Blastema Cells Research Articles

The art of fin regeneration in zebrafish.

The zebrafish fin provides a valuable model to study the epimorphic type of regeneration, whereby the amputated part of the appendage is nearly perfectly replaced. To accomplish fin regeneration, two reciprocally interacting domains need to be established at the injury site, namely a wound epithelium and a blastema. The wound epithelium provides a supporting niche for the blastema, which contains mesenchyme‐derived progenitor cells for the regenerate. The fate of blastemal daughter cells depends on their relative position with respect to the fin margin. The apical compartment of the outgrowth maintains its undifferentiated character, whereas the proximal descendants of the blastema progressively switch from the proliferation program to the morphogenesis program. A delicate balance between self‐renewal and differentiation has to be continuously adjusted during the course of regeneration. This review summarizes the current knowledge about the cellular and molecular mechanisms of blastema formation, and discusses several studies related to the regulation of growth and morphogenesis during fin regeneration. A wide range of canonical signaling pathways has been implicated during the establishment and maintenance of the blastema. Epigenetic mechanisms play a crucial role in the regulation of cellular plasticity during the transition between differentiation states. Ion fluxes, gap‐junctional communication and protein phosphatase activity have been shown to coordinate proliferation and tissue patterning in the caudal fin. The identification of the downstream targets of the fin regeneration signals and the discovery of mechanisms integrating the variety of input pathways represent exciting future aims in this fascinating field of research.

Unique gene expression profile of the proliferating Xenopus tadpole tail blastema cells deciphered by RNA-sequencing analysis.

Organ regenerative ability depends on the animal species and the developmental stage. The molecular bases for variable organ regenerative ability, however, remain unknown. Previous studies have identified genes preferentially expressed in the blastema tissues in various animals, but transcriptome analysis of the isolated proliferating blastema cells has not yet been reported. In the present study, we used RNA-sequencing analysis to analyze the gene expression profile of isolated proliferating blastema cells of regenerating Xenopus laevis tadpole tails. We used flow cytometry to isolate proliferating cells, and non-proliferating blastema cells, from regenerating tadpole tails as well as proliferating tail bud cells from tail bud embryos, the latter two of which were used as control cells, based on their DNA content. Among the 28 candidate genes identified by RNA-sequencing analysis, quantitative reverse transcription-polymerase chain reaction identified 10 genes whose expression was enriched in regenerating tadpole tails compared with non-regenerating tadpole tails or tails from the tail bud embryos. Among them, whole mount in situ hybridization revealed that chromosome segregation 1-like and interleukin 11 were expressed in the broad area of the tail blastema, while brevican, lysyl oxidase, and keratin 18 were mainly expressed in the notochord bud in regenerating tails. We further combined whole mount in situ hybridization with immunohistochemistry for the incorporated 5-bromo-2-deoxyuridine to confirm that keratin 18 and interleukin 11 were expressed in the proliferating tail blastema cells. Based on the proposed functions of their homologs in other animal species, these genes might have roles in the extracellular matrix formation in the notochord bud (brevican and lysyl oxidase), cell proliferation (chromosome segregation 1-like and keratin 18), and in the maintenance of the differentiation ability of proliferating blastema cells (interleukin 11) in regenerating tadpole tails.

Vasa, PL10, and Piwi gene expression during caudal regeneration of the polychaete annelid Alitta virens

Polychaetes are famous for their outstanding ability to regenerate lost body parts. Moreover, these worms possess a number of ancestral features in anatomy, development, and genetics, making them particularly suitable for comparative studies. Thus, fundamental as well as new undisclosed so far features of regenerative processes may be revealed, using polychaetes as a model. In the present work, we aimed to analyze the molecular basis of caudal regeneration in the nereid polychaete Alitta virens (formerly Nereis virens). We focused on homologues genes of RNA helicases Vasa and PL10 and ncRNA-binding proteins Piwi. These markers are suggested to play a significant role in maintenance of undifferentiated state of primordial germ cells and multipotent stem cells across invertebrates. In normal conditions, A. virens homologues of Vasa, PL10, and Piwi were differentially expressed in the subterminal growth zone and germline cells. Caudal amputation induced expression of studied genes de novo, which further accompanies all steps of regeneration. An early appearance of the transcripts in wound epithelium and internal blastemal cells suggests involvement of these genes in the well-known cell dedifferentiation events that assure polychaete regeneration. Provided interpretation of the gene expression dynamics implies the primary restoration of the pygidium and growth zone, which promotes following segment formation. Obtained results are valuable as a molecular fingerprint of the alterations occurring in regulatory state of locally regenerating tissues.

