RESUMO
BACKGROUND: Regeneration of the damaged central nervous system is one of the most interesting post-embryonic developmental phenomena. Two distinct cellular events have been implicated in supplying regenerative neurogenesis with cellular material - generation of new cells through cell proliferation and recruitment of already existing cells through cell migration. The relative contribution and importance of these two mechanisms is often unknown. METHODS: Here, we use the regenerating radial nerve cord (RNC) of the echinoderm Holothuria glaberrima as a model of extensive post-traumatic neurogenesis in the deuterostome central nervous system. To uncouple the effects of cell proliferation from those of cell migration, we treated regenerating animals with aphidicolin, a specific inhibitor of S-phase DNA replication. To monitor the effect of aphidicolin on DNA synthesis, we used BrdU immunocytochemistry. The specific radial glial marker ERG1 was used to label the regenerating RNC. Cell migration was tracked with vital staining with the lipophilic dye DiI. RESULTS: Aphidicolin treatment resulted in a significant 2.1-fold decrease in cell proliferation. In spite of this, the regenerating RNC in the treated animals did not differ in histological architecture, size and cell number from its counterpart in the control vehicle-treated animals. DiI labeling showed extensive cell migration in the RNC. Some cells migrated from as far as 2 mm away from the injury plane to contribute to the neural outgrowth. CONCLUSIONS: We suggest that inhibition of cell division in the regenerating RNC of H. glaberrima is compensated for by recruitment of cells, which migrate into the RNC outgrowth from deeper regions of the neuroepithelium. Neural regeneration in echinoderms is thus a highly regulative developmental phenomenon, in which the size of the cell pool can be controlled either by cell proliferation or cell migration, and the latter can neutralize perturbations in the former.
RESUMO
Adult neurogenesis, generation of new functional cells in the mature central nervous system (CNS), has been documented in a number of diverse organisms, ranging from humans to invertebrates. However, the origin and evolution of this phenomenon is still poorly understood for many of the key phylogenetic groups. Echinoderms are one such phylum, positioned as a sister group to chordates within the monophyletic clade Deuterostomia. They are well known for the ability of their adult organs, including the CNS, to completely regenerate after injury. Nothing is known, however, about production of new cells in the nervous tissue under normal physiological conditions in these animals. In this study, we show that new cells are continuously generated in the mature radial nerve cord (RNC) of the sea cucumber Holothuria glaberrima. Importantly, this neurogenic activity is not evenly distributed, but is significantly more extensive in the lateral regions of the RNC than along the midline. Some of the new cells generated in the apical region of the ectoneural neuroepithelium leave their place of origin and migrate basally to populate the neural parenchyma. Gene expression analysis showed that generation of new cells in the adult sea cucumber CNS is associated with transcriptional activity of genes known to be involved in regulation of various aspects of neurogenesis in other animals. Further analysis of one of those genes, the transcription factor Myc, showed that it is expressed, in some, but not all radial glial cells, suggesting heterogeneity of this CNS progenitor cell population in echinoderms.
RESUMO
BACKGROUND: Adult echinoderms can completely regenerate major parts of their central nervous system even after severe injuries. Even though this capacity has long been known, the molecular mechanisms that drive fast and complete regeneration in these animals have remained uninvestigated. The major obstacle for understanding these molecular pathways has been the lack of functional genomic studies on regenerating adult echinoderms. RESULTS: Here, we employ RNA interference-mediated gene knockdown to characterize the role of Myc during the early (first 48 hours) post-injury response in the radial nerve cord of the sea cucumber Holothuria glaberrima. Our previous experiments identified Myc as the only pluripotency-associated factor, whose expression significantly increased in the wounded CNS. The specific function(s) of this gene, however, remained unknown. Here we demonstrate that knockdown of Myc inhibits dedifferentiation of radial glia and programmed cell death, the two most prominent cellular events that take place in the regenerating sea cucumber nervous system shortly after injury. CONCLUSIONS: In this study, we show that Myc overexpression is required for proper dedifferentiation of radial glial cells and for triggering the programmed cell death in the vicinity of the injury. Myc is thus the first transcription factor, whose functional role has been experimentally established in echinoderm regeneration.
