regeneração , Proteínas Wnt/metabolismo , Animais , Northern Blotting , Diferenciação Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Hibridização In Situ , Proteínas Wnt/genética , Proteína Wnt-5aRESUMO
The explosive growth of information from genetics and genomics has led to an appreciation of the conservation of gene regulatory networks between organisms, and between development and regeneration. With ever increasing knowledge, it will be possible to intervene therapeutically to regulate these networks, which will lead to new therapies to induce regeneration. The question then becomes how to do this, rather then when to try. Our thesis is that the time is now, and that this feat can be achieved by combining the insights provided by developmental biologists with the technologies being developed by biomaterial engineers, to achieve the goal of engineering regeneration. We thus envision regenerative engineering as the next step toward achieving the goal of human regeneration. Among the most important discoveries about regeneration from studies of salamanders that regenerate exceptionally well, is that both pattern-following and pattern-forming cells are required. Much progress is being made toward understanding the former cells, but little is known about the cells that control positional information and pattern formation. Within the near future, it will become possible to provide the information needed for regeneration exogenously in the form of an engineered extracellular matrix that is a biomimetic of the endogenous information. Since growth factors (morphogens) can control pattern formation, an engineered grid could be based on spatially organized patterns of sulfation of glycosaminoglycans that control the behavior of cells by modulating morphogen activity. Progress in engineering the positional information grid for regeneration will necessitate learning the sulfation codes associated with successful regeneration in animals such as salamanders.
Assuntos
Ambystoma mexicanum/fisiologia , Extremidades/fisiologia , Regeneração/fisiologia , Salamandridae/fisiologia , Animais , Diferenciação Celular/fisiologia , Linhagem da Célula/fisiologia , Proliferação de Células/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Modelos Biológicos , Regeneração/efeitos dos fármacosRESUMO
Successful development depends on the creation of spatial gradients of transcription factors within developing fields, and images of graded distributions of gene products populate the pages of developmental biology journals. Therefore the challenge is to understand how the graded levels of intracellular transcription factors are generated across fields of cells. We propose that transcription factor gradients are generated as a result of an underlying gradient of cell cycle lengths. Very long cell cycles will permit accumulation of a high level of a gene product encoded by a large transcription unit, whereas shorter cell cycles will permit progressively fewer transcripts to be completed due to gating of transcription by the cell cycle. We also propose that the gradients of cell cycle lengths are generated by gradients of extracellular morphogens/growth factors. The model of cell cycle gated transcriptional regulation brings focus back to the functional role of morphogens as cell cycle regulators, and proposes a specific and testable mechanism by which morphogens, in their roles as growth factors (how they were originally discovered), also determine cell fate.
RESUMO
The existence of multipotent cells in the adult tissues and organs of those vertebrates that are capable of regeneration has been accepted for decades. Although studies of vertebrate limb regeneration have yet to identify many of the specific molecules involved in regeneration, numerous tissue grafting experiments and studies of cell lineage have contributed significantly to an understanding of the origin, activation, proliferation and cell-cell interactions of these progenitor cells. This has allowed the development of ideas about the regulation of pattern formation to restore the structure and function of lost tissues and organs. An understanding of the molecular mechanisms controlling these processes has lagged behind the dramatic advances achieved with other model organisms. However, given the intense, new research interest in stem cells over the past few years, there is good reason to be encouraged that insights about the biology of mammalian stem cells will accelerate progress in understanding the biology of regeneration in organisms that can regenerate. Advances in regeneration research will then feed back in terms of devising new strategies for therapies to induce regeneration in organisms such as humans that have traditionally been viewed as incapable of regeneration.
Assuntos
Extremidades/fisiologia , Regeneração/fisiologia , Células-Tronco/fisiologia , Vertebrados/fisiologia , Animais , Vasos Sanguíneos/fisiologia , Regeneração Óssea/fisiologia , Tecido Conjuntivo/fisiologia , Expressão Gênica/fisiologia , Camundongos , Músculos/fisiologia , Regeneração Nervosa/fisiologia , Transdução de Sinais/fisiologia , Cicatrização/fisiologiaRESUMO
The axolotl is one of the few tetrapods that are capable of regenerating complicated biological structures, such as complete limbs, throughout adulthood. Upon injury the axolotl generates a population of regeneration-competent limb progenitor cells known as the blastema, which will grow, establish pattern, and differentiate into the missing limb structures. In this review we focus on the crucial early events that occur during wound healing, the neural-epithelial interactions that drive the formation of the early blastema, and how these mechanisms differ from those of other species that have restricted regenerative potential, such as humans. We also discuss how the presence of cells from the different axes of the limb is required for the continued growth and establishment of pattern in the blastema as described in the polar coordinate model, and how this positional information is reprogrammed in blastema cells during regeneration. Multiple cell types from the mature limb stump contribute to the blastema at different stages of regeneration, and we discuss the contribution of these types to the regenerate with reference to whether they are "pattern-forming" or "pattern-following" cells. Lastly, we explain how an engineering approach will help resolve unanswered questions in limb regeneration, with the goal of translating these concepts to developing better human regenerative therapies.
