Apical Epidermal Cap Research Articles

Cellular responses in the FGF10-mediated improvement of hindlimb regenerative capacity in Xenopus laevis revealed by single-cell transcriptomics.

Xenopus laevis tadpoles possess regenerative capacity in their hindlimb buds at early developmental stages (stages ~52-54); they can regenerate complete hindlimbs with digits after limb bud amputation. However, they gradually lose their regenerative capacity as metamorphosis proceeds. Tadpoles in late developmental stages regenerate fewer digits (stage ~56), or only form cartilaginous spike without digits or joints (stage ~58 or later) after amputation. Previous studies have shown that administration of fibroblast growth factor 10 (FGF10) in late-stage (stage 56) tadpole hindlimb buds after amputation can improve their regenerative capacity, which means that the cells responding to FGF10 signaling play an important role in limb bud regeneration. In this study, we performed single-cell RNA sequencing (scRNA-seq) of hindlimb buds that were amputated and administered FGF10 by implanting FGF10-soaked beads at a late stage (stage 56), and explored cell clusters exhibiting a differential gene expression pattern compared with that in controls treated with phosphate-buffered saline. The scRNA-seq data showed expansion of fgf8-expressing cells in the cluster of the apical epidermal cap of FGF10-treated hindlimb buds, which was reported previously, indicating that the administration of FGF10 was successful. On analysis, in addition to the epidermal cluster, a subset of myeloid cells and a newly identified cluster of steap4-expressing cells showed remarkable differences in their gene expression profiles between the FGF10- or phosphate-buffered saline-treatment conditions, suggesting a possible role of these clusters in improving the regenerative capacity of hindlimbs via FGF10 administration.

Nerves and Proliferation of Progenitor Cells in Limb Regeneration.

Nerves, in conjunction with the apical epidermal cap (AEC), play an important role in the proliferation of the mesenchymal progenitor cells comprising the blastema of regenerating urodele amphibian limbs. Reinnervation after amputation requires factors supplied by the forming blastema, and neurotrophic factors must be present at or above a quantitative threshold for mitosis of the blastema cells. The AEC forms independently of nerves, but requires nerves to be maintained. Urodele limb buds are independent of nerves for regeneration, but innervation imposes a regenerative requirement for nerve factors on their cells as they differentiate. There are three main ideas on the functional relationship between nerves, AEC, and blastema cells: (1) nerves and AEC produce factors with different roles in maintaining progenitor status and mitosis; (2) the AEC produces the factors that promote blastema cell mitosis, but requires nerves to express them; (3) blastema cells, nerves, and AEC all produce the same factor(s) that additively attain the required threshold for mitosis.

Mechanisms of urodele limb regeneration.

This review explores the historical and current state of our knowledge about urodele limb regeneration. Topics discussed are (1) blastema formation by the proteolytic histolysis of limb tissues to release resident stem cells and mononucleate cells that undergo dedifferentiation, cell cycle entry and accumulation under the apical epidermal cap. (2) The origin, phenotypic memory, and positional memory of blastema cells. (3) The role played by macrophages in the early events of regeneration. (4) The role of neural and AEC factors and interaction between blastema cells in mitosis and distalization. (5) Models of pattern formation based on the results of axial reversal experiments, experiments on the regeneration of half and double half limbs, and experiments using retinoic acid to alter positional identity of blastema cells. (6) Possible mechanisms of distalization during normal and intercalary regeneration. (7) Is pattern formation is a self‐organizing property of the blastema or dictated by chemical signals from adjacent tissues? (8) What is the future for regenerating a human limb?

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.

Microscopic observations show invasion of inflammatory cells in the limb blastema and epidermis in pre-metamorphic frog tadpoles which destroy the Apical Epidermal CAP and impede regeneration

Some limb regeneration in tadpoles of Rana dalmatina occurs at stages 44–48 when small hind-limbs are present while scarring occurs at stages 51–52 when forelimbs have developed and metamorphosis is approaching. Ultrastructural analysis of cells forming the regenerating blastema detects mesenchymal cells and an Apical Epidermal Cap (AEC) in regenerating limb blastema 5–6 days post-amputation at stages 46–48. In contrast, granulocytes and numerous macrophages and lymphocytes prevail over mesenchymal cells in limb blastema at stages 51–52, which are destined to form scars. An increase in inflammatory cells in limb blastema prior to metamorphosis suggests a negative influence of immune cells on limb regeneration. Inflammatory cells invade the apical wound epidermis where stem keratinocytes are likely destroyed, impeding the formation of an AEC, the microregion which leads to limb regeneration. The invasion of immune cells, however, may also represent a physiological consequence of the death of cell populations in the tadpoles occurring with approaching metamorphosis. The passage from an aquatic to a terrestrial life in this frog elicits the typical amniote scarring reaction after wounding, and the limb cannot regenerate. The present observations sustain the hypothesis that the evolution of the adaptive immunity in tetrapods while efficiently preserving adult self-condition, determined the loss of tissue regeneration since the embryonic antigens evocated in blastema cells are removed by immune cells of the adult.

