Apical Epithelial Cap Research Articles

Tail regeneration at different ontogenetic stages of the tiger salamander Ambystoma tigrinum suggests possible changes in regeneration between larval and metamorphic individuals

AbstractUrodele amphibians possess remarkable regenerative abilities, allowing them to rebuild lost body parts. Contrary to lizards, salamanders can fully restore their tails, including the neural spine and components of the vertebral column. The axolotl (Ambystoma mexicanum) is the vertebrate model organism for regeneration research due to its ease of breeding in captivity. However, axolotls are paedomorphic, retaining larval somatic features throughout adulthood and do not naturally undergo metamorphosis, a transition phase from aquatic larvae to terrestrial adults with profound morphological and physiological changes. We investigate the influence of metamorphosis on salamander tail regeneration after conspecific biting in the metamorphosing sister taxon Ambystoma tigrinum using histological analysis to answer two key questions: (1) Does regeneration continue during metamorphosis, or is it halted? (2) How does regeneration differ histologically among larval, metamorphosing and postmetamorphic individuals? Our findings demonstrate that regeneration continues even during metamorphic climax, indicating the simultaneous coordination of metamorphosis and regeneration. Additionally, notable distinctions were observed between developmental stages concerning the speed of regeneration and structural differences in the formation of an apical epithelial cap (AEC). While the approach taken in this study necessarily restricts sample size, it offers valuable insights into regeneration in a metamorphosing species under natural conditions.

The Accessory Limb Model Regenerative Assay and Its Derivatives.

When the Accessory Limb Model (ALM) regenerative assay was first published by Endo, Bryant, and Gardiner in 2004, it provided a robust system for testing the cellular and molecular contributions during each of the basic steps of regeneration: the formation of the wound epithelium, neural induction of the apical epithelial cap, and the formation of a positional disparity between blastema cells. The basic ALM procedure was developed in the axolotl and involves deviating a limb nerve into a lateral wound and grafting skin from the opposing side of the limb axis into the site of injury. In this chapter, we will review the studies that lead to the conception of the ALM, as well as the studies that have followed the development of this assay. We will additionally describe in detail the standard ALM surgery and how to perform this surgery on different limb positions.

Midkine is a dual regulator of wound epidermis development and inflammation during the initiation of limb regeneration.

Formation of a specialized wound epidermis is required to initiate salamander limb regeneration. Yet little is known about the roles of the early wound epidermis during the initiation of regeneration and the mechanisms governing its development into the apical epithelial cap (AEC), a signaling structure necessary for outgrowth and patterning of the regenerate. Here, we elucidate the functions of the early wound epidermis, and further reveal midkine (mk) as a dual regulator of both AEC development and inflammation during the initiation of axolotl limb regeneration. Through loss- and gain-of-function experiments, we demonstrate that mk acts as both a critical survival signal to control the expansion and function of the early wound epidermis and an anti-inflammatory cytokine to resolve early injury-induced inflammation. Altogether, these findings unveil one of the first identified regulators of AEC development and provide fundamental insights into early wound epidermis function, development, and the initiation of limb regeneration.

Cell type-specific transcriptome analysis unveils secreted signaling molecule genes expressed in apical epithelial cap during appendage regeneration.

