RESUMO
In amniote limbs, Fibroblast Growth Factor 10 (FGF10) is essential for limb development, but whether this function is broadly conserved in tetrapods and/or involved in adult limb regeneration remains unknown. To tackle this question, we established Fgf10 mutant lines in the newt Pleurodeles waltl which has amazing regenerative ability. While Fgf10 mutant forelimbs develop normally, the hindlimbs fail to develop and downregulate FGF target genes. Despite these developmental defects, Fgf10 mutants were able to regenerate normal hindlimbs rather than recapitulating the embryonic phenotype. Together, our results demonstrate an important role for FGF10 in hindlimb formation, but little or no function in regeneration, suggesting that different mechanisms operate during limb regeneration versus development.
Assuntos
Fator 10 de Crescimento de Fibroblastos , Animais , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Membro Posterior/crescimento & desenvolvimento , Regeneração , Pleurodeles/genética , Pleurodeles/crescimento & desenvolvimento , Pleurodeles/metabolismoRESUMO
Newts can regenerate functional elbow joints after amputation at the joint level. Previous studies have suggested the potential contribution of cells from residual tendon tissues to joint cartilage regeneration. A serum-free tissue culture system for tendons was established to explore cell dynamics during joint regeneration. Culturing isolated tendons in this system, stimulated by regeneration-related factors, such as fibroblast growth factor (FGF) and platelet-derived growth factor, led to robust cell migration and proliferation. Moreover, cells proliferating in an FGF-rich environment differentiated into Sox9-positive chondrocytes upon BMP7 introduction. These findings suggest that FGF-stimulated cells from tendons may aid in joint cartilage regeneration during functional elbow joint regeneration in newts.
Assuntos
Proteína Morfogenética Óssea 7 , Condrócitos , Fatores de Crescimento de Fibroblastos , Animais , Diferenciação Celular , Condrócitos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/metabolismo , Salamandridae/metabolismo , Tendões/metabolismo , Proteína Morfogenética Óssea 7/metabolismo , Proteína Morfogenética Óssea 7/farmacologiaRESUMO
Preparing viable single cells is critical for conducting single-cell RNA sequencing (scRNA-seq) because the presence of ambient RNA from dead or damaged cells can interfere with data analysis. Here, we developed a method for isolating viable single cells from adult planarian bodies using fluorescence-activated cell sorting (FACS). This method was then applied to both adult pluripotent stem cells (aPSCs) and differentiating/differentiated cells. Initially, we employed a violet instead of ultraviolet (UV) laser to excite Hoechst 33342 to reduce cellular damage. After optimization of cell staining conditions and FACS compensation, we generated FACS profiles similar to those created using a previous method that employed a UV laser. Despite successfully obtaining high-quality RNA sequencing data for aPSCs, non-aPSCs produced low-quality RNA reads (i.e., <60% of cells possessing barcoding mRNAs). Subsequently, we identified an effective FACS gating condition that excluded low-quality cells and tissue debris without staining. This non-staining isolation strategy not only reduced post-dissociation time but also enabled high-quality scRNA-seq results for all cell types (i.e., >80%). Taken together, these findings imply that the non-staining FACS strategy may be beneficial for isolating viable cells not only from planarians but also from other organisms and tissues for scRNA-seq studies.
