Blastema Cell Proliferation Research Articles

The Regulatory Role of miRNAs in Zebrafish Fin Regeneration

Since Teleostei fins have a strong regenerative capacity, further research was conducted on the regulation of gene expression during fin regeneration. This research focuses on miRNA, which is a key post-transcriptional regulatory molecule. In this study, a miRNA library for the fin regeneration of zebrafish was constructed to reveal the differential expression of miRNA during fin regeneration and to explore the regulatory pathway for fin regeneration. Following the injection of miRNA agomir into zebrafish, the proliferation of blastema cells and the overall fin regeneration area were significantly reduced. It was observed that the miRNAs impaired blastocyte formation by affecting fin regeneration through the inhibition of the expressions of genes and proteins associated with blastocyte formation (including yap1 and Smad1/5/9), which is an effect associated with the Hippo pathway. Furthermore, it has been demonstrated that miRNAs can impair the patterns and mineralization of newly formed fin rays. The miRNAs influenced fin regeneration by inhibiting the expression of a range of bone-related genes and proteins in osteoblast lineages, including sp7, runx2a, and runx2b. This study provides a valuable reference for the further exploration of morphological bone reconstruction in aquatic vertebrates.

A myogenic regulatory factor is required for myogenesis during limb regeneration in the Chinese mitten crab

Myogenic regulatory factors (MRFs) are a group of transcription factors that regulate the activity of skeletal muscle cells during embryonic development and postnatal myogenesis in various vertebrate species. However, the role of MRFs in limb regeneration remains poorly understood in crustaceans. In this study, we identified a full-length cDNA encoding a myogenic regulatory factor from Eriocheir sinensis (EsMRF) and evaluated its mRNA expression profile during muscle development, growth, and regeneration. The expression of EsMRF was found to correlate with the onset of muscle formation during development and with the regeneration process following limb autotomy. To elucidate the function of MRF during limb regeneration in E. sinensis, we assessed regenerative efficiency using RNA interference (RNAi) targeting EsMRF. Our findings revealed that the blockade of MRF delayed limb regeneration by disrupting the proliferation and myogenesis of blastema cells at the basal growth stage. Furthermore, luciferase assays results demonstrated that EsMRF can transcriptionally activate target myogenic genes, either through direct binding to their promoters or by interacting with co-regulators such as EsHEB or EsMEF2. This study identifies a novel MRF in E. sinensis and elucidates its function during limb regeneration, thereby contributing to our understanding of muscle growth and regeneration mechanisms in crustaceans.

MTOR mutation disrupts larval zebrafish tail fin regeneration via regulating proliferation of blastema cells and mitochondrial functions

BackgroundThe larval zebrafish tail fin can completely regenerate in 3 days post amputation. mTOR, the main regulator of cell growth and metabolism, plays an essential role in regeneration. Lots of studies have documented the role of mTOR in regeneration. However, the mechanisms involved are still not fully elucidated.Materials and resultsThis study aimed to explore the role and mechanism of mTOR in the regeneration of larval zebrafish tail fins. Initially, the spatial and temporal expression of mTOR signaling in the larval fin was examined, revealing its activation following tail fin amputation. Subsequently, a mTOR knockout (mTOR-KO) zebrafish line was created using CRISPR/Cas9 gene editing technology. The investigation demonstrated that mTOR depletion diminished the proliferative capacity of epithelial and mesenchymal cells during fin regeneration, with no discernible impact on cell apoptosis. Insight from SMART-seq analysis uncovered alterations in the cell cycle, mitochondrial functions and metabolic pathways when mTOR signaling was suppressed during fin regeneration. Furthermore, mTOR was confirmed to enhance mitochondrial functions and Ca2 + activation following fin amputation. These findings suggest a potential role for mTOR in promoting mitochondrial fission to facilitate tail fin regeneration.ConclusionIn summary, our results demonstrated that mTOR played a key role in larval zebrafish tail fin regeneration, via promoting mitochondrial fission and proliferation of blastema cells.

ERK-activated CK-2 triggers blastema formation during appendage regeneration.