Wound epidermis (WE) and the apical epithelial cap (AEC) are believed to trigger regeneration of amputated appendages such as limb and tail in amphibians by producing certain secreted signaling molecules. To date, however, only limited information about the molecular signatures of these epidermal structures is available. Here we used a transgenic Xenopus laevis line harboring the enhanced green fluorescent protein (egfp) gene under control of an es1 gene regulatory sequence to isolate WE/AEC cells by performing fluorescence-activated cell sorting during the time course of tail regeneration (day 1, day 2, day 3 and day 4 after amputation). Time-course transcriptome analysis of these isolated WE/AEC cells revealed that more than 8,000 genes, including genes involved in signaling pathways such as those of reactive oxygen species, fibroblast growth factor (FGF), canonical and non-canonical Wnt, transforming growth factor β (TGF β) and Notch, displayed dynamic changes of their expression during tail regeneration. Notably, this approach enabled us to newly identify seven secreted signaling molecule genes (mdk, fstl, slit1, tgfβ1, bmp7.1, angptl2 and egfl6) that are highly expressed in tail AEC cells. Among these genes, five (mdk, fstl, slit1, tgfβ1 and bmp7.1) were also highly expressed in limb AEC cells but the other two (angptl2 and egfl6) are specifically expressed in tail AEC cells. Interestingly, there was no expression of fgf8 in tail WE/AEC cells, whose expression and pivotal role in limb AEC cells have been reported previously. Thus, we identified common and different properties between tail and limb AEC cells.

Regeneration of caudal fin in Poecilia latipinna: Insights into the progressive tissue morphogenesis

ABSTRACTStudies using fish fin as a model to understand the nuance of epimorphosis are gaining interest of lately. This study illustrates for the first time the daily changes in the tissue architecture of regenerating tail fin of Poecilia latipinna. Wound epithelium is formed within 24 hpa that eventually gets stratified into apical epithelial cap by 48 hpa. In the subsequent day, proliferating cells accumulate in front of each fin-ray marking the beginning of blastema. Distally these cells express signs of cartilage condensation by 4 dpa. However, ossification and subsequent transformation of actinotrichia to lepidotrichia was observed on 5 dpa. Subsequently, the regenerate grew at variable rate until it achieved the original size on 25 dpa. This result would serve as a worthwhile standard reference for further explorative studies that demand manipulation of a regulatory signal at a defined time point.

Variation in expression and activity pattern of mmp2 and mmp9 on different time scales in the regenerating caudal fin of Poecilia latipinna.

Alteration in the expression pattern of matrix metalloproteinase (MMP)2 and MMP9 was studied in the regenerating caudal fin of Poecilia latipinna immediately following amputation until the new tissues gained structural integrity. Timed expression pattern of these two MMPs was studied at enzyme, transcript as well as protein levels. Additionally, both the gelatinases were localized in the regenerating caudal fin during three specific stages of regeneration. The results revealed a progressive increase in the expression of MMP2 starting at 1h post amputation (hpa), indicating its possible role in the remodelling of extracellular matrix early on during caudal-fin regeneration. Nevertheless, a reduction in transcript level expression of MMP2 at 6 hpa and 12 hpa stages, points towards a possible transcriptional regulation, to further moderate its activity. As observed in the case of MMP2, expression of MMP9 too increased from 1 hpa and remained elevated until 5 dpa. However, the active MMP9 revealed its presence only 12 hpa onwards. Moreover, both the gelatinases were localised in the apical epithelial cap and in the progress zone at wound epithelium (1 dpa) and blastema (60 hpa) stages respectively. Further, during early differentiation stage (5 dpa), high intensities of MMP2 and MMP9 were localized in the newly formed actinotrichia as compared with the tissue proximal to it. Based on the results, it could be construed that the controlled up-regulation of MMP2 and MMP9 from 1 hpa until the early differentiation stage ensures a regulated digestion of extracellular matrix, perhaps to facilitate the recruitment, proliferation, morphogenesis and re-patterning of resident stem cells during caudal fin regeneration in P. latipinna.

TISSUE REPAIR AND EPIMORPHIC REGENERATION: AN OVERVIEW.

This manuscript discusses wound healing as a component of epimorphic regeneration and the role of the immune system in this process. Epimorphic regeneration involves formation of a blastema, a mass of undifferentiated cells capable of giving rise to the regenerated tissues. The apical epithelial cap plays an important role in blastemal formation. True regeneration is rarely observed in mammals. With the exception of transgenic strains, tissue repair in mammals usually leads to non-functional fibrotic tissue formation. In contrast, a number of lower order species including planarians, salamanders, and reptiles, have the ability to overcome the burden of scarring and tissue loss through complex adaptations that allow them to regenerate various anatomic structures through epimorphic regeneration. Blastemal cells have been suggested to originate via various mechanisms including de-differentiation, transdifferentiation, migration of pre-existing adult stem cell niches, and combinations of these.