DNA Methylation Dynamics Regulate the Formation of a Regenerative Wound Epithelium during Axolotl Limb Regeneration.

The formation of a blastema during regeneration of an axolotl limb involves important changes in the behavior and function of cells at the site of injury. One of the earliest events is the formation of the wound epithelium and subsequently the apical epidermal cap, which involves in vivo dedifferentiation that is controlled by signaling from the nerve. We have investigated the role of epigenetic modifications to the genome as a possible mechanism for regulating changes in gene expression patterns of keratinocytes of the wound and blastema epithelium that are involved in regeneration. We report a modulation of the expression DNMT3a, a de novo DNA methyltransferase, within the first 72 hours post injury that is dependent on nerve signaling. Treatment of skin wounds on the upper forelimb with decitabine, a DNA methyltransferase inhibitor, induced changes in gene expression and cellular behavior associated with a regenerative response. Furthermore, decitabine-treated wounds were able to participate in regeneration while untreated wounds inhibited a regenerative response. Elucidation of the specific epigenetic modifications that mediate cellular dedifferentiation likely will lead to insights for initiating a regenerative response in organisms that lack this ability.

The role of peripheral nerves in urodele limb regeneration

Nerve axons and the apical epidermal cap (AEC) are both essential for the formation of an accumulation blastema by amputated limbs of urodele salamanders. The AEC forms in the absence of axons, but is not maintained, and blastema formation fails. Growth stages of the blastema become nerve-independent for morphogenesis, but remain dependent on the nerve for blastema growth. Denervated growth stage blastemas form smaller than normal skeletal parts, owing to diminished mitosis, but form the full proximodistal array of skeletal elements. This difference in nerve dependency of morphogenesis and proliferation is hypothesized to be the result of a dependence of the AEC on nerves for blastema cell proliferation but not for blastema morphogenesis. Regenerating axons induce the synthesis and secretion of the anterior gradient protein (AGP) by distal Schwann cells during dedifferentiation and by the gland cells of the AEC during blastema growth stages. AGP promotes the regeneration of a denervated limb to digit stages when electroporated into the limb during dedifferentiation. Once a critical mass of blastema cells has been attained, the blastema can undergo morphogenesis in the absence of the nerve, but the regenerate will be a miniature, because the nerve is no longer inducing the AEC to carry out its AGP-mediated proliferative function. AGP expression by both Schwann cells and the AEC is induced by axons, but the nature of the inductive agent is unclear.

Histological study of the dynamics in epidermis regeneration of the carp tail fin (Cyprinus carpio, Linnaeus, 1758)

Teleostean fins when partially amputated suffer a regenerative process called epimorphic regeneration, characterized by the following stages: healing, based on the formation of a multistratified epidermal layer, the formation of a mass of pluripotent cells known as blastema, the differentiation of these cells, the synthesis and disposition of the extracellular matrix, morphological growth and restoration. The epidermis has a fundamental role in the regenerative process of fish fins, as the healing time of this structure leads it to a faster regenerative process and it also works as a defense against the external environment. In this sense, due to the fast regeneration shown by the epidermis, the aim of this paper is to study the histology of the regenerative dynamics of the carp fin tail (Cyprinus carpio), under the light and transmission electron microscope. Epidermic regeneration begins right in the first hours after the fin amputation and it continues throughout the regenerative process. After 24 hours, an apical epidermal cap is established. Cytoplasmatic prolongations and intercellular junctions are observed and the cells of the basal layer of the epidermis change from the cubic form to the cylindrical, due to the development of the cytoplasmatic organelles responsible for the synthesis of the basal membrane, lost after amputation. These results show the importance of histological studies in regenerative processes. We believe that the association of molecular biology with histological studies can throw further light onto these regenerative dynamics.