Time point-based integrative analyses of deep-transcriptome identify four signal pathways in blastemal regeneration of zebrafish lower jaw.

There has been growing interest in applying tissue engineering to stem cell-based regeneration therapies. We have previously reported that zebrafish can faithfully regenerate complicated tissue structures through blastemal cell type conversions and tissue reorganization. To unveil the regenerative factors and engineering arts of blastemal regeneration, we conducted transcriptomal analyses at four time points corresponding to preamputation, re-epitheliation, blastemal formation, and respecification. By combining the hierarchical gene ontology term network, the DAVID annotation system, and Euclidean distance clustering, we identified four signaling pathways: foxi1-foxo1b-pou3f1, pax3a-mant3a-col11/col2, pou5f1-cdx4-kdrl, and isl1-wnt11 PCP-sox9a. Results from immunohistochemical staining and promoter-driven transgenic fish suggest that these pathways, respectively, define wound epidermis reconstitution, cell type conversions, blastemal angiogenesis/vasculogenesis, and cartilage matrix-orientation. Foxi1 morpholino-knockdown caused expansions of Foxo1b- and Pax3a-expression in the basal layer-blastemal junction region. Moreover, foxi1 morphants displayed increased sox9a and hoxa2b transcripts in the embryonic pharyngeal arches. Thus, a Foxi1 signal switch is required to establish correct tissue patterns, including re-epitheliation and blastema formation. This study provides novel insight into a blastema regeneration strategy devised by epithelial cell transdifferentiation, blood vessel engineering, and cartilage matrix deposition.

Involvement of dachshund and Distal-less in distal pattern formation of the cricket leg during regeneration.

Cricket nymphs have the remarkable ability to regenerate a functional leg following amputation, indicating that the regenerating blastemal cells contain information for leg morphology. However, the molecular mechanisms that underlie regeneration of leg patterns remain poorly understood. Here, we analyzed phenotypes of the tibia and tarsus (three tarsomeres) obtained by knockdown with regeneration-dependent RNA interference (rdRNAi) against Gryllus dachshund (Gb'dac) and Distal-less (Gb'Dll). We found that depletion of Gb'Dll mRNA results in loss of the tarsal segments, while rdRNAi against Gb'dac shortens the tibia at the two most distal tarsomeres. These results indicate that Gb'Dll expression is indispensable for formation of the tarsus, while Gb'dac expression is necessary for elongation of the tibia and formation of the most proximal tarsomere. These findings demonstrate that mutual transcriptional regulation between the two is indispensable for formation of the tarsomeres, whereas Gb'dac is involved in determination of tibial size through interaction with Gb'ds/Gb'ft.

Leg regeneration is epigenetically regulated by histone H3K27 methylation in the cricket Gryllus bimaculatus.