A transgenic reporter under control of an es1 promoter/enhancer marks wound epidermis and apical epithelial cap during tail regeneration in Xenopus laevis tadpole

Rapid wound healing and subsequent formation of the apical epithelial cap (AEC) are believed to be required for successful appendage regeneration in amphibians. Despite the significant role of AEC in limb regeneration, its role in tail regeneration and the mechanisms that regulate the wound healing and AEC formation are not well understood. We previously identified Xenopus laevis es1, which is preferentially expressed in wounded regions, including the AEC after tail regeneration. In this study we established and characterized transgenic Xenopus laevis lines harboring the enhanced green fluorescent protein (EGFP) gene under control of an es1 gene regulatory sequence (es1:egfp).The EGFP reporter expression was clearly seen in several regions of the embryo and then declined to an undetectable level in larvae, recapitulating the endogenous es1 expression. After amputation of the tadpole tail, EGFP expression was re-activated at the edge of the stump epidermis and then increased in the wound epidermis (WE) covering the amputation surface. As the stump started to regenerate, the EGFP expression became restricted to the most distal epidermal region, including the AEC. EGFP was preferentially expressed in the basal or deep cells but not in the superficial cells of the WE and AEC.We performed a small-scale pharmacological screening for chemicals that affected the expression of EGFP in the stump epidermis after tail amputation. The EGFP expression was attenuated by treatment with an inhibitor for ERK, TGF-β or reactive oxygen species (ROS) signaling. These treatments also impaired wound closure of the amputation surface, suggesting that the three signaling activities are required for es1 expression in the WE and successful wound healing after tail amputation.These findings showed that es1:egfp Xenopus laevis should be a useful tool to analyze molecular mechanisms regulating wound healing and appendage regeneration.

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.

Going out on a limb about regrowing an arm

Starting with the observations that fetuses effortlessly grow limbs, fetuses heal wounds without scar and children up to the age of two can partially regrow amputated digits, the potential for adult humans to regrow limbs is explored. The process of limb growth in amphibians is reviewed with these steps summarizing the process: blood vessels contract to minimize bleeding; the injury site is covered by skin cells transforming into the apical epithelial cap which sends signals important for the next phases of the regrowth; resident fibroblasts leave the surrounding extracellular matrix and migrate across the amputation surface; migratory fibroblasts proliferate and dedifferentiate to form an aggregation of stemlike cells called the blastema; and the blastema coordinates the formation of a new limb. Other factors contributing to this process are: innervation, cell spatial "memory," chemical signals between cells, gene up and down regulation, cell differentiation (or dedifferentiation) and inflammatory cells. Remarkable discoveries have been made in all these areas in the last few years that might be integrated into technology for limb regeneration. In particular, the demonstration of the plasticity of supposedly "terminally differentiated" cells speak to the idea that mature cells at the amputation site might be harnessed for limb regrowth. Also, the demonstration that macrophages can be driven to a regenerative phenotype (M2) and they may also be stem-like is promising for complex regenerations. This article posits that scientific discoveries useful for limb regeneration have been made and now it is time to develop technology exploiting these discoveries to regrow limbs.