Assuntos
Planárias , Células-Tronco Pluripotentes , Animais , Citometria de Fluxo/métodos , Planárias/genética , Análise da Expressão Gênica de Célula Única , RNA MensageiroRESUMO
Planarians show outstanding regenerative ability due to the proliferation of neoblasts. Hence the method to isolate planarian neoblasts is important to understand the regeneration process. In our previous study, we reported a method to isolate planarian neoblasts of Dugesia japonica using fluorescence-activated cell sorting (FACS). However, we have not yet succeeded in cultivating these cells even under in vivo conditions after transplantation into x-ray-irradiated planarians. This suggests that dissociated cells might enter apoptotic or necrotic states in the process of fluorescent dye staining and sorting. Here, we developed a new method to isolate viable neoblasts, which can proliferate in the x-ray-irradiated planarians. First, the toxicity of various fluorescence dyes was investigated. All nuclear fluorescent dyes such as Hoechst 33342, DRAQ5, and DyeCycle, showed, more or less, toxicity to mammalian culture cells. In contrast, cytoplasmic fluorescent dye for live cells, calcein AM, was less toxic on these cells. Next, we stained the dissociated planarian cells with only calcein AM, and then collected the x-ray-sensitive fraction. Although the purity of neoblasts was slightly lower than that of the original staining method (ca. 97% â ca. 89%), the sorted cells could actively proliferate when they were injected into x-ray-irradiated planarians. This simple staining and sorting method will provide new opportunities to isolate viable neoblasts and understand regenerating processes.
Assuntos
Planárias , Animais , Citometria de Fluxo/métodos , Raios X , Corantes Fluorescentes/farmacologia , MamíferosRESUMO
Transgenic techniques have greatly increased our understanding of the transcriptional regulation of target genes through live reporter imaging, as well as the spatiotemporal function of a gene using loss- and gain-of-function constructs. In Xenopus species, two well-established transgenic methods, restriction enzyme-mediated integration and I-SceI meganuclease-mediated transgenesis, have been used to generate transgenic animals. However, donor plasmids are randomly integrated into the Xenopus genome in both methods. Here, we established a new and simple targeted transgenesis technique based on CRISPR/Cas9 in Xenopus laevis. In this method, Cas9 ribonucleoprotein (RNP) targeting a putative harbor site (the transforming growth factor beta receptor 2-like (tgfbr2l) locus) and a preset donor plasmid DNA were co-injected into the one-cell stage embryos of X. laevis. Approximately 10% of faithful reporter expression was detected in F0 crispants in a promoter/enhancer-specific manner. Importantly, efficient germline transmission and stable transgene expression were observed in the F1 offspring. The simplicity of this method only required preparation of a donor vector containing the tgfbr2l genome fragment and Cas9 RNP targeting this site, which are common experimental procedures used in Xenopus laboratories. Our improved technique allows the simple generation of transgenic X. laevis, so is expected to become a powerful tool for reporter assay and gene function analysis.
Assuntos
Sistemas CRISPR-Cas , Técnicas de Transferência de Genes , Animais , Animais Geneticamente Modificados , Sistemas CRISPR-Cas/genética , Ribonucleoproteínas/genética , Transgenes , Xenopus laevis/genéticaRESUMO
Urodele amphibians, Pleurodeles waltl and Ambystoma mexicanum, have organ-level regeneration capability, such as limb regeneration. Multipotent cells are induced by an endogenous mechanism in amphibian limb regeneration. It is well known that dermal fibroblasts receive regenerative signals and turn into multipotent cells, called blastema cells. However, the induction mechanism of the blastema cells from matured dermal cells was unknown. We previously found that BMP2, FGF2, and FGF8 (B2FF) could play sufficient roles in blastema induction in urodele amphibians. Here, we show that B2FF treatment can induce dermis-derived cells that can participate in multiple cell lineage in limb regeneration. We first established a newt dermis-derived cell line and confirmed that B2FF treatment on the newt cells provided plasticity in cellular differentiation in limb regeneration. To clarify the factors that can provide the plasticity in differentiation, we performed the interspecies comparative analysis between newt cells and mouse cells and found the Pde4b gene was upregulated by B2FF treatment only in the newt cells. Blocking PDE4B signaling by a chemical PDE4 inhibitor suppressed dermis-to-cartilage transformation and the mosaic knockout animals showed consistent results. Our results are a valuable insight into how dermal fibroblasts acquire multipotency during the early phase of limb regeneration via an endogenous program in amphibian limb regeneration.