Assuntos
Apoptose/fisiologia , Diferenciação Celular , Genes myc , Neuroglia/citologia , Nervo Radial/lesões , Animais , Equinodermos , Eletroporação , Técnicas de Silenciamento de Genes , Interferência de RNA , RNA Mensageiro/genéticaRESUMO
Cell dedifferentiation is an integral component of post-traumatic regeneration in echinoderms. As dedifferentiated cells become multipotent, we asked if this spontaneous broadening of developmental potential is associated with the action of the same pluripotency factors (known as Yamanaka factors) that were used to induce pluripotency in specialized mammalian cells. In this study, we investigate the expression of orthologs of the four Yamanaka factors in regeneration of two different organs, the radial nerve cord and the digestive tube, in the sea cucumber Holothuria glaberrima. All four pluripotency factors are expressed in uninjured animals, although their expression domains do not always overlap. In regeneration, the expression levels of the four genes were not regulated in a coordinated way, but instead showed different dynamics for individual genes and also were different between the radial nerve and the gut. SoxB1, the ortholog of the mammalian Sox2, was drastically downregulated in the regenerating intestine, suggesting that this factor is not required for dedifferentiation/regeneration in this organ. On the other hand, during the early post-injury stage, Myc, the sea cucumber ortholog of c-Myc, was significantly upregulated in both the intestine and the radial nerve cord and is therefore hypothesized to play a central role in dedifferentiation/regeneration of various tissue types.
Assuntos
Holothuria/fisiologia , Células-Tronco Pluripotentes/metabolismo , Regeneração , Animais , Evolução Molecular , Regulação da Expressão Gênica , Regeneração Nervosa/fisiologia , Filogenia , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Fatores de Tempo , Fatores de Transcrição/química , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Echinoderms are emerging as important models in regenerative biology. Significant amount of data are available on cellular mechanisms of post-traumatic repair in these animals, whereas studies of gene expression are rare. In this study, we employ high-throughput sequencing to analyze the transcriptome of the normal and regenerating radial nerve cord (a homolog of the chordate neural tube), in the sea cucumber Holothuria glaberrima. RESULTS: Our de novo assembly yielded 70,173 contigs, of which 24,324 showed significant similarity to known protein-coding sequences. Expression profiling revealed large-scale changes in gene expression (4,023 and 3,257 up-regulated and down-regulated transcripts, respectively) associated with regeneration. Functional analysis of sets of differentially expressed genes suggested that among the most extensively over-represented pathways were those involved in the extracellular matrix (ECM) remodeling and ECM-cell interactions, indicating a key role of the ECM in regeneration. We also searched the sea cucumber transcriptome for homologs of factors known to be involved in acquisition and/or control of pluripotency. We identified eleven genes that were expressed both in the normal and regenerating tissues. Of these, only Myc was present at significantly higher levels in regeneration, whereas the expression of Bmi-1 was significantly reduced. We also sought to get insight into which transcription factors may operate at the top of the regulatory hierarchy to control gene expression in regeneration. Our analysis yielded eleven putative transcription factors, which constitute good candidates for further functional studies. The identified candidate transcription factors included not only known regeneration-related genes, but also factors not previously implicated as regulators of post-traumatic tissue regrowth. Functional annotation also suggested that one of the possible adaptations contributing to fast and efficient neural regeneration in echinoderms may be related to suppression of excitotoxicity. CONCLUSIONS: Our transcriptomic analysis corroborates existing data on cellular mechanisms implicated in regeneration in sea cucumbers. More importantly, however, it also illuminates new aspects of echinoderm regeneration, which have been scarcely studied or overlooked altogether. The most significant outcome of the present work is that it lays out a roadmap for future studies of regulatory mechanisms by providing a list of key candidate genes for functional analysis.