Early Regulation of Axolotl Limb Regeneration

Amphibian limb regeneration has been studied for a long time. In amphibian limb regeneration, an undifferentiated blastema is formed around the region damaged by amputation. The induction process of blastema formation has remained largely unknown because it is difficult to study the induction of limb regeneration. The recently developed accessory limb model (ALM) allows the investigation of limb induction and reveals early events of amphibian limb regeneration. The interaction between nerves and wound epidermis/epithelium is an important aspect of limb regeneration. During early limb regeneration, neurotrophic factors act on wound epithelium, leading to development of a functional epidermis/epithelium called the apical epithelial cap (AEC). AEC and nerves create a specific environment that inhibits wound healing and induces regeneration through blastema formation. It is suggested that FGF-signaling and MMP activities participate in creating a regenerative environment. To understand why urodele amphibians can create such a regenerative environment and humans cannot, it is necessary to identify the similarities and differences between regenerative and nonregenerative animals. Here we focus on ALM to consider limb regeneration from a new perspective and we also reported that focal adhesion kinase (FAK)-Src signaling controlled fibroblasts migration in axolotl limb regeneration.

Spatiotemporal regulation of keratin 5 and 17 in the axolotl limb

Amphibians have greater regeneration capability than higher vertebrates. They can regenerate their limbs after an amputation. As a limb is regenerated, a regeneration-specific epithelium called the apical epithelial cap (AEC) is induced. The AEC is an essential structure for limb regeneration. Despite the importance of the AEC, molecular marker genes have not been well studied at the molecular level. In the present study, keratin5 (KRT5) and KRT17 were investigated in an axolotl-regenerating limb. KRT5 and KRT17 were expressed in a regenerating limb but down-regulated in a differentiating limb. KRT5 showed characteristic regulation in a regenerating blastema. KRT5 was suppressed in the basal layer of the AEC. This KRT5 suppression was correlated to the blastema differentiation and nerve presence. Simple skin wounding could also upregulate both KRT5 and KRT17 gene expression. But these genes were suppressed within a shorter time than in limb regeneration. The KRT5 and KRT17 gene profile can be a useful marker gene to investigate AEC in limb regeneration.

Activation of germline-specific genes is required for limb regeneration in the Mexican axolotl

The capacity for tissue and organ regeneration in humans is dwarfed by comparison to that of salamanders. Emerging evidence suggests that mechanisms learned from the early phase of salamander limb regeneration—wound healing, cellular dedifferentiation and blastemal formation–will reveal therapeutic approaches for tissue regeneration in humans. Here we describe a unique transcriptional fingerprint of regenerating limb tissue in the Mexican axolotl (Ambystoma mexicanum) that is indicative of cellular reprogramming of differentiated cells to a germline-like state. Two genes that are required for self-renewal of germ cells in mice and flies, Piwi-like 1 (PL1) and Piwi-like 2 (PL2), are expressed in limb blastemal cells, the basal layer keratinocytes and the thickened apical epithelial cap in the wound epidermis in the regenerating limb. Depletion of PL1 and PL2 by morpholino oligonucleotides decreased cell proliferation and increased cell death in the blastema leading to a significant retardation of regeneration. Examination of key molecules that are known to be required for limb development or regeneration further revealed that FGF8 is transcriptionally downregulated in the presence of the morpholino oligos, indicating PL1 and PL2 might participate in FGF signaling during limb regeneration. Given the requirement for FGF signaling in limb development and regeneration, the results suggest that PL1 and PL2 function to establish a unique germline-like state that is associated with successful regeneration.

Collagen Reconstitution is Inversely Correlated with Induction of Limb Regeneration inAmbystoma mexicanum

Amphibians can regenerate missing body parts, including limbs. The regulation of collagen has been considered to be important in limb regeneration. Collagen deposition is suppressed during limb regeneration, so we investigated collagen deposition and apical epithelial cap (AEC) formation during axolotl limb regeneration. The accessory limb model (ALM) has been developed as an alternative model for studying limb regeneration. Using this model, we investigated the relationship between nerves, epidermis, and collagen deposition. We found that Sp-9, an AEC marker gene, was upregulated by direct interaction between nerves and epidermis. However, collagen deposition hindered this interaction, and resulted in the failure of limb regeneration. During wound healing, an increase in deposition of collagen caused a decrease in the blastema induction rate in ALM. Wound healing and limb regeneration are alternate processes.