RESUMO
The 5'Hox genes play crucial roles in limb development and specify regions in the proximal-distal axis of limbs. However, there is no direct genetic evidence that Hox genes are essential for limb development in non-mammalian tetrapods or for limb regeneration. Here, we produced single to quadruple Hox13 paralog mutants using the CRISPR/Cas9 system in newts (Pleurodeles waltl), which have strong regenerative capacities, and also produced germline mutants. We show that Hox13 genes are essential for digit formation in development, as in mice. In addition, Hoxa13 has a predominant role in digit formation, unlike in mice. The predominance is probably due to the restricted expression pattern of Hoxd13 in limb buds and the strong dependence of Hoxd13 expression on Hoxa13. Finally, we demonstrate that Hox13 genes are also necessary for digit formation in limb regeneration. Our findings reveal that the general function of Hox13 genes is conserved between limb development and regeneration, and across taxa. The predominance of Hoxa13 function both in newt limbs and fish fins, but not in mouse limbs, suggests a potential contribution of Hoxa13 function in fin-to-limb transition.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio , Animais , Extremidades , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Botões de Extremidades/metabolismo , Camundongos , Salamandridae/genética , Salamandridae/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The migration of adult stem cells in vivo is an important issue, but the complex tissue structures involved, and limited accessibility of the cells hinder a detailed investigation. To overcome these problems, the freshwater planarian Dugesia japonica was used because it has a simple body plan and abundant adult pluripotent stem cells (neoblasts) distributed uniformly throughout its body. To investigate the migratory mechanisms of neoblasts, two planarian homologous genes of metastatic tumor antigen (MTA-A and MTA-B), a protein involved in cancer metastasis that functions through histone deacetylation, were identified, and their function was analyzed using RNA interference (RNAi). MTA-A or MTA-B knockdown disrupted homeostatic tissue turnover and regeneration in planarians. Whereas neoblasts in MTA-A (RNAi) and MTA-B (RNAi) animals were maintained, neoblast differentiation was inhibited. Furthermore, the normal uniform neoblast distribution pattern changed to a branch-like pattern in MTA-A (RNAi) and MTA-B (RNAi) animals. To examine the neoblast migratory ability, a partial X-ray irradiation assay was performed in D. japonica. Using this assay system, the MTA-A knockdown neoblasts migrated collectively in a branch-like pattern, and the MTA-B knockdown neoblasts were not able to migrate. These results indicated that MTA-A was required for the exit of neoblasts from the branch-like region, and that MTA-B was required for neoblast migration. Thus, the migration mediated by MTA-A and MTA-B enabled uniform neoblast distribution and was required for neoblast differentiation to achieve tissue homeostasis and regeneration.
Assuntos
Células-Tronco Adultas , Planárias , Células-Tronco Pluripotentes , Animais , Diferenciação Celular/genéticaRESUMO
The regenerative ability of planarians relies on their adult pluripotent stem cell population. Although all stem cells express a piwi homolog, recently it has become possible to classify the piwi+ stem cell population into specialized subpopulations according to the expression of genes related to differentiation. However, piwi+ stem cells behave practically as a homogeneous population after amputation, during which stem cells show accelerated proliferation, named 'induced hyperproliferation'. Here, we show that plac8-A was expressed in almost all of the stem cells, and that a decrease of the plac8-A expression level led to induced hyperproliferation uniformly in a broad stem cell subpopulation after amputation. This reduction of plac8-A expression was caused by activated JNK signaling after amputation. Pharmacological inhibition of JNK signaling caused failure to induce hyperproliferation and resulted in regenerative defects. Such defects were abrogated by simultaneous knockdown of plac8-A expression. Thus, JNK-dependent suppression of plac8-A expression is indispensable for stem cell dynamics involved in regeneration. These findings suggest that plac8-A acts as a molecular switch of piwi+ stem cells for entry into the regenerative state after amputation.