Hemimetabolous insects such as the cricket Gryllus bimaculatus regenerate lost tissue parts using blastemal cells, a population of dedifferentiated proliferating cells. The expression of several factors that control epigenetic modification is upregulated in the blastema compared with differentiated tissue, suggesting that epigenetic changes in gene expression might control the differentiation status of blastema cells during regeneration. To clarify the molecular basis of epigenetic regulation during regeneration, we focused on the function of the Gryllus Enhancer of zeste [Gb'E(z)] and Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (Gb'Utx) homologues, which regulate methylation and demethylation of histone H3 lysine 27 (H3K27), respectively. Methylated histone H3K27 in the regenerating leg was diminished by Gb'E(z)(RNAi) and was increased by Gb'Utx(RNAi). Regenerated Gb'E(z)(RNAi) cricket legs exhibited extra leg segment formation between the tibia and tarsus, and regenerated Gb'Utx(RNAi) cricket legs showed leg joint formation defects in the tarsus. In the Gb'E(z)(RNAi) regenerating leg, the Gb'dac expression domain expanded in the tarsus. By contrast, in the Gb'Utx(RNAi) regenerating leg, Gb'Egfr expression in the middle of the tarsus was diminished. These results suggest that regulation of the histone H3K27 methylation state is involved in the repatterning process during leg regeneration among cricket species via the epigenetic regulation of leg patterning gene expression.

Osteoblast de- and redifferentiation are controlled by a dynamic response to retinoic acid during zebrafish fin regeneration.

Zebrafish restore amputated fins by forming tissue-specific blastema cells that coordinately regenerate the lost structures. Fin amputation triggers the synthesis of several diffusible signaling factors that are required for regeneration, raising the question of how cell lineage-specific programs are protected from regenerative crosstalk between neighboring fin tissues. During fin regeneration, osteoblasts revert from a non-cycling, mature state to a cycling, preosteoblastic state to establish a pool of progenitors within the blastema. After several rounds of proliferation, preosteoblasts redifferentiate to produce new bone. Blastema formation and proliferation are driven by the continued synthesis of retinoic acid (RA). Here, we find that osteoblast dedifferentiation and redifferentiation are inhibited by RA signaling, and we uncover how the bone regenerative program is achieved against a background of massive RA synthesis. Stump osteoblasts manage to contribute to the blastema by upregulating expression of the RA-degrading enzyme cyp26b1. Redifferentiation is controlled by a presumptive gradient of RA, in which high RA levels towards the distal tip of the blastema suppress redifferentiation. We show that this might be achieved through a mechanism involving repression of Bmp signaling and promotion of Wnt/β-catenin signaling. In turn, cyp26b1(+) fibroblast-derived blastema cells in the more proximal regenerate serve as a sink to reduce RA levels, thereby allowing differentiation of neighboring preosteoblasts. Our findings reveal a mechanism explaining how the osteoblast regenerative program is protected from adverse crosstalk with neighboring fibroblasts that advances our understanding of the regulation of bone repair by RA.

Generation of aneurogenic larvae by parabiosis of salamander embryos.

Limb regeneration of salamanders is nerve dependent, and the removal of the nerves in early stages of limb regeneration severely curtails the proliferation of the blastemal cells and growth of the regenerate. The removal of the neural tube from a developing salamander embryo results in an aneurogenic larva and the aneurogenic limb (ANL) develops independently without innervation. Paradoxically, the limb in an ANL is capable of regeneration in a nerve-independent manner. Here, we describe a detailed method for the generation of ANL in the spotted salamander, Ambystoma maculatum, for regeneration studies.

Retinoic acid signaling spatially restricts osteoblasts and controls ray-interray organization during zebrafish fin regeneration.

The zebrafish caudal fin consists of repeated units of bony rays separated by soft interray tissue, an organization that must be faithfully re-established during fin regeneration. How and why regenerating rays respect ray-interray boundaries, thus extending only the existing bone, has remained unresolved. Here, we demonstrate that a retinoic acid (RA)-degrading niche is established by Cyp26a1 in the proximal basal epidermal layer that orchestrates ray-interray organization by spatially restricting osteoblasts. Disruption of this niche causes preosteoblasts to ignore ray-interray boundaries and to invade neighboring interrays where they form ectopic bone. Concomitantly, non-osteoblastic blastema cells and regenerating blood vessels spread into the interrays, resulting in overall disruption of ray-interray organization and irreversible inhibition of fin regeneration. The cyp26a1-expressing niche plays another important role during subsequent regenerative outgrowth, where it facilitates the Shha-promoted proliferation of osteoblasts. Finally, we show that the previously observed distal shift of ray bifurcations in regenerating fins upon RA treatment or amputation close to the bifurcation can be explained by inappropriate preosteoblast alignment and does not necessarily require putative changes in proximodistal information. Our findings uncover a mechanism regulating preosteoblast alignment and maintenance of ray-interray boundaries during fin regeneration.