A novel Xenopus laevis larval keratin gene, xlk2: Its gene structure and expression during regeneration and metamorphosis of limb and tail

A novel cytokeratin (CK) gene, xlk2, was cloned from a cDNA library prepared from regenerating limbs of Xenopus larvae. The deduced amino acid sequence indicated that its product, XLK2, is a 48 kDa type I (acidic) CK and has a high similarity to CK13, 15, and 19 with the highest homology (58%) to mouse CK15. The gene of xlk2 exclusively expressed in basal cells of the bi-layered larval epidermis, but not in other cells in larvae and not in other periods of life. Its expression was down-regulated during spontaneous and thyroid hormone-induced metamorphosis. The basal cells of the apical epidermal cap (AEC) formed on the regenerate of larval limbs terminated the expression of xlk2, whereas those of the adjacent normal epidermis continued to express it. The AEC-basal cells did not re-express the gene in the regenerate. In contrast, the basal cells of the tail regenerate also once terminated the expression of xlk2, but was able to re-express xlk2 later, supporting a notion that the “de-differentiated” basal cells of the tail epidermal regenerate re-differentiate into larval normal epidermal cells.

Amphibian regeneration and stem cells.

Larval and adult urodeles and anuran tadpoles readily regenerate their limbs via a process of histolysis and dedifferentiation of mature cells local to the amputation surface that accumulate under the wound epithelium as a blastema of stem cells. These stem cells require growth and trophic factors from the apical epidermal cap (AEC) and the nerves that re-innervate the blastema for their survival and proliferation. Members of the fibroblast growth factor (FGF) family synthesized by both AEC and nerves, and glial growth factor, substance P, and transferrin of nerves are suspected survival and proliferation factors. Stem cells derived from fibroblasts and muscle cells can transdifferentiate into other cell types during regeneration. The regeneration blastema is a self-organizing system based on positional information inherited from parent limb cells. Retinoids, which act through nuclear receptors, have been used in conjunction with assays for cell adhesivity to show that positional identity of blastema cells is encoded in the cell surface. These molecules are involved in the cell-cell signaling network that re-establishes the original structural pattern of the limb. Other systems of interest that regenerate by histolysis and dedifferentiation of pigmented epithelial cells are the neural retina and lens. Members of the FGF family are also important to the regeneration of these structures. The mechanism of amphibian regeneration by dedifferentiation is of importance to the development of a regenerative medicine, since understanding this mechanism may offer insights into how we might chemically induce the regeneration of mammalian tissues.

Unique expression patterns of matrix metalloproteinases in regenerating newt limbs.

The process of regeneration of urodele limbs includes a drastic remodeling of extracellular matrices (ECMs) that is induced by matrix metalloproteinases (MMPs) and is thought to be one of the triggers of the regeneration. We studied this remodeling in limbs of Japanese newt, Cynops pyrrhogaster, by using five genes of newt MMPs (nMMPs) as probes: nMMP9, nMMP3/10-a, nMMP3/10-b, and nMMP13 that had been characterized previously, and nMMPe that was newly cloned in the present study. nMMPe was 502 amino acid residues long and showed a low homology to other known vertebrate MMPs. Reverse transcriptase-polymerase chain reactions analysis localized the transcript of nMMPe in the apical epidermal cap (AEC) and the non-blastemal wound epidermis but not in the blastemal mesenchyme or the normal epidermis. Northern blot analysis localized the transcripts of nMMP9, nMMP3/10-a, and nMMP13 in the bone of regenerating limbs, whereas those of nMMP3/10-b in AEC. mRNA in situ hybridization experiments identified the nMMP-expressing cells. nMMP9 gene was strongly expressed in chondrocytes of the cartilage of epiphysis. Of interest, basal cells of AEC, but not those of the normal skin, expressed nMMP3/10-b intensely. Immunohistochemical analysis showed that the nMMP9 proteins synthesized by chondrocytes were secreted and distributed widely in the basement membrane of bone and ECMs of the amputation plane. These nMMPs characterized in the present study might cooperatively work to remodel ECMs of regenerating limbs.

Cell proliferation during blastema formation in the regenerating teleost fin.

Epimorphic regeneration in teleost fins occurs through the establishment of a balanced growth state in which a blastema gives rise to all the mesenchymal cells, whereas definite areas of the epidermis proliferate leading to its extension, thus, allowing the enlargement of the whole structure. This type of regeneration involves specific mechanisms that temporally and spatially regulate cell proliferation. To understand how the blastema is formed and how this growth situation is set up, we investigated cell proliferation patterns in the regenerating fin of the goldfish Carassius auratus from the time of amputation to that of blastema formation by using proliferating cell nuclear antigen immunostaining and bromodeoxyuridine labeling. Wound closure and apical epidermal cap formation took place by epidermal migration and re-arrangement, without the contribution of cell proliferation. As soon as the apical cap had formed, the epidermis started to proliferate at its lateral surfaces, in which all layers maintained cycling for the duration of the studied process. The distal epidermal cap, on the contrary, presented very few cycling cells, and its cytoarchitecture was indicative of continuous remodeling due to ray growth. The basal layer of this epidermal cap showed a typical morphology and remained nonproliferative whilst in contact with the proliferating blastema. Proliferation in the mesenchymal compartment of the ray started far from the amputation plane. Subsequently, cycling cells approached that location, until they formed the blastema in contact with the apical epidermal cap. Differences observed between the epidermis and mesenchyma, regarding activation of the cell cycle and the establishment of proliferative patterns, suggest that differential mechanisms regulate cell proliferation in each of these compartments during the initial stages of regeneration.

