Blastema Stage Research Articles

Axolotl mandible regeneration occurs through mechanical gap closure and a shared regenerative program with the limb.

The mandible plays an essential part in human life and, thus, defects in this structure can dramatically impair the quality of life in patients. Axolotls, unlike humans, are capable of regenerating their lower jaws; however, the underlying mechanisms and their similarities to those in limb regeneration are unknown. In this work, we used morphological, histological and transcriptomic approaches to analyze the regeneration of lateral resection defects in the axolotl mandible. We found that this structure can regenerate all missing tissues in 90 days through gap minimization, blastema formation and, finally, tissue growth, differentiation and integration. Moreover, transcriptomic comparisons of regenerating mandibles and limbs showed that they share molecular phases of regeneration, that these similarities peak during blastema stages and that mandible regeneration occurs at a slower pace. Altogether, our study demonstrates the existence of a shared regenerative program used in two different regenerating body structures with different embryonic origins in the axolotl and contributes to our understanding of the minimum requirements for a successful regeneration in vertebrates, bringing us closer to understand similar lesions in human mandibles.

Transcriptomic landscape of posterior regeneration in the annelid Platynereis dumerilii

BackgroundRestorative regeneration, the capacity to reform a lost body part following amputation or injury, is an important and still poorly understood process in animals. Annelids, or segmented worms, show amazing regenerative capabilities, and as such are a crucial group to investigate. Elucidating the molecular mechanisms that underpin regeneration in this major group remains a key goal. Among annelids, the nereididae Platynereis dumerilii (re)emerged recently as a front-line regeneration model. Following amputation of its posterior part, Platynereis worms can regenerate both differentiated tissues of their terminal part as well as a growth zone that contains putative stem cells. While this regeneration process follows specific and reproducible stages that have been well characterized, the transcriptomic landscape of these stages remains to be uncovered.ResultsWe generated a high-quality de novo Reference transcriptome for the annelid Platynereis dumerilii. We produced and analyzed three RNA-sequencing datasets, encompassing five stages of posterior regeneration, along with blastema stages and non-amputated tissues as controls. We included two of these regeneration RNA-seq datasets, as well as embryonic and tissue-specific datasets from the literature to produce a Reference transcriptome. We used this Reference transcriptome to perform in depth analyzes of RNA-seq data during the course of regeneration to reveal the important dynamics of the gene expression, process with thousands of genes differentially expressed between stages, as well as unique and specific gene expression at each regeneration stage. The study of these genes highlighted the importance of the nervous system at both early and late stages of regeneration, as well as the enrichment of RNA-binding proteins (RBPs) during almost the entire regeneration process.ConclusionsIn this study, we provided a high-quality de novo Reference transcriptome for the annelid Platynereis that is useful for investigating various developmental processes, including regeneration. Our extensive stage-specific transcriptional analysis during the course of posterior regeneration sheds light upon major molecular mechanisms and pathways, and will foster many specific studies in the future.

Downregulation of Yap1 during limb regeneration results in defective bone formation in axolotl

The Hippo pathway plays an imperative role in cellular processes such as differentiation, regeneration, cell migration, organ growth, apoptosis, and cell cycle. Transcription coregulator component of Hippo pathway, YAP1, promotes transcription of genes involved in cell proliferation, migration, differentiation, and suppressing apoptosis. However, its role in epimorphic regeneration has not been fully explored. The axolotl is a well-established model organism for developmental biology and regeneration studies. By exploiting its remarkable regenerative capacity, we investigated the role of Yap1 in the early blastema stage of limb regeneration. Depleting Yap1 using gene-specific morpholinos attenuated the competence of axolotl limb regeneration evident in bone formation defects. To explore the affected downstream pathways from Yap1 down-regulation, the gene expression profile was examined by employing LC-MS/MS technology. Based on the generated data, we provided a new layer of evidence on the putative roles of increased protease inhibition and immune system activities and altered ECM composition in diminished bone formation capacity during axolotl limb regeneration upon Yap1 deficiency. We believe that new insights into the roles of the Hippo pathway in complex structure regeneration were granted in this study.