Assuntos
Proteínas de Helminto/genética , Planárias/fisiologia , Regeneração/fisiologia , Animais , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Proliferação de Células , Regulação para Baixo , Proteínas de Helminto/antagonistas & inibidores , Proteínas de Helminto/metabolismo , Sistema de Sinalização das MAP Quinases , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Interferência de RNA , RNA de Cadeia Dupla/metabolismoRESUMO
Research on individual behaviors can help to reveal the processes and mechanisms that mediate an animal's habits and interactions with the environment. Importantly, individual behaviors arise as outcomes of genetic programs, morphogenesis, physiological processes, and neural functions; thus, behavioral analyses can be used to detect disorders in these processes. Planarians belong to an early branching bilateral group of organisms that possess a simple central nervous system. Furthermore, planarians display various behavioral responses to the environment via their nervous system. Planarians also have remarkable regenerative abilities, including whole-brain regeneration. Therefore, the combination of planarians' phylogenetic position, behavioral properties, regenerative ability, and genetic accessibility provides a unique opportunity to understand the basic mechanisms underlying the anatomical properties of neural morphogenesis and the dynamic physiological processes and neural function. Here, we describe a step-by-step protocol for conducting simple behavioral analyses in planarians with the aim of helping to introduce researchers to the utility of performing behavioral analyses in planarians. Since the conditions of planarians impact experimental results and reproducibility, this protocol begins with a method for maintaining planarians. Next, we introduce the behavioral tests as well as the methods for quantifying them using minimal and cost-effective equipment and materials. Finally, we present a unique RNAi technique that enables conditional silencing of neural activity in the brain of planarians.
Assuntos
Planárias , Animais , Encéfalo , Sistema Nervoso Central , Filogenia , Planárias/genética , Reprodutibilidade dos TestesRESUMO
The coincidence of cell cycle exit and differentiation has been described in a wide variety of stem cells and organisms for decades, but the causal relationship is still unclear due to the complicated regulation of the cell cycle. Here, we used the planarian Dugesia japonica since they may possess a simple cell cycle regulation in which Cdh1 is one of the factors responsible for exiting the cell cycle. When cdh1 was functionally inhibited, the planarians could not maintain their tissue homeostasis and could not regenerate their missing body parts. While the knockdown of cdh1 caused pronounced accumulation of the stem cells, the progenitor and differentiated cells were decreased. Further analyses indicated that the stem cells with cdh1 knockdown did not undergo differentiation even though they received ERK signaling activation as an induction signal. These results suggested that stem cells could not acquire differentiation competence without cell cycle exit. Thus, we propose that cell cycle regulation determines the differentiation competence and that cell cycle exit to G0 enables stem cells to undergo differentiation.
Assuntos
Proteínas Cdh1/genética , Ciclo Celular/fisiologia , Planárias/crescimento & desenvolvimento , Regeneração/genética , Animais , Proteínas Cdh1/metabolismo , Diferenciação Celular/fisiologia , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Planárias/citologia , Interferência de RNA , Regeneração/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
Hemimetabolous insects, such as the two-spotted cricket Gryllus bimaculatus, can recover lost tissues, in contrast to the limited regenerative abilities of human tissues. Following cricket leg amputation, the wound surface is covered by the wound epidermis, and plasmatocytes, which are insect macrophages, accumulate in the wound region. Here, we studied the function of Toll-related molecules identified by comparative RNA sequencing during leg regeneration. Of the 11 Toll genes in the Gryllus genome, expression of Toll2-1, Toll2-2 and Toll2-5 was upregulated during regeneration. RNA interference (RNAi) of Toll, Toll2-1, Toll2-2, Toll2-3 or Toll2-4 produced regeneration defects in more than 50% of crickets. RNAi of Toll2-2 led to a decrease in the ratio of S- and M-phase cells, reduced expression of JAK/STAT signalling genes, and reduced accumulation of plasmatocytes in the blastema. Depletion of plasmatocytes in crickets using clodronate also produced regeneration defects, as well as fewer proliferating cells in the regenerating legs. Plasmatocyte depletion also downregulated the expression of Toll and JAK/STAT signalling genes in the regenerating legs. These results suggest that Spz-Toll-related signalling in plasmatocytes promotes leg regeneration through blastema cell proliferation by regulating the Upd-JAK/STAT signalling pathway.