Assuntos
Sistema Nervoso Central/fisiologia , Regeneração Nervosa , Pepinos-do-Mar/fisiologia , Transcriptoma , Animais , Apoptose , Análise por Conglomerados , Mapeamento de Sequências Contíguas , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Anotação de Sequência Molecular , Neurogênese/genética , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pepinos-do-Mar/citologia , Análise de Sequência de DNARESUMO
BACKGROUND: Unlike the mammalian central nervous system (CNS), the CNS of echinoderms is capable of fast and efficient regeneration following injury and constitutes one of the most promising model systems that can provide important insights into evolution of the cellular and molecular events involved in neural repair in deuterostomes. So far, the cellular mechanisms of neural regeneration in echinoderm remained obscure. In this study we show that radial glial cells are the main source of new cells in the regenerating radial nerve cord in these animals. RESULTS: We demonstrate that radial glial cells of the sea cucumber Holothuria glaberrima react to injury by dedifferentiation. Both glia and neurons undergo programmed cell death in the lesioned CNS, but it is the dedifferentiated glial subpopulation in the vicinity of the injury that accounts for the vast majority of cell divisions. Glial outgrowth leads to formation of a tubular scaffold at the growing tip, which is later populated by neural elements. Most importantly, radial glial cells themselves give rise to new neurons. At least some of the newly produced neurons survive for more than 4 months and express neuronal markers typical of the mature echinoderm CNS. CONCLUSIONS: A hypothesis is formulated that CNS regeneration via activation of radial glial cells may represent a common capacity of the Deuterostomia, which is not invoked spontaneously in higher vertebrates, whose adult CNS does not retain radial glial cells. Potential implications for biomedical research aimed at finding the cure for human CNS injuries are discussed.
Assuntos
Holothuria/fisiologia , Regeneração Nervosa/fisiologia , Neuroglia/citologia , Animais , Bromodesoxiuridina/metabolismo , Morte Celular , Proliferação de Células , Holothuria/citologia , Holothuria/crescimento & desenvolvimento , Marcação In Situ das Extremidades Cortadas , Mitose , Neuroglia/metabolismo , Neurônios/metabolismo , Neurônios/patologiaRESUMO
BACKGROUND: Retrotransposons are mobile genetic elements that constitute a sizable proportion of eukaryote genomes. Although retroelements are known to play significant roles in embryogenesis, stress reactions, and disease progression, they have never been studied in the context of animal regeneration. RESULTS: In this study, high-throughput transcriptome analysis revealed unexpectedly large-scale changes in transcriptional activity of retrotransposons in regenerating radial organs of the sea cucumber Holothuria glaberrima. In particular, we identified 36 long terminal repeat (LTR) retroelements, of which 20 showed significant changes in their expression during regeneration (11 up-regulated, 8 down-regulated, and one was initially up-regulated, but later down-regulated). We then studied in detail the most significantly up-regulated element, Gypsy-1_Hg. This transposon showed a drastic (>50-fold) increase in expression in regeneration and started to return to the normal levels only after the anatomical organization of the injured tissues was restored. All cells expressing Gypsy-1_Hg were located in the vicinity of the wound and included glia and neurons of the radial nerve. The retrotransposon-expressing cells survived programmed cell death and contributed to regeneration. CONCLUSIONS: Our findings demonstrate considerable changes in transcriptional activity of retrotransposons (both over-expression and down-regulation) associated with posttraumatic regeneration in an echinoderm.
Assuntos
Regeneração/fisiologia , Retroelementos/genética , Pepinos-do-Mar/fisiologia , Sequências Repetidas Terminais/genética , Animais , Extremidades/fisiologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Regeneração/genéticaRESUMO
Research on the involvement of retroelements in developmental processes has been gaining momentum recently; however, most of the studies published so far have been focused on embryonic development. This commentary presents two recent papers, which document significant changes in transcriptional activity of retroelements in two different model systems, salamander limb regeneration and regeneration of radial organs in the sea cucumber Holothuria glaberrima. We hypothesize that transcriptional activity of the retrotransposons can be specifically controlled by the host and may play some hitherto unrecognized role in regeneration.