Blastema induction in aneurogenic state and Prrx-1 regulation by MMPs and FGFs in Ambystoma mexicanum limb regeneration

Urodele amphibians can regenerate amputated limbs. It has been considered that differentiated dermal tissues generate multipotent and undifferentiated cells called blastema cells during limb regeneration. In early phases of limb regeneration, blastema cells are induced by nerves and the apical epithelial cap (AEC). We had previously investigated the role of neurotrophic factors in blastema or blastema-like formation consisting of Prrx-1 positive cells. A new system suitable for investigating early phases of limb regeneration, called the accessory limb model (ALM), was recently developed. In this study, we performed a comparative transcriptome analysis between a blastema and wound using ALM. Matrix metalloproteinase (MMP) and fibroblast growth factor (FGF) signaling components were observed to be predominantly expressed in ALM blastema cells. Furthermore, we found that MMP activity induced a blastema marker gene, Prrx-1, in vitro, and FGF signaling pathways worked in coordination to maintain Prrx-1 expression and ALM blastema formation. Furthermore, we demonstrated that these two activities were sufficient to induce an ALM blastema in the absence of a nerve in vivo.

Two different regenerations accomplish limb regeneration cooperatively

We have developed a model called accessory limb model, which has a mystery. If an accessory limb is induced in a middle of stylopod, an induced limb possesses autopod and zeugopod, no stylopod. In theory, the induced limb should have had half of stylopod. However, none of the accessory limb possesses such a half of stylopod. If a stylopod was injured enough to damage a bone, an induced accessory limb exhibits half of stylopod. We found that this stylopod regeneration was AEC (apical epithelial cap) independent even though distal structures (autopod and zeugopod) were AEC dependent. These AEC dependent and independent mechanisms coordinately achieve limb regeneration. This coordinated regeneration system also works in regular limb regeneration (amputated limb). Furthermore, we also found how to create the distal structure in AEC free environment. Fgf-signaling can trigger intercalary responses as reported in Xenopus. When we provide the distal in an amputated limb without AEC, no intercalation takes place, resulting in no intermediate structure. However, when we activated fgf-signaling with the same condition, intermediate structures were induced. Those findings solve the mystery of the accessory limb model and propose an entire new model for limb regeneration study. Those findings warm many of the running molecular analysis because all of them have not paid attention to an existence of those two regenerations. For a finer analysis of the epimorphic regeneration, we think that it is necessary to distinguish two types of regeneration.

Identification of genes associated with regenerative success of Xenopus laevis hindlimbs.

BackgroundEpimorphic regeneration is the process by which complete regeneration of a complex structure such as a limb occurs through production of a proliferating blastema. This type of regeneration is rare among vertebrates but does occur in the African clawed frog Xenopus laevis, traditionally a model organism for the study of early development. Xenopus tadpoles can regenerate their tails, limb buds and the lens of the eye, although the ability of the latter two organs to regenerate diminishes with advancing developmental stage. Using a heat shock inducible transgene that remains silent unless activated, we have established a stable line of transgenic Xenopus (strain N1) in which the BMP inhibitor Noggin can be over-expressed at any time during development. Activation of this transgene blocks regeneration of the tail and limb of Xenopus tadpoles.ResultsIn the current study, we have taken advantage of the N1 transgenic line to directly compare morphology and gene expression in same stage regenerating vs. BMP signalling deficient non-regenerating hindlimb buds. The wound epithelium of N1 transgenic hindlimb buds, which forms over the cut surface of the limb bud after amputation, does not transition normally into the distal thickened apical epithelial cap. Instead, a basement membrane and dermis form, indicative of mature skin. Furthermore, the underlying mesenchyme remains rounded and does not expand to form a cone shaped blastema, a normal feature of successful regeneration.Using Affymetrix Gene Chip analysis, we have identified genes linked to regenerative success downstream of BMP signalling, including the BMP inhibitor Gremlin and the stress protein Hsp60 (no blastema in zebrafish). Gene Ontology analysis showed that genes involved in embryonic development and growth are significantly over-represented in regenerating early hindlimb buds and that successful regeneration in the Xenopus hindlimb correlates with the induction of stress response pathways.ConclusionN1 transgenic hindlimbs, which do not regenerate, do not form an apical epithelial cap or cone shaped blastema following amputation. Comparison of gene expression in stage matched N1 vs. wild type hindlimb buds has revealed several new targets for regeneration research.