Appendage regeneration relies on the formation of blastema, a heterogeneous cellular structure formed at the injury site. However, little is known about the early injury-activated signaling pathways that trigger blastema formation during appendage regeneration. Here, we provide compelling evidence that the extracellular signal-regulated kinase (ERK)-activated casein kinase 2 (CK-2), which has not been previously implicated in appendage regeneration, triggers blastema formation during leg regeneration in the American cockroach, Periplaneta americana. After amputation, CK-2 undergoes rapid activation through ERK-induced phosphorylation within blastema cells. RNAi knockdown of CK-2 severely impairs blastema formation by repressing cell proliferation through down-regulating mitosis-related genes. Evolutionarily, the regenerative role of CK-2 is conserved in zebrafish caudal fin regeneration via promoting blastema cell proliferation. Together, we find and demonstrate that the ERK-activated CK-2 triggers blastema formation in both cockroach and zebrafish, helping explore initiation factors during appendage regeneration.

Hedgehog signaling is essential in the regulation of limb regeneration in the Chinese mitten crab, Eriocheir sinensis.

Tissue autotomy is a unique adaptive response to environmental stress, followed by regeneration process compensating for the loss of body parts. The crustaceans present remarkable activity of appendage autotomy and regeneration, however, the molecular mechanism is still unclear. In this study, the Eriocheir sinensis Hedgehog (EsHH) and Smoothened (EsSMO) were identified in the regenerative limbs, and the function of Hedgehog signaling pathway on limb regeneration was evaluated. At the blastema growth stage of limb regeneration, the expression of EsHH and EsSMO was up-regulated in response to limb autotomy stress, and down-regulated at blastema differentiation stage. To clarify the effect of Hedgehog pathway during limb regeneration, the regenerative efficiency was evaluated with Smoothened inhibitor cyclopamine or RNAi (ds-HH) injection. We observed that the regenerative efficiency was significantly repressed with blockage of Hedgehog pathway at both the basal growth stage and the proecdysial growth stage, which was indicated by the delay of wound healing and blastema growth, as well as a decrease in the size of newly formed limbs. In addition, gene expression and BrdU incorporation assay showed that the proliferation and myogenic differentiation of blastema cells were suppressed with either cyclopamine or ds-HH injection. Thus, these results suggest that Hedgehog signaling pathway is essential for the establishment of limb regeneration in E. sinensis through promoting the proliferation and myogenic differentiation of blastema cells.

A small noncoding RNA links ribosome recovery and translation control to dedifferentiation during salamander limb regeneration.

Building a blastema from the stump is a key step of salamander limb regeneration. Stump-derived cells temporarily suspend their identity as they contribute to the blastema by a process generally referred to as dedifferentiation. Here, we provide evidence for a mechanism that involves an active inhibition of protein synthesis during blastema formation and growth. Relieving this inhibition results in a higher number of cycling cells and enhances the pace of limb regeneration. By small RNA profiling and fate mapping of skeletal muscle progeny as a cellular model for dedifferentiation, we find that the downregulation of miR-10b-5p is critical for rebooting the translation machinery. miR-10b-5p targets ribosomal mRNAs, and its artificial upregulation causes decreased blastema cell proliferation, reduction in transcripts that encode ribosomal subunits, diminished nascent protein synthesis, and retardation of limb regeneration. Taken together, our data identify a link between miRNA regulation, ribosome biogenesis, and protein synthesis during newt limb regeneration.

Hippo Signaling Regulates Blastema Formation During Limb Regeneration in Chinese Mitten Crab (Eriocheir sinensis).

Limb autotomy and regeneration are specific adaptations of crustaceans in response to external stress and attacks, which make them a suitable model to investigate the mechanism of organ regeneration in invertebrates. In this study, the Hippo gene of Eriocheir sinensis (EsHPO) was identified, and the effects of Hippo signaling on limb regeneration were evaluated. The expression of EsHPO and other key components of Hippo signaling was down-regulated during the basal growth phase in response to limb autotomy stress and then up-regulated during the proecdysial growth phase. The descending expression patterns of Hippo signal components were correlated with transcriptional activation of YKI and downstream target genes during the blastema formation stage, which suggested that Hippo signaling plays a key role during limb regeneration in E. sinensis. To further test the hypothesis, the transcription factor YKI was blocked via verteporfin injection after autotomy, which disrupted limb regeneration by repressing wound healing and preventing blastema emergence. Furthermore, our experiments revealed that the proliferation of blastema cells was blocked by verteporfin. In addition, the expression of genes related to ECM remodeling, cell cycle progression, and apoptosis resistance was down-regulated following the injection of verteporfin. Our findings therefore indicate that Hippo signaling is essential for successful wound healing and limb regeneration in E. sinensis by inducing ECM remodeling, as well as promoting the proliferation and repressing the apoptosis of blastema cells.