Mechanistic target of rapamycin complex 1 signaling regulates cell proliferation, cell survival, and differentiation in regenerating zebrafish fins.

BackgroundThe mechanistic target of rapamycin complex1 (mTORC1) signaling pathway has been implicated in functions of multicellular processes, including cell growth and metabolism. Although recent reports showed that many signaling pathways, including Activin, Bmp, Fgf, sonic hedgehog, Insulin-like growth factor (IGF), Notch, retinoic acid, and Wnt, are implicated in non-mammalian vertebrate regeneration, also known as epimorphic regeneration, mTORC1 function remains unknown.ResultsTo investigate the role of mTORC1 signaling pathway in zebrafish caudal fin, we examined the activation and function of mTORC1 signaling using an antibody against phosphorylated S6 kinase and a specific inhibitor, rapamycin. mTORC1 signaling is activated in proliferative cells of intra-ray and wound epidermal cells before blastema formation, as well as in proliferative blastema cells, wound epidermal cells, and osteoblasts during regenerative outgrowth. Before blastema formation, proliferation of intra-ray and wound epidermal cells is suppressed, but cell death is not affected by mTORC1 signaling inhibition with rapamycin. Moreover, rapamycin treatment inhibits blastema and wound epidermal cell proliferation and survival during blastema formation and regenerative outgrowth, as well as osteoblast proliferation and differentiation during regenerative outgrowth. We further determined that mTORC1 signaling is regulated through IGF-1 receptor/phosphatidylinositol-3 kinase and Wnt pathways during fin regeneration.ConclusionTaken together, our findings reveal that mTORC1 signaling regulates proliferation, survival, and differentiation of intra-ray cells, wound epidermis, blastema cells, and/or osteoblasts in various fin regeneration stages downstream of IGF and Wnt signaling pathways.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-014-0042-9) contains supplementary material, which is available to authorized users.

Denervation impairs regeneration of amputated zebrafish fins.

BackgroundZebrafish are able to regenerate many of its tissues and organs after damage. In amphibians this process is regulated by nerve fibres present at the site of injury, which have been proposed to release factors into the amputated limbs/fins, promoting and sustaining the proliferation of blastemal cells. Although some candidate factors have been proposed to mediate the nerve dependency of regeneration, the molecular mechanisms involved in this process remain unclear.ResultsWe have used zebrafish as a model system to address the role of nerve fibres in fin regeneration. We have developed a protocol for pectoral fin denervation followed by amputation and analysed the regenerative process under this experimental conditions. Upon denervation fins were able to close the wound and form a wound epidermis, but could not establish a functional apical epithelial cap, with a posterior failure of blastema formation and outgrowth, and the accumulation of several defects. The expression patterns of genes known to be key players during fin regeneration were altered upon denervation, suggesting that nerves can contribute to the regulation of the Fgf, Wnt and Shh pathways during zebrafish fin regeneration.ConclusionsOur results demonstrate that proper innervation of the zebrafish pectoral fin is essential for a successful regenerative process, and establish this organism as a useful model to understand the molecular and cellular mechanisms of nerve dependence, during vertebrate regeneration.Electronic supplementary materialThe online version of this article (doi:10.1186/s12861-014-0049-2) contains supplementary material, which is available to authorized users.

Blastema cells derived from New Zealand white rabbit's pinna carry stemness properties as shown by differentiation into insulin producing, neural, and osteogenic lineages representing three embryonic germ layers.