Assuntos
Gryllidae/metabolismo , Membro Posterior/fisiologia , Proteínas de Insetos/biossíntese , Regeneração , Transdução de Sinais , Receptores Toll-Like/biossíntese , Animais , Regulação da Expressão Gênica , Gryllidae/genética , Proteínas de Insetos/genética , Receptores Toll-Like/genéticaRESUMO
Planarian Dugesia japonica is a flatworm that can autonomously regenerate its own body after an artificial amputation. A recent report showed the role of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathway in the head morphogenesis during the planarian regeneration process after amputation; however, neuron-specific regeneration mechanisms have not yet been reported. Here, whether MEK/ERK pathway was involved in the dopaminergic neuronal regeneration in planarians was investigated. Planarians regenerated their body within 14 days after amputation; however, the head region morphogenesis was inhibited by MEK inhibitor U0126 (3 or 10 µM). Furthermore, the number of planarian tyrosine hydroxylase (DjTH)-positive dopaminergic neurons in the regenerated head region was also decreased by U0126. The 6-hydroxydopamine (6-OHDA), a dopaminergic neurotoxin, can decrease the number of dopaminergic neurons; however, planarians can regenerate dopaminergic neurons after injecting 6-OHDA into the intestinal tract. MEK inhibitor PD98059 (30 µM) or U0126 (10 µM) significantly decreased dopaminergic neurons 5 days after the 6-OHDA injection. During the regeneration process of dopaminergic neurons, phosphorylated histone H3 (H3P)-positive stem cells known as "neoblasts" were increased in the head region; however, MEK inhibitors significantly decreased the number of H3P-positive neoblasts. These results suggested that dopaminergic neuronal regeneration in planarian was regulated by the MEK/ERK pathway.
Assuntos
Planárias , Animais , Dopamina/fisiologia , Neurônios Dopaminérgicos , Quinases de Proteína Quinase Ativadas por Mitógeno , Oxidopamina/toxicidade , Planárias/fisiologiaRESUMO
Planarians belong to the phylum Platyhelminthes and can regenerate their missing body parts after injury via activation of somatic pluripotent stem cells called neoblasts. Previous studies suggested that fibroblast growth factor (FGF) signaling plays a crucial role in the regulation of head tissue differentiation during planarian regeneration. To date, however, no FGF homologues in the Platyhelminthes have been reported. Here, we used a planarian Dugesia japonica model and identified an fgf gene termed Djfgf, which encodes a putative secreted protein with a core FGF domain characteristic of the FGF8/17/18 subfamily in bilaterians. Using Xenopus embryos, we found that DjFGF has FGF activity as assayed by Xbra induction. We next examined Djfgf expression in non-regenerating intact and regenerating planarians. In intact planarians, Djfgf was expressed in the auricles in the head and the pharynx. In the early process of regeneration, Djfgf was transiently expressed in a subset of differentiated cells around wounds. Notably, Djfgf expression was highly induced in the process of head regeneration when compared to that in the tail regeneration. Furthermore, assays of head regeneration from tail fragments revealed that combinatorial actions of the anterior extracellular signal-regulated kinase (ERK) and posterior Wnt/ß-catenin signaling restricted Djfgf expression to a certain anterior body part. This is the region where neoblasts undergo active proliferation to give rise to their differentiating progeny in response to wounding. The data suggest the possibility that DjFGF may act as an anterior counterpart of posteriorly localized Wnt molecules and trigger neoblast responses involved in planarian head regeneration.