Evi5 is required for Xenopus limb and tail regeneration.

Amphibians such as salamanders and the African clawed frog Xenopus are great models for regeneration studies because they can fully regenerate their lost organs. While axolotl can regenerate damaged organs throughout its lifetime, Xenopus has a limited regeneration capacity after metamorphosis. The ecotropic viral integrative factor 5 (Evi5) is of great interest because its expression is highly upregulated in the limb blastema of axolotls, but remains unchanged in the fibroblastema of post-metamorphic frogs. Yet, its role in regeneration-competent contexts in Xenopus has not been fully analyzed. Here we show that Evi5 is upregulated in Xenopus tadpoles after limb and tail amputation, as in axolotls. Down-regulation of Evi5 with morpholino antisense oligos (Mo) impairs limb development and limb blastema formation in Xenopus tadpoles. Mechanistically, we show that Evi5 knockdown significantly reduces proliferation of limb blastema cells and causes apoptosis, blocking the formation of regeneration blastema. RNA-sequencing analysis reveals that in addition to reduced PDGFα and TGFβ signaling pathways that are required for regeneration, evi5 Mo downregulates lysine demethylases Kdm6b and Kdm7a. And knockdown of Kdm6b or Kdm7a causes defective limb regeneration. Evi5 knockdown also impedes tail regeneration in Xenopus tadpoles and axolotl larvae, suggesting a conserved function of Evi5 in appendage regeneration. Thus, our results demonstrate that Evi5 plays a critical role in appendage regeneration in amphibians.

En1 and Lmx1b do not recapitulate embryonic dorsal-ventral limb patterning functions during mouse digit tip regeneration.

The mouse digit tip regenerates following amputation. How the regenerate is patterned is unknown, but a long-standing hypothesis proposes developmental patterning mechanisms are re-used during regeneration. The digit tip bone exhibits dorsal-ventral (DV) polarity, so we focus on En1 and Lmx1b, two factors necessary for DV patterning during limb development. We investigate whether they are re-expressed during regeneration in a developmental-like pattern and whether they direct DV morphology of the regenerate. We find that both En1 and Lmx1b are expressed in the regenerating digit tip epithelium and mesenchyme, respectively, but without DV polarity. Conditional genetics and quantitative analysis of digit tip bone morphology determine that genetic deletion of En1 or Lmx1b in adult digit tip regeneration modestly reduces bone regeneration but does not affect DV patterning. Collectively, our data suggest that, while En1 and Lmx1b are re-expressed during mouse digit tip regeneration, they do not define the DV axis during regeneration.

Regulatory T cells regulate blastemal proliferation during zebrafish caudal fin regeneration.

The role of T cells in appendage regeneration remains unclear. In this study, we revealed an important role for regulatory T cells (Tregs), a subset of T cells that regulate tolerance and tissue repair, in the epimorphic regeneration of zebrafish caudal fin tissue. Upon amputation, fin tissue-resident Tregs infiltrate into the blastema, a population of progenitor cells that produce new fin tissues. Conditional genetic ablation of Tregs attenuates blastemal cell proliferation during fin regeneration. Blastema-infiltrating Tregs upregulate the expression of igf2a and igf2b, and pharmacological activation of IGF signaling restores blastemal proliferation in Treg-ablated zebrafish. These findings further extend our understandings of Treg function in tissue regeneration and repair.

Exploring Macrophage-Dependent Wound Regeneration During Mycobacterial Infection in Zebrafish.