Stem cells (SCs) are known as undifferentiated cells with self-renewal and differentiation capacities. Regeneration is a phenomenon that occurs in a limited number of animals after injury, during which blastema tissue is formed. It has been hypothesized that upon injury, the dedifferentiation of surrounding tissues leads into the appearance of cells with SC characteristics. In present study, stem-like cells (SLCs) were obtained from regenerating tissue of New Zealand white rabbit's pinna and their stemness properties were examined by their capacity to differentiate toward insulin producing cells (IPCs), as well as neural and osteogenic lineages. Differentiation was induced by culture of SLCs in defined medium, and cell fates were monitored by specific staining, RT-PCR and flow cytometry assays. Our results revealed that dithizone positive cells, which represent IPCs, and islet-like structures appeared 1week after induction of SLCs, and this observation was confirmed by the elevated expression of Ins, Pax6 and Glut4 at mRNA level. Furthermore, SLCs were able to express neural markers as early as 1week after retinoic acid treatment. Finally, SLCs were able to differentiate into osteogenic lineage, as confirmed by Alizarin Red S staining and RT-PCR studies. In conclusion, SLCs, which could successfully differentiate into cells derived from all three germ layers, can be considered as a valuable model to study developmental biology and regenerative medicine.

Childhood pleuropulmonary blastoma: a clinicopathologic study of 16 cases

To study the clinicopathological and immunohistochemical features, histogenesis and prognosis of pleuropulmonary blastoma (PPB) in children. PPB specimens from 16 pediatric cases with an age ranging from 1 year and 7 months to 5 years and 3 months (mean age of 3 years) were retrieved and analyzed by routine histological, immunohistochemical and electron methods. Among 16 patients, there were 2 type I, 7 type II and 7 type III PPB cases. Type I PPB as multilocular cystic structure, consisted of thin fibrous wall lining the respiratory epithelium, subepithelial primitive blastema or immature mesenchymal cells, with or without rhabdomyoblastic differentiation or cartilage; Type II PPB as cystic-solid tumor, comparing with type I, consisted of intracystic components with appearance of anaplastic tumor cells. Type III PPB consisted of completely solid mass, the same as the solid region of type II, had mixed pattern including blastema, undifferentiated spindle-cell proliferations and sarcomas. In addition, anaplastic tumor cells and intra-and extra- cytoplasmic eosinophilic globules were also commonly present. Epithelial components in PPB were benign. Immunohistochemical study showed primitive mesenchymal differentiation of tumors. All cases were positive for vimentin, desmin, myogenin and SMA in tumors with skeletal muscle differentiation, S-100 was positive in tumors with cartilage differentiation. All tumors were negative for synaptophysin, CD99, and CD117. Benign epithelial components were positive for AE1/AE3 and EMA. In 12 cases, electron microscopy revealed few organelles in the primitive mesenchymal cells and rich heterochromatin in mesenchymal cells, the latter also demonstrating cytoplasmic myofilament dysplasia. Nine cases had clinical follow-up ranging from 5 to 48 months, of which 4 patients died. PPB is a rare lung neoplasm of children under the age of 6 years, with distinct pathological morphology. PPB may arise from lung or pleura mesenchymal cells and has a poor clinical outcome.

Early events in annelid regeneration: a cellular perspective.