Assuntos
Fatores de Crescimento de Fibroblastos/genética , Animais , Fatores de Crescimento de Fibroblastos/metabolismo , Filogenia , Planárias/genéticaRESUMO
Planarians have a remarkable regenerative ability owing to their adult pluripotent stem cells (aPSCs), which are called "neoblasts." Planarians maintain a considerable number of neoblasts throughout their adulthood to supply differentiated cells for the maintenance of tissue homeostasis and asexual reproduction (fission followed by regeneration). Thus, planarians serve as a good model to study the regulatory mechanisms of in vivo aPSCs. In asexually reproducing invertebrates, such as sponge, Hydra, and planaria, piwi family genes are the markers most commonly expressed in aPSCs. While piwi family genes are known as guardians against transposable elements in the germline cells of animals that only sexually propagate, their functions in the aPSC system have remained elusive. In this review, we introduce recent knowledge on the PIWI family proteins in the aPSC system in planarians and other organisms and discuss how PIWI family proteins contribute to the regulation of the aPSC system.
Assuntos
Proteínas Argonautas/metabolismo , Planárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Reprodução Assexuada , Animais , Proteínas Argonautas/genética , Planárias/genética , Células-Tronco Pluripotentes/citologia , Reprodução Assexuada/genéticaRESUMO
Extracellular matrix (ECM) plays key roles in shaping fates of stem cells, not only by providing a suitable niche but also by mediating physical and biochemical cues. Despite intensive investigations on regeneration, the roles of ECM in fate determination of stem cells in animals with great regenerative potency, such as planarian, have remained unclear. Here, we developed a method for decellularizing and isolating extracellular matrix from planarians. Although the isolated scaffold appears translucent, it contains all the internal features resembling those of the structure of intact planarians, and we thus called it the "ECM-body". Nuclear staining demonstrated that the ECM-body contains very few or no remaining cells. Histological sections displayed well-preserved morphological integrity of the specimen. Scanning electron microscopy showed a porous surface on the ECM-body, potentially suitable for housing cells. Furthermore, our preliminary experiment suggested that ECM-body can be utilized as a biomimetic scaffold for cell culture as it may support survival of injected neoblasts.
Assuntos
Materiais Biomiméticos , Sistema Livre de Células , Matriz Extracelular , Planárias/fisiologia , Animais , Alicerces TeciduaisRESUMO
We previously revealed that the mechanism of demosponge skeleton construction is self-organization by multiple rounds of sequential mechanical reactions of player cells. In these reactions, "transport cells" dynamically carry fine skeletal elements (spicules) on epithelia surrounding the inner body space of sponges (basal epithelium (basopinacoderm) and the endodermal epithelium (ENCM)). Once spicules pierce ENCM and apical pinacoderm, subsequently they are cemented to the substratum under the sponge body, or connected to other skeleton-constructing spicules. Thus, the "pierce" step is the key to holding up spicules in the temporary periphery of growing sponges' bodies. Since sponges can regress as well as grow, here we asked how skeleton construction occurs during local regression of the body. We found that prior to local basopinacoderm retraction (and thus body regression), the body became thinner. Some spicules that were originally carried outward stagnated for a while, and were then carried inwards either on ENCM or basopinacoderm. Spicules that were carried inwards on ENCM pierced epithelia after a short transport, and thus became held up at relatively inward positions compared to spicules carried on outwardly extending basopinacoderm. The switch of epithelia on which transport cells migrate efficiently occurred in thinner body spaces where basopinacoderm and ENCM became close to each other. Thus, the mechanisms underlying this phenomenon are rather mechanical: the combination of sequential reactions of skeleton construction and the narrowed body space upon local retraction of basopinacoderm cause spicules to be held up at more-inward positions, which might strengthen the basopinacoderm's attachment to substratum.