RESUMO
We employ non-radioactive in situ hybridization techniques, which combine good tissue morphology preservation with high sensitivity of transcript detection, to map gene expression in the regenerating digestive tube of the sea cucumber Holothuriaglaberrima. We investigated localization of transcripts of Wnt9, TCTP, and Bmp1/Tll, the genes that have been previously known to be implicated in embryogenesis and cancer. The choice was determined by our long-term goal of trying to understand how the developmental regulatory pathways known to be involved in tumor development can be activated in post-traumatic regeneration without leading to malignant growth. The gene expression data combined with the available morphological information highlight the gut mesothelium (the outer layer of the digestive tube) as a highly dynamic tissue, whose cells undergo remarkable changes in their phenotype and gene expression in response to injury. This reversible transition of the gut mesothelium from a complex specialized tissue to a simple epithelium composed of rapidly proliferating multipotent cells seems to depend on the expression of genes from multiple developmental/cancer-related pathways.
Assuntos
Biomarcadores Tumorais/metabolismo , Proteína Morfogenética Óssea 1/metabolismo , Regeneração , Pepinos-do-Mar/crescimento & desenvolvimento , Proteínas Wnt/metabolismo , Sequência de Aminoácidos , Estruturas Animais/citologia , Estruturas Animais/crescimento & desenvolvimento , Estruturas Animais/metabolismo , Animais , Biomarcadores Tumorais/genética , Proteína Morfogenética Óssea 1/genética , Desdiferenciação Celular , Epitélio/crescimento & desenvolvimento , Epitélio/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Modelos Biológicos , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Pepinos-do-Mar/citologia , Pepinos-do-Mar/metabolismo , Alinhamento de Sequência , Proteína Tumoral 1 Controlada por Tradução , Proteínas Wnt/genéticaRESUMO
BACKGROUND: The proper balance of cell division and cell death is of crucial importance for all kinds of developmental processes and for maintaining tissue homeostasis in mature tissues. Dysregulation of this balance often results in severe pathologies, such as cancer. There is a growing interest in understanding the factors that govern the interplay between cell death and proliferation under various conditions. Survivin and mortalin are genes that are known to be implicated in both mitosis and apoptosis and are often expressed in tumors. RESULTS: The present study takes advantage of the ability of the sea cucumber Holothuria glaberrima Selenka, 1867 (Holothuroidea, Aspidochirota) to discard its viscera and completely regrow them. This visceral regeneration involves an extensive expression of survivin and mortalin transcripts in the gut mesothelium (the outer tissue layer of the digestive tube), which coincides in time with drastic de-differentiation and a burst in cell division and apoptosis. Double labeling experiments (in situ hybridization combined with TUNEL assay or with BrdU immunohistochemistry) suggest that both genes support cell proliferation, while survivin might also be involved in suppression of the programmed cell death. CONCLUSIONS: Visceral regeneration in the sea cucumber H. glaberrima is accompanied by elevated levels of cell division and cell death, and, moreover, involves expression of pro-cancer genes, such as survivin and mortalin, which are known to support proliferation and inhibit apoptosis. Nevertheless, once regeneration is completed and the expression pattern of both genes returns to normal, the regrown digestive tube shows no anomalies. This strongly suggests that sea cucumbers must possess some robust cancer-suppression mechanisms that allow rapid re-growth of the adult tissues without leading to runaway tumor development.