The regenerating tail of lizard transits through a tumour‐like stage represented by the regenerative blastema

AbstractReview. The regenerating tail of lizard transits through a tumour‐like stage represented by the regenerative blastema. Acta Zoologica (Stockolm). Molecular studies on lizard tail regeneration indicate that the blastema stage is a tumour‐like outgrowth capable of self‐regulate to produce a new tail. Various oncogenes and tumour suppressors are expressed, and their proteins are localized in specific regions of the growing blastema. SnoRNAs are exclusively overexpressed in the tail blastema suggesting changes in ribosome translation efficiency in blastema cells, like in cancer. Blastema cells secrete high levels of hyaluronate and adopt an anaerobic metabolism (Warburg effect). These studies indicate that the lizard blastema represents a unique case among terrestrial vertebrates of physiological tumour remission. Mesenchymal cells and fibroblasts forming the blastema are turned within 1–2 months into a functional organ, the tail. In vitro studies on isolated mesenchymal cells from the regenerative blastema shows that these cells do not undergo contact inhibition but continue proliferation after confluence, and contain nestin, vimentin and K17. After 2–3 weeks they stratify into 5–7 layers forming a pellicle of loose connective tissue. Future molecular studies on genes and proteins that allow the control of growth in the lizard blastema may help to determine how lizards turn a tumour into a new organ with numerous differentiated and functional tissues, providing clues on cancer growth regulation.

Timing Does Matter: Nerve-Mediated HDAC1 Paces the Temporal Expression of Morphogenic Genes During Axolotl Limb Regeneration.

Sophisticated axolotl limb regeneration is a highly orchestrated process that requires highly regulated gene expression and epigenetic modification patterns at precise positions and timings. We previously demonstrated two waves of post-amputation expression of a nerve-mediated repressive epigenetic modulator, histone deacetylase 1 (HDAC1), at the wound healing (3 days post-amputation; 3 dpa) and blastema formation (8 dpa onward) stages in juvenile axolotls. Limb regeneration was profoundly inhibited by local injection of an HDAC inhibitor, MS-275, at the amputation sites. To explore the transcriptional response of post-amputation axolotl limb regeneration in a tissue-specific and time course-dependent manner after MS-275 treatment, we performed transcriptome sequencing of the epidermis and soft tissue (ST) at 0, 3, and 8 dpa with and without MS-275 treatment. Gene Ontology (GO) enrichment analysis of each coregulated gene cluster revealed a complex array of functional pathways in both the epidermis and ST. In particular, HDAC activities were required to inhibit the premature elevation of genes related to tissue development, differentiation, and morphogenesis. Further validation by Q-PCR in independent animals demonstrated that the expression of 5 out of 6 development- and regeneration-relevant genes that should only be elevated at the blastema stage was indeed prematurely upregulated at the wound healing stage when HDAC1 activity was inhibited. WNT pathway-associated genes were also prematurely activated under HDAC1 inhibition. Applying a WNT inhibitor to MS-275-treated amputated limbs partially rescued HDAC1 inhibition, resulting in blastema formation defects. We propose that post-amputation HDAC1 expression is at least partially responsible for pacing the expression timing of morphogenic genes to facilitate proper limb regeneration.

Expression of Piwi Genes during the Regeneration of Lineus sanguineus (Nemertea, Pilidiophora, Heteronemertea).

The transposon silencer piwi genes play important roles in germline determination and maintenance, gametogenesis, and stem-cell self-renewal, and the expression of certain piwi genes is indispensable for regeneration. Knowledge about piwi genes is needed for phylum Nemertea, which contains members (e.g., Lineus sanguineus) with formidable regeneration capacity. By searching the L. sanguineus genome, we identified six Argonaute genes including three ago (Ls-Ago2, Ls-Ago2a, and Ls-Ago2b) and three piwi (Ls-piwi1, Ls-piwi2, and Ls-piwi3) genes. In situ hybridization revealed that, in intact females, Ls-piwi2 and Ls-piwi3 were not expressed, while Ls-piwi1 was expressed in ovaries. During regeneration, Ls-piwi1 and Ls-pcna (proliferating cell nuclear antigen) had strong and similar expressions. The expression of Ls-piwi1 became indetectable while Ls-pcna continued to be expressed when the differentiation of new organs was finished. During anterior regeneration, expression signals of Ls-piwi2 and Ls-piwi3 were weak and only detected in the blastema stage. During posterior regeneration, no expression was observed for Ls-piwi2. To date, no direct evidence has been found for the existence of congenital stem cells in adult L. sanguineus. The “pluripotent cells” in regenerating tissues are likely to be dedifferentiated from other type(s) of cells.

Functional Characterization of the Lin28/let-7 Circuit During Forelimb Regeneration in Ambystoma mexicanum and Its Influence on Metabolic Reprogramming.

The axolotl (Ambystoma mexicanum) is a caudate amphibian, which has an extraordinary ability to restore a wide variety of damaged structures by a process denominated epimorphosis. While