Assuntos
Epitélio/metabolismo , Poríferos , Pele/metabolismo , AnimaisRESUMO
BACKGROUND: Even subtle changes in environmental factors can exert behavioral effects on creatures, which may alter interspecific interactions and eventually affect the ecosystem. However, how changes in environmental factors impact complex behaviors regulated by neural processes is largely unknown. The freshwater planarian Dugesia japonica, a free-living flatworm, displays distinct behavioral traits mediated by sensitive perception of environmental cues. Planarians are thus useful organisms for examining interactions between environmental changes and specific behaviors of animals. RESULTS: Here we found that feeding behavior was suppressed when the concentration of ions in the breeding water was low, while other behaviors were unaffected, resulting in differences in population size. Notably, the decline in feeding behavior was reversed in an ion-concentration-dependent manner soon after the planarians were moved to ion-containing water, which suggests that ions in environmental water rapidly promote feeding behavior in planarians. Moreover, the concentration of ions in the environmental water affected the feeding behavior by modulating the sensitivity of the response to foods. Finally, we found that calcium ions in the aquatic environment were required for the feeding behavior, and exposure to higher levels of calcium ions enhanced the feeding behavior, showing that there was a good correlation between the concentration of calcium ions and the responsiveness of planarians to foods. CONCLUSIONS: Environmental calcium ions are indispensable for and potentiate the activity level of the feeding behavior of planarians. Our findings suggest that the ions in the aquatic environment profoundly impact the growth and survival of aquatic animals via modulating their neural activities and behaviors.
RESUMO
Urodele newts have unique biological properties, notably including prominent regeneration ability. The Iberian ribbed newt, Pleurodeles waltl, is a promising model amphibian distinguished by ease of breeding and efficient transgenic and genome editing methods. However, limited genetic information is available for P. waltl. We conducted an intensive transcriptome analysis of P. waltl using RNA-sequencing to build and annotate gene models. We generated 1.2 billion Illumina reads from a wide variety of samples across 12 different tissues/organs, unfertilized egg, and embryos at eight different developmental stages. These reads were assembled into 1,395,387 contigs, from which 202,788 non-redundant ORF models were constructed. The set is expected to cover a large fraction of P. waltl protein-coding genes, as confirmed by BUSCO analysis, where 98% of universal single-copy orthologs were identified. Ortholog analyses revealed the gene repertoire evolution of urodele amphibians. Using the gene set as a reference, gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Our transcriptome resource is expected to enhance future research employing this emerging model animal for regeneration research as well as for investigations in other areas including developmental biology, stem cell biology, and cancer research. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Pleurodeles/genética , Regeneração/genética , Transcriptoma , Animais , Feminino , Perfilação da Expressão Gênica , Masculino , Especificidade de Órgãos , Filogenia , Análise de Sequência de RNARESUMO
Early embryonic cells are capable of acquiring numerous developmental fates until they become irreversibly committed to specific lineages depending on intrinsic determinants and/or regional interactions. From fertilization to gastrulation, such pluripotent cells first increase in number and then turn to undergoing differentiation. Mechanisms regulating pluripotency in each species attract great interest in developmental biology. Also, outlining the evolutionary background of pluripotency can enhance our understanding of mammalian pluripotency and provide a broader view of early development of vertebrates. Here, we introduce integrative models of pluripotent states in amniotes (mammals, birds and reptiles) to offer a comprehensive overview of widely accepted knowledge about mammalian pluripotency and our recent findings in non-mammalian amniotes, such as chicken and gecko. In particular, we describe 1) the IL6/Stat3 signaling pathway as a positive regulator of naive pluripotency, 2) Fgf/Erk signaling as a process that prepares cells for differentiation, 3) the role of the interactions between these two signaling pathways during the transition from pluripotency to differentiation, and 4) functional diversification of two transcription factors, Class V POUs and Nanog. In the last section, we also briefly discuss possible relationships of unique cell cycle properties of early embryonic cells with signaling pathways and developmental potentials in the pluripotent cell states.