Assuntos
Pepinos-do-Mar/citologia , Pepinos-do-Mar/fisiologia , Animais , Proliferação de Células , Epitélio/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Regeneração , Pepinos-do-Mar/genéticaRESUMO
The nervous system of echinoderms has long been considered too unique to be directly comparable to the nervous system of other Deuterostomia. Using two novel monoclonal antibodies in combination with epifluorescence, confocal, and electron microscopy, we demonstrate here that the central nervous system of the sea cucumber Holothuria glaberrima possesses a major non-neuronal cell type, which shares striking similarities with the radial glia of chordates. The basic features in common include (a) an elongated shape, (b) long radial processes, (c) short lateral protrusions branching off the main processes and penetrating into the surrounding neuropile, (d) prominent orderly oriented bundles of intermediate filaments, and (e) ability to produce Reissner's substance. Radial glia account for the majority of glia cells in echinoderms and constitutes more than half of the total cell population in the radial nerve cord and about 45% in the circumoral nerve ring. The difference in glia cell number between those regions is significant, suggesting structural specialization within the seemingly simple echinoderm nervous system. Both cell death and proliferation are seen under normal physiological conditions. Although both glia and neurons undergo apoptosis, most of the mitotic cells are identified as radial glia, indicating a key role of this cell type in cell turnover in the nervous system. A hypothesis is proposed that the radial glia could be an ancestral feature of the deuterostome nervous system, and the origin of this cell type might have predated the diversification of the Chordata and Ambulacraria lineages.
Assuntos
Sistema Nervoso Central/citologia , Neuroglia/fisiologia , Pepinos-do-Mar/anatomia & histologia , Animais , Bromodesoxiuridina/metabolismo , Contagem de Células/métodos , Morte Celular , Proliferação de Células , Marcação In Situ das Extremidades Cortadas/métodos , Microscopia Imunoeletrônica/métodos , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neuroglia/ultraestruturaRESUMO
BACKGROUND: Echinoderms and chordates belong to the same monophyletic taxon, the Deuterostomia. In spite of significant differences in body plan organization, the two phyla may share more common traits than was thought previously. Of particular interest are the common features in the organization of the central nervous system. The present study employs two polyclonal antisera raised against bovine Reissner's substance (RS), a secretory product produced by glial cells of the subcomissural organ, to study RS-like immunoreactivity in the central nervous system of sea cucumbers. RESULTS: In the ectoneural division of the nervous system, both antisera recognize the content of secretory vacuoles in the apical cytoplasm of the radial glia-like cells of the neuroepithelium and in the flattened glial cells of the non-neural epineural roof epithelium. The secreted immunopositive material seems to form a thin layer covering the cell apices. There is no accumulation of the immunoreactive material on the apical surface of the hyponeural neuroepithelium or the hyponeural roof epithelium. Besides labelling the supporting cells and flattened glial cells of the epineural roof epithelium, both anti-RS antisera reveal a previously unknown putative glial cell type within the neural parenchyma of the holothurian nervous system. CONCLUSION: Our results show that: a) the glial cells of the holothurian tubular nervous system produce a material similar to Reissner's substance known to be synthesized by secretory glial cells in all chordates studied so far; b) the nervous system of sea cucumbers shows a previously unrealized complexity of glial organization. Our findings also provide significant clues for interpretation of the evolution of the nervous system in the Deuterostomia. It is suggested that echinoderms and chordates might have inherited the RS-producing radial glial cell type from the central nervous system of their common ancestor, i.e., the last common ancestor of all the Deuterostomia.
RESUMO
After a complete transection, the radial nerve cord (RNC) of the adult sea cucumber Eupentacta fraudatrix quickly regrows and reconnects. The description of the major cellular events that accompany this regeneration is derived from light and transmission electron microscopy. Shortly after lesioning, the extensive nerve fiber degeneration and neuronal apoptosis occur. The gap in the cord created by the transection is rapidly bridged, at first by connective tissue and subsequently by regenerating nerve tissue. On either side of the wound, the ectoneural and hyponeural components of the injured RNC form separate tubular rudiments, whose epithelial walls are composed mostly of dedifferentiated glial cells, capable of mitotic division, but also contain some nerve fibers and occasional neuronal perikarya. It is suggested that the glial cells play a crucial role in regeneration not only by providing the supporting guiding scaffold for regrowing nerve fibers, but also by producing new neurons. Other mechanisms of post-traumatic neurogenesis may involve proliferation and/or migration of existing perikarya. The anterior and posterior regenerates grow towards one another and eventually fuse to restore the anatomical continuity of the RNC. Re-differentiation of gliocytes and accumulation of nerve cells in the newly formed regions of the nervous tissue make histological organization of the fully regenerated RNC indistinguishable from that of the intact cord. The authors suggest that the holothurian RNC provides a valuable experimental model, which opens new possibilities for exploring the fundamental mechanisms underlying regeneration of the nervous system in deuterostomes.