Expression and biological effect of urodele fibroblast growth factor 1: relationship to limb regeneration.

Fibroblast growth factors (FGFs) have been previously implicated in urodele limb regeneration. Here, we examined expression of FGF-1 by blastema cells and neurons and investigated its involvement in wound epithelial formation and function and in the trophic effect of nerves. Neurons innervating the limb and blastema cells in vivo and in vitro expressed the FGF-1 gene. The peptide was present in blastemas in vivo. Wound epithelium thickened when recombinant newt FGF-1 was provided on heparin-coated beads, demonstrating that the FGF-1 was biologically active and that the wound epithelium is a possible target tissue of FGF. FGF-1 did not stimulate accessory limb formation. FGF-1 was as effective as 10% fetal bovine serum in maintaining proliferative activity of blastema cells in vitro but was unable to maintain growth of denervated, nerve-dependent stage blastemas when provided on beads or by injection. FGF-1 had a strong stimulating effect on blastema cell accumulation and proliferation of limbs inserted into the body cavity that were devoid of an apical epithelial cap (AEC). These results show that FGF-1 can signal wound epithelium cap formation and/or function and can stimulate mesenchyme accumulation/proliferation in the absence of the AEC but that FGF-1 is not directly involved in the neural effect on blastema growth.

Vasculature in pre-blastema and nerve-dependent blastema stages of regenerating forelimbs of the adult newt, Notophthalmus viridescens.

Immunocytochemistry utilizing a monoclonal antibody (BV1; blood vessel 1) highly reactive to the vasculature of the adult newt showed that a developing vasculature was present during early, pre-blastema, and early-bud blastema stages of forelimb regeneration in this species. Infusion of Prussian Blue and DiI into the brachial artery further delineated the intactness of this early vasculature. Finally, macroscopic observations of vascular flow underneath the apical epithelial cap (AEC) and microsurgical removal of the AEC and observation of subsequent bleeding buttressed the conclusion that an intact vasculature exists during early nerve-dependent stages of newt forelimb regeneration. The results suggest that this process of neovascular formation is angiogenesis, i.e., the formation of new vessels from pre-existing vessels in the stump. Furthermore, angiogenesis is an ongoing process initiated early after amputation. Blastema cells and the AEC are likely sourcesof factors that stimulate neovascularization.

Effect of retinoic acid onFgf‐8expression in regenerating urodele amphibian limbs

In our previous study, we have shown that Fgf‐8 is expressed in the basal layer of the apical epithelial cap (AEC) and in the underlying thin layer of mesenchymal tissue of the regenerating limbs of Mexican axolotl, Amby‐stoma mexicanum. Our present RT‐PCR data also demonstrate that Fgf‐8 transcript is localized both in the mesenchymal and epidermal tissues. To understand the effect of retinoic acid (RA) on the expression of Fgf‐8 in the regenerating axolotl limbs, RA was injected intraperitoneally at the dediffer‐entiation stage of limb regeneration. The RA treatment caused a change in the Fgf‐8 expression profile of the regenerating limbs. In RA‐treated limbs, duration of Fgf‐8 expression was prolonged and a high level of expression was maintained during dedifferentiation and blastema formation stages. These results suggest that Fgf‐8 is an important molecule in the process of pattern duplication of regenerating salamander limbs evoked by RA treatment.