The ability to regenerate extensive portions of the body is widespread among the phylum Annelida and this group includes some of the most highly regenerative animals known. Knowledge of the cellular and molecular basis of regeneration in this group is thus important for understanding how regenerative processes have evolved both within the group and across animal phyla. Here, the cellular basis of annelid regeneration is reviewed, with a focus on the earliest steps of regeneration, namely wound-healing and formation of the blastema. Information from a wide range of annelids is compiled in order to identify common and variable elements. There is a large body of valuable older literature on the cellular basis of regeneration in annelids and an effort is made to review this literature in addition to more recent studies. Annelids typically seal the wound through muscular contraction and undergo some autolysis of tissue at the site of the wound. Bodily injury elicits extensive cell migration toward the wound, involving several different types of cells. Some migrating cells form a tissue-clot and phagocytize damaged tissues, whereas others are inferred to contribute to regenerated tissue, specifically mesodermal tissue. In one annelid subgroup, the clitellates, a group of mesodermal cells, sometimes referred to as neoblasts, is inferred to migrate over considerable distances, with cells moving to the wound from several segments away. Epidermis and gut epithelia severed upon amputation typically heal by fusing with like tissue, although not always. After amputation, cellular contacts with the extracellular matrix are disrupted and major changes in cell morphology and adhesion occur within tissues near the wound. Interactions of tissues at the wound appear key for initiating a blastema, with a particularly important role suggested for the ventral nerve cord, although species are variable in this regard; longer-distance effects mediated by the brain are also reported. The anterior-posterior polarity of the blastema can be mis-assigned, leading most commonly to double-headed worms, and the dorsal-ventral polarity of the blastema appears to be induced by the ventral nerve cord. The blastema is thought to arise from contributions of all three tissue layers, with each layer replacing itself in a tissue-specific manner. Blastemal cells originate mostly locally, although some long-distance migration of source-cells is suggested in clitellates. A number of important questions remain about the cellular basis of regeneration in annelids and addressing many of these would be greatly aided by developing approaches to identify and isolate specific cell types and techniques to image and trace cells in vivo.

Evaluation of five different suture materials in the skin of the earthworm (Lumbricus terrestris).

The purpose of this study was to determine which suture material is the most appropriate for dermal closure of terrestrial annelids. This paper describes the tissue reactions of the earthworm, Lumbricus terrestris, to five different types of suture materials in order to determine which suture material is the most appropriate for dermal closure. Silk, monofilament nylon, polydiaxonone, polyglactin 910, and chromic gut were studied. There was mild to moderate tissue reaction to all five suture materials. In all of the biopsies wound-healing reaction consisted of aggregates of blastemal cells which appeared in various stages of dedifferentiation from the body wall. Inflammatory cells infiltrated the wound sites, reminiscent of the typical foreign body reaction in vertebrates. The results indicate polyglactin 910 would be the best suture material with regards to tissue security and reaction scores. Chromic gut occupies the next position but there were problems with suture security over time. This appears to be the first suture material performance study on a terrestrial invertebrate. The earthworm, Lumbricus terrestris, was chosen for its wide availability, size, and the extensive species knowledge base. The earthworm may prove to be a good surgical/suture model for economically important invertebrates such as mollusks, tunicates, and insect larval stages.

Neoblast Specialization in Regeneration of the Planarian Schmidtea mediterranea

SummaryPlanarians can regenerate any missing body part in a process requiring dividing cells called neoblasts. Historically, neoblasts have largely been considered a homogeneous stem cell population. Most studies, however, analyzed neoblasts at the population rather than the single-cell level, leaving the degree of heterogeneity in this population unresolved. We combined RNA sequencing of neoblasts from wounded planarians with expression screening and identified 33 transcription factors transcribed in specific differentiated cells and in small fractions of neoblasts during regeneration. Many neoblast subsets expressing distinct tissue-associated transcription factors were present, suggesting candidate specification into many lineages. Consistent with this possibility, klf, pax3/7, and FoxA were required for the differentiation of cintillo-expressing sensory neurons, dopamine-β-hydroxylase-expressing neurons, and the pharynx, respectively. Together, these results suggest that specification of cell fate for most-to-all regenerative lineages occurs within neoblasts, with regenerative cells of blastemas being generated from a highly heterogeneous collection of lineage-specified neoblasts.

Wilms’ Tumor Blastemal Stem Cells Dedifferentiate to Propagate the Tumor Bulk

SummaryAn open question remains in cancer stem cell (CSC) biology whether CSCs are by definition at the top of the differentiation hierarchy of the tumor. Wilms’ tumor (WT), composed of blastema and differentiated renal elements resembling the nephrogenic zone of the developing kidney, is a valuable model for studying this question because early kidney differentiation is well characterized. WT neural cell adhesion molecule 1-positive (NCAM1+) aldehyde dehydrogenase 1-positive (ALDH1+) CSCs have been recently isolated and shown to harbor early renal progenitor traits. Herein, by generating pure blastema WT xenografts, composed solely of cells expressing the renal developmental markers SIX2 and NCAM1, we surprisingly show that sorted ALDH1+ WT CSCs do not correspond to earliest renal stem cells. Rather, gene expression and proteomic comparative analyses disclose a cell type skewed more toward epithelial differentiation than the bulk of the blastema. Thus, WT CSCs are likely to dedifferentiate to propagate WT blastema.