Assuntos
Modelos Animais de Doenças , Regeneração Nervosa/fisiologia , Nervo Radial/fisiologia , Pepinos-do-Mar , Traumatismos do Sistema Nervoso , Animais , Cones de Crescimento/ultraestrutura , Microscopia Eletrônica de Transmissão , Nervo Radial/ultraestruturaRESUMO
In adult echinoderms, the nervous system includes the ectoneural and hyponeural subsystems. The former has been believed to develop from the ectoderm, whereas the latter is considered to be mesodermal in origin. However, this view has not been substantially supported by embryological examinations. Our study deals with the developmental origin of the nervous system in the direct-developing sea cucumber Eupentacta fraudatrix. The rudiment of the adult nervous system develops from ectodermally derived cells, which ingress into the primary body cavity from the floor of the vestibule. At the earliest stages, only the rudiment of the ectoneural nerve ring is laid down. The radial nerve cords and tentacular nerves grow out from this subcutaneous rudiment. The ectoneural cords do not develop simultaneously but make their appearance in the following order: unpaired mid-ventral cord, paired dorsal lateral cords, and ventral lateral cords. These transitional developmental stages probably recapitulate the evolution of the echinoderm body plan. The holothurian hyponeural subsystem, as other regions of the metazoan nervous system, has an ectodermal origin. It originally appears as a narrow band of tissue, which bulges out of the basal region of the ectoneural neuroepithelium. Our data combined with those of other workers strongly suggest that the adult nervous tissue in echinoderms develops separately from the superficial larval system of ciliary nerves. Therefore, our data are neither in strict accordance with Garstang's hypothesis nor do they allow to refuse it. Nevertheless, in addition to ciliary bands, other areas of neurogenetic epidermis must be taken into account.
Assuntos
Sistema Nervoso Central/embriologia , Pepinos-do-Mar/embriologia , Animais , Sistema Nervoso Central/ultraestrutura , Ectoderma/fisiologia , Ectoderma/ultraestrutura , Epiderme/fisiologia , Epiderme/ultraestrutura , Pepinos-do-Mar/ultraestruturaRESUMO
Transmission electron microscopy was employed to study structural changes in the lantern muscles occurring during the transition from young to adult in the sea urchin Strongylocentrotus nudus. A comparative examination of four major lantern muscles (compass depressors, compass elevators, protractors and retractors) suggests that myogenesis involves four consecutive stages. At the initial stage, the muscles show the organization of a mesentery delimited by pseudostratified coelomic epithelia, which are composed of peritoneal cells spanning the whole height of each epithelium, and myoepithelial cells, which are clustered together to fill the interstices between the basal processes of the peritoneal cells. During the next stage, the clusters of myoepithelial cells partly "sink" into the underlying connective tissue. At the third stage of muscularization, the myoepithelial cells increase in size and further invade the underlying connective tissue so that the myoepithelium splits into an apical peritoneal layer and a deeper mass of myoepithelial cells immersed in the connective tissue. However, these two layers are connected by a continuous basal lamina. This is thus the first description of an intermediate developmental stage between pseudostratified myoepithelim and genuine echinoderm muscles. For such a myoepithelium, we propose the term "immersed myoepithelium". At the most advanced stage of myogenesis, the myocytes detach completely from the epithelium to form subepithelial muscle bundles. Myogenesis in the sea urchin takes a long time during which continuous myogenic differentiation occurs in the coelomic epithelium and the newly formed myocytes and associated neurons penetrate into the underlying connective tissue.