RESUMO
Urodele amphibians are unique among adult vertebrates in their ability to regenerate complex body structures after traumatic injury. In salamander regeneration, the cells maintain a memory of their original position and use this positional information to recreate the missing pattern. We used an in vivo gain-of-function assay to determine whether components of the extracellular matrix (ECM) have positional information required to induce formation of new limb pattern during regeneration. We discovered that salamander limb ECM has a position-specific ability to either inhibit regeneration or induce de novo limb structure, and that this difference is dependent on heparan sulfates that are associated with differential expression of heparan sulfate sulfotransferases. We also discovered that an artificial ECM containing only heparan sulfate was sufficient to induce de novo limb pattern in salamander limb regeneration. Finally, ECM from mouse limbs is capable of inducing limb pattern in axolotl blastemas in a position-specific, developmental-stage-specific, and heparan sulfate-dependent manner. This study demonstrates a mechanism for positional information in regeneration and establishes a crucial functional link between salamander regeneration and mammals.
RESUMO
BACKGROUND: Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories. RESULTS: Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human - Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species. CONCLUSIONS: Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.
Assuntos
Ambystoma/genética , Etiquetas de Sequências Expressas , Ambystoma mexicanum/genética , Animais , Mapeamento Cromossômico/métodos , Mapeamento de Sequências Contíguas , Biblioteca Gênica , Humanos , Polimorfismo Genético , Ratos , Regeneração/genética , Homologia de Sequência do Ácido Nucleico , Peixe-ZebraRESUMO
Gene expression has been studied in considerable detail in the developing vertebrate brain, neural crest, and some placode-derived organs. As a further investigation of vertebrate head morphogenesis, expression patterns of several homeobox-containing genes were examined using whole-mount in situ hybridization in a sensory system primitive for the vertebrate subphylum: the axolotl lateral lines and the placodes from which they develop. Axolotl Msx-2 and Dlx-3 are expressed in all of the lateral line placodes. Both genes are expressed throughout development of the lateral line system and their expression continues in the fully developed neuromasts. Expression within support cells is highly polarized. In contrast to most other observations of Msx genes in vertebrate organogenesis, expression of Msx-2 in developing lateral line organs is exclusively epithelial and is not associated with epithelial-mesenchymal interactions. A Hox-complex gene, Hoxb-3, is shown to be expressed in the embryonic hindbrain and in a lateral line placode at the same rostrocaudal level, but not in other placodes nor in mature lateral line organs. A Hox gene of a separate paralog group, Hoxa-4, is expressed in a more posterior hindbrain domain in the embryo, but is not expressed in the lateral line placode at that rostrocaudal level. These data provide the first test of the hypothesis that the neurogenic placodes develop in two rostrocaudal series aligned with the rhombomeric segments and patterned by combinations of Hox genes in parallel with the central nervous system.
RESUMO
1. The regulative ability of the regeneration blastema of the newt limb (Notophthalmus viridescens) was tested by operationsin situ. Either the anterior, posterior, dorsal, or ventral half of the blastema was removed at various stages during regeneration. 2. All blastemas operated on prior to the stage of four early digits showed a delay in reaching the subsequent stages of regeneration. 3. The blastema is capable of extensive regulation in the anterior-posterior and dorsoventral axes even after many of its cells have begun to differentiate. 4. Early digital stages of regeneration were found to be defective in regulative ability. Additional skeletal elements were present in limbs which had been operated on at the stage of three early digits. Supernumerary digits as well as additional skeletal elements were present in limbs which had been operated on at the stage of four early digits. Removal of the posterior half of the regenerate at one of these late stages resulted in more severe abnormalities than did removal of the anterior half. 5. Either the anterior or the posterior half of a mature limb was removed back to the level of the wrist. In several cases, an almost complete autopodium developed alongside the remaining half autopodium. 6. Removal of half of a regenerate at digital stages gave results similar to those obtained following removal of half of a mature limb. 7. The results are discussed in the context of other experiments on regenerating limbs, and of experiments on other developing systems. It is concluded that amphibian blastemas in common with a number of other systems can develop according to the presumptive fates of their cells, or they can regulate when they are given the opportunity for growth and cell division.