The molecular and cellular mechanisms associated with tissue degradation or regeneration in an infectious context are poorly defined. Herein, we explored the role of macrophages in orchestrating either tissue regeneration or degradation in zebrafish embryos pre-infected with the fish pathogen Mycobacterium marinum. Zebrafish were inoculated with different infectious doses of M. marinum prior to fin resection. While mild infection accelerated fin regeneration, moderate or severe infection delayed this process by reducing blastemal cell proliferation and impeding tissue morphogenesis. This was correlated with impaired macrophage recruitment at the wound of the larvae receiving high infectious doses. Macrophage activation characterized, in part, by a high expression level of tnfa was exacerbated in severely infected fish during the early phase of the regeneration process, leading to macrophage necrosis and their complete absence in the later phase. Our results demonstrate how a mycobacterial infection influences the macrophage response and tissue regenerative processes.

Toll signalling promotes blastema cell proliferation during cricket leg regeneration via insect macrophages.

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.

PGE2 facilitates tail regeneration via activation of Wnt signaling in Gekko japonicus.

Tail regeneration is a distinguishing feature of lizards; however, the mechanisms underlying tail regeneration remain elusive. Prostaglandin E2 (PGE2) is an arachidonic acid metabolite that has been extensively investigated in the inflammatory response under both physiological and pathological conditions. PGE2 also act as a regulator of hematopoietic stem cell homeostasis by interacting with Wnt signaling molecules. The present study aims to identify the effects of PGE2 on tail regeneration and the molecular mechanisms behind it. We initially found that PGE2 levels increased during the early stages of tail regeneration, accompanied by the up-regulated expression of cyclooxygenase 1 and cyclooxygenase 2. Next, we demonstrated that reduced PGE2 production leads to the retardation of tail regeneration. Subsequent experiments demonstrated that this effect is likely mediated by Wnt signaling, which proposing that the activation of the Wnt pathway is essential for the initiation of regeneration. The results showed that inhibition of PGE2 production could suppress Wnt activation and inhibit the proliferation of both epithelial and blastema cells. Furthermore, our findings indicated that forced activation of Wnt signaling could rescue the inhibitory effect of Cox antagonist on regeneration, suggesting a positive role of PGE2 on tail regeneration via a non-inflammatory mechanism.

BMP signaling regulates the skeletal and connective tissue differentiation during caudal fin regeneration in sailfin molly (Poecilia latipinna).

Caudal fin regeneration in sailfin molly, Poecilia latipinna (Lesueur 1821) involves an initial wound healing stage, followed by blastema that is formed of fast proliferating cells. In order to replicate the lost fin, correct differentiation of the blastemal cells into various tissues is the prime essence. Among the molecular signals governing proper differentiation of blastemal cells, members of the bone morphogenetic protein (BMP) family are crucial. Herein, we investigated the specific effects of inhibition of BMP signaling using LDN193189 on skeletal and connective tissue formation in the regenerating tail fin of P.latipinna during early differentiation phase. It was observed that BMP inhibition leads to reduction in the length of regeneration, which can be correlated with compromised proliferation of blastemal cells. Decreased expression of cell proliferation marker like pcna together with reduced BrdU positive cells consolidate the above observation. Further, histological analysis revealed stunted progression of skeletal tissues and this correlated with the reduced expression of sox9, runx2 and dlx5, Osc and Osn genes in response to BMP inhibition. Also, defective bone patterning was observed due to BMP inhibition, which was associated with diminished levels of shh, ptc-1, gli2 and other BMP ligands. Moreover, histochemical analysis revealed that collagen, one of the most prominent components of connective tissue, was formed below par in treated fin tissues which was subsequently confirmed by biochemical and transcript level analyses. Overall our results highlight the importance of the BMP pathway in proper differentiation of skeletal and connective tissues during the differentiation stage of regenerating caudal fin.

Transcriptome analysis of the regenerating tail vs. the scarring limb in lizard reveals pathways leading to successful vs. unsuccessful organ regeneration in amniotes.