Cell proliferation and movement during early fin regeneration in zebrafish

Cell proliferation and cell movement during early regeneration of zebrafish caudal fins were examined by injecting BrdU and Di-I, respectively. In normal fins of adult fish, a small number of proliferating cells are observed in the epidermis only. Shortly following amputation, epithelial cells covered the wound to form the epidermal cap but did not proliferate. However, by 24 hr, epithelial cells proximal to the level of amputation were strongly labeled with BrdU. Label incorporation was also detected in a few mesenchymal cells. Proliferating cells in the basal epithelial layer were first observed at 48 hr at the level of the newly formed lepidotrichia. At 72 hr, proliferating mesenchymal cells were found distal to the plane of amputation whereas more proximal labeled cells included mainly those located between the lepidotrichia and the basal membrane. When BrdU-injected fins were allowed to regenerate for longer periods, labeled cells were observed in the apical epidermal cap, a location where cells are not thought to proliferate. This result is suggestive of cell migration. Epithelial cells, peripheral to the rays or in the tissue between adjacent rays, were labeled with Di-I and were shown to quickly migrate towards the site of amputation, the cells closer to the wound migrating faster. Amputation also triggered migration of cells of the connective tissue located between the hemirays. Although cell movement was induced up to seven segments proximal from the level of amputation, cells located within two segments from the wound provided the main contribution to the blastema. Thus, cell proliferation and migration contribute to the early regeneration of zebrafish fins.

Identification and characterization of newt rad (ras associated with diabetes), a gene specifically expressed in regenerating limb muscle

Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription-PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry-over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration. © 2001 Wiley-Liss, Inc.

Identification and characterization of newt rad (ras associated with diabetes), a gene specifically expressed in regenerating limb muscle.

Formation of the blastema is a key event for limb regeneration in urodele amphibians, and skeletal muscle has been thought to be a major origin of the multipotent blastemal mesenchyme. In the present study, we used differential display to identify the genes expressed differentially in the muscle at the amputation site. We have isolated a cDNA clone that was upregulated during limb regeneration of the Japanese newt, Cynops pyrrhogaster. Deduced amino acid sequence revealed that the cloned cDNA was a newt homolog of rad (ras associated with diabetes), a gene overexpressed in skeletal muscle of Type II diabetic patients. Expression of newt rad (nrad) was not observed in unamputated normal limb muscle, increased within 4 hr after amputation, and then decreased to the level of normal muscle between 11 and 21 days after amputation. In situ hybridization showed that the transcripts of nrad were localized around most of the nuclei of skeletal muscle near the amputation site, indicating the expression of nrad in the multinucleate myotubes. This expression gradually decreased along the distal to proximal axis. No signals were observed in apical epidermal cap or blastemal mesenchyme. However, reverse transcription-PCR analysis detected a very low level of nrad expression in blastema, suggesting the carry-over of nrad expression in blastema from muscle. Administration of retinoic acid, which has been shown to cause an enhanced dedifferentiation in the regenerating limbs, increased nrad expression in more proximally located limb muscle tissues and prolonged the expression period. Thus, it was strongly suggested that the nrad expression is correlated with the dedifferentiation of myotubes of regenerating limbs. We also analyzed the expression of nrad during development. Transcripts were observed in immature oocytes, seen faintly or not seen thereafter until stage 57 when its expression increased again. These results indicated that nrad may play a role(s) in the developmental process as well as limb regeneration.

Expression of Msx-2 during development, regeneration, and wound healing in axolotl limbs.

Msx genes are transcription factors that are expressed during embryogenesis of developing appendages in regions of epithelial-mesenchymal interactions. Various lines of evidence indicate that these genes function to maintain embryonic tissues in an undifferentiated, proliferative state. We have identified the axolotl homolog of Msx-2, and investigated its expression during limb development, limb regeneration, and wound healing. As in limb buds of higher vertebrates, axolotl Msx-2 is expressed in the apical epidermis and mesenchyme; however, its expression domain is more extensive, reflecting the broader region of the apical epidermal cap in amphibians. Msx-2 expression is downregulated at late stages of limb development, but is reexpressed within one hour after limb amputation. Msx-2 is also reexpressed during wound healing, and may be essential in the early stages of initiation of the limb regeneration cascade.