Specific NuRD components are required for fin regeneration in zebrafish.

BackgroundEpimorphic regeneration of a missing appendage in fish and urodele amphibians involves the creation of a blastema, a heterogeneous pool of progenitor cells underneath the wound epidermis. Current evidence indicates that the blastema arises by dedifferentiation of stump tissues in the vicinity of the amputation. In response to tissue loss, silenced developmental programs are reactivated to form a near-perfect copy of the missing body part. However, the importance of chromatin regulation during epimorphic regeneration remains poorly understood.ResultsWe found that specific components of the Nucleosome Remodeling and Deacetylase complex (NuRD) are required for fin regeneration in zebrafish. Transcripts of the chromatin remodeler chd4a/Mi-2, the histone deacetylase hdac1/HDAC1/2, the retinoblastoma-binding protein rbb4/RBBP4/7, and the metastasis-associated antigen mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration, and these transcripts displayed a similar spatial and temporal expression patterns. In addition, chemical inhibition of Hdac1 and morpholino-mediated knockdown of chd4a, mta2, and rbb4 impaired regenerative outgrowth, resulting in reduction in blastema cell proliferation and in differentiation defects.ConclusionAltogether, our data suggest that specialized NuRD components are induced in the blastema during fin regeneration and are involved in blastema cell proliferation and redifferentiation of osteoblast precursor cells. These results provide in vivo evidence for the involvement of key epigenetic factors in the cellular reprogramming processes occurring during epimorphic regeneration in zebrafish.

Characterization of in vitro transcriptional responses of dorsal root ganglia cultured in the presence and absence of blastema cells from regenerating salamander limbs

During salamander limb regeneration, nerves provide signals that induce the formation of a mass of proliferative cells called the blastema. To better understand these signals, we developed a blastema−dorsal root ganglia (DRG) co‐culture model system to test the hypothesis that nerves differentially express genes in response to cues provided by the blastema. DRG with proximal and distal nerve trunks were isolated from axolotls (Ambystoma mexicanum), cultured for 5 days, and subjected to microarray analysis. Relative to freshly isolated DRG, 1541 Affymetrix probe sets were identified as differentially expressed and many of the predicted genes are known to function in injury and neurodevelopmental responses observed for mammalian DRG. We then cultured 5‐day DRG explants for an additional 5 days with or without co‐cultured blastema cells. On day 10, we identified 27 genes whose expression in cultured DRG was significantly affected by the presence or absence of blastema cells. Overall, our study established a DRG−blastema in vitro culture system and identified candidate genes for future investigations of axon regrowth, nerve−blastema signaling, and neural regulation of limb regeneration.

V-ATPase Proton Pumping Activity Is Required for Adult Zebrafish Appendage Regeneration

The activity of ion channels and transporters generates ion-specific fluxes that encode electrical and/or chemical signals with biological significance. Even though it is long known that some of those signals are crucial for regeneration, only in recent years the corresponding molecular sources started to be identified using mainly invertebrate or larval vertebrate models. We used adult zebrafish caudal fin as a model to investigate which and how ion transporters affect regeneration in an adult vertebrate model. Through the combined use of biophysical and molecular approaches, we show that V-ATPase activity contributes to a regeneration-specific H+ ef`flux. The onset and intensity of both V-ATPase expression and H+ efflux correlate with the different regeneration rate along the proximal-distal axis. Moreover, we show that V-ATPase inhibition impairs regeneration in adult vertebrate. Notably, the activity of this H+ pump is necessary for aldh1a2 and mkp3 expression, blastema cell proliferation and fin innervation. To the best of our knowledge, this is the first report on the role of V-ATPase during adult vertebrate regeneration.