Lizards are amniotes regenerating the tail but not the limb, and no information on their different gene expression is available. Transcriptomes of regenerating tail and limb blastemas show differences in gene expression between the two organs. In tail blastemal, snoRNAs and Wnt signals appear up-regulated probably in association with the apical epidermal peg (AEP), an epithelial region that sustains tail regeneration but is absent in the limb. A balance between pro-oncogenes and tumor suppressors is likely present in tail blastema allowing a regulated proliferation. Small collagens, protease inhibitors, embryonic keratins are up-regulated in the regenerating tail blastema but not in the limb where Wnt inhibitors, inflammation-immune and extracellular matrix proteins depress cell growth. The AEP and the spinal cord in the tail maintains Wnt and fibroblast growth signaling that stimulate blastema cell proliferation and growth while these signals are absent in the limb as a consequence of the intense inflammation. Regeneration of amniote appendages requires a control of cell proliferation and inflammatory-immune reactions to form an apical epidermal cap. Genes that control cell proliferation and inflammation, addressing regeneration and not tumor formation in the tail and scarring in the limb are discussed for future studies. Developmental Dynamics 246:116-134, 2017. © 2016 Wiley Periodicals, Inc.

Fgf signalling controls diverse aspects of fin regeneration.

Studies have shown that fibroblast growth factor (Fgf) signalling is necessary for appendage regeneration, but its exact function and the ligands involved during regeneration have not yet been elucidated. Here, we performed comprehensive expression analyses and identified fgf20a and fgf3/10a as major Fgf ligands in the wound epidermis and blastema, respectively. To reveal the target cells and processes of Fgf signalling, we performed a transplantation experiment of mesenchymal cells that express the dominant-negative Fgf receptor 1 (dnfgfr1) under control of the heat-shock promoter. This mosaic knockdown analysis suggested that Fgf signalling is directly required for fin ray mesenchyme to form the blastema at the early pre-blastema stage and to activate the regenerative cell proliferation at a later post-blastema stage. These results raised the possibility that the early epidermal Fgf20a and the later blastemal Fgf3/10a could be responsible for these respective processes. We demonstrated by gain-of-function analyses that Fgf20a induces the expression of distal blastema marker junbl, and that Fgf3 promotes blastema cell proliferation. Our study highlights that Fgfs in the wound epidermis and blastema have distinct functions to regulate fin regeneration cooperatively.

Mechanism of Action of Secreted Newt Anterior Gradient Protein.

Anterior gradient (AG) proteins have a thioredoxin fold and are targeted to the secretory pathway where they may act in the ER, as well as after secretion into the extracellular space. A newt member of the family (nAG) was previously identified as interacting with the GPI-anchored salamander-specific three-finger protein called Prod1. Expression of nAG has been implicated in the nerve dependence of limb regeneration in salamanders, and nAG acted as a growth factor for cultured newt limb blastemal (progenitor) cells, but the mechanism of action was not understood. Here we show that addition of a peptide antibody to Prod1 specifically inhibit the proliferation of blastema cells, suggesting that Prod1 acts as a cell surface receptor for secreted nAG, leading to S phase entry. Mutation of the single cysteine residue in the canonical active site of nAG to alanine or serine leads to protein degradation, but addition of residues at the C terminus stabilises the secreted protein. The mutation of the cysteine residue led to no detectable activity on S phase entry in cultured newt limb blastemal cells. In addition, our phylogenetic analyses have identified a new Caudata AG protein called AG4. A comparison of the AG proteins in a cell culture assay indicates that nAG secretion is significantly higher than AGR2 or AG4, suggesting that this property may vary in different members of the family.

Regulation of Axolotl (Ambystoma mexicanum) Limb Blastema Cell Proliferation by Nerves and BMP2 in Organotypic Slice Culture.

We have modified and optimized the technique of organotypic slice culture in order to study the mechanisms regulating growth and pattern formation in regenerating axolotl limb blastemas. Blastema cells maintain many of the behaviors that are characteristic of blastemas in vivo when cultured as slices in vitro, including rates of proliferation that are comparable to what has been reported in vivo. Because the blastema slices can be cultured in basal medium without fetal bovine serum, it was possible to test the response of blastema cells to signaling molecules present in serum, as well as those produced by nerves. We also were able to investigate the response of blastema cells to experimentally regulated changes in BMP signaling. Blastema cells responded to all of these signals by increasing the rate of proliferation and the level of expression of the blastema marker gene, Prrx-1. The organotypic slice culture model provides the opportunity to identify and characterize the spatial and temporal co-regulation of pathways in order to induce and enhance a regenerative response.

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.