A conceptual framework for analyzing axial patterning in regenerating urodele limbs.

This review describes what we have learned about mechanisms of patterning in regenerating urodele limbs. Experimental evidence for three concepts is presented. First, the regeneration blastema is a self-organizing system based on positional memories inherited from parent limb cells. Second, the autonomous patterning mechanism involves local cell interactions that determine patterns of Hox gene activation. The effector molecules for positional identity reside in or on the cell surface, and can be altered by retinoic acid. Third, proximodistal patterning of the blastema is linked to blastemal growth, which in turn is dependent on a signal from the apical epidermal cap and on the non-uniformity of positional identity in the transverse axes. Lastly, the question of the degrees of similarity between the mechanisms of urodele limb regeneration, urodele limb embryogenesis, and the embryogenesis of other tetrapod limbs is discussed.

Regeneration of fin rays in teleosts: a histochemical, radioautographic, and ultrastructural study.

Regeneration followed partial amputation of the tail fin in three representative species of teleosts. A systematic study, using histochemical methods, radioautography and transmission electron microscopy, disclosed the essentials of the natural history of regeneration of the fin soft-rays. At about 24 hours, epidermal cells had completely covered the cut edge. By the second day an apical epidermal cap was established. Beneath this cap a blastema was formed by about three days after amputation. A collagenous lepidotrichial matrix had begun to develop by 4 days after excision. Actinotrichia were first detected in the regenerates at 5 days. During these processes, the basal lamina of the epidermis played an important morphogenetic role. The cells responsible for the regenerate underwent regressive changes after form and function had been restored.

Glycoconjugates in normal wound tissue matrices during the initiation phase of limb regeneration in adult Ambystoma

The present study identifies, localizes, and reports the relative composition of specific glycosaminoglycans within tissue matrices during the initiation phase of limb regeneration. The regenerate tissues were harvested and assayed morphologically, histochemically, and chemically. We observed 1) a population of cells interspersed among the cells of the dermis, epimysium, perimysium, perichondrium, and periosteum. 2) This population was distinguishable by a unique pattern of glycoconjugate staining, i.e., intracellular and pericellular heparan sulfate and glycoproteins and extracellularly associated hyaluronate and glycoproteins. 3) Cells with these staining characteristics aggregated to a position directly beneath the apical epidermal cap. 4) Extracellular hyaluronate and glycoproteins colocalized with undifferentiated tissues. And 5) extracellular chondroitin sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans colocalized with differentiated tissues. The correlations of distinct glycoconjugate compositions with specific regeneration morphologies suggest the possibility that these components may be related to the phenotypic expression of tissues during regeneration.

Histological analysis of limb regeneration in postmetamorphic adult Ambystoma.

Previous investigation into the regenerative ability of postmetamorphic adult land phase Ambystoma has revealed that these species have the capacity to completely regenerate a limb, given optimal environmental conditions, and the gross morphological characteristics of limb regeneration in these species compared favorably with the external regeneration morphology of aquatic phase forms. The present study concerns a histological and histochemical examination of the regenerating limb tissues and their respective extracellular and intracellular tissue matrices. Postmetamorphic adult Ambystoma were amputated through the forearm, placed within optimal environmental conditions, and allowed to regenerate. The tissues were harvested at designated intervals after amputation and prepared for light microscopic examination. The limb tissues were assayed histologically for similarities to and differences from previously established regeneration morphologies. It was noted that specific correlations (i.e., apical epidermal cap formation, but outgrowth and elongation, palette formation, and digit formation) existed between regeneration histologies in these species and those previously reported for the aquatic urodeles, newt, axolotl, and larval salamander. By utilizing the histological and histochemical characteristics of the tissue, the regenerate limb was divided into five tissue units: epidermal, blastemal, soft, hard, and neuro/vascular. Based on the unique morphology of their extracellular matrices and respective histochemical staining patterns, four distinct blastemal regions were delineated within the blastemal units: subregenerate epidermal blastema, soft-tissue blastema, hard-tissue blastema, and core blastema. Histochemically, changing patterns of highly sulfated, weakly sulfated, and carboxylated polysaccharides and glycosylated compounds were located within both the extra- and intracellular stump and regenerate tissue matrices during regeneration. In addition, these patterns of intra- and extracellular macromolecular material correlated to previous reports of similar-type compounds assayed during regeneration in aquatic urodeles. With this in mind, the adult land phase Ambystoma can be considered an appropriate model system for studies concerning normal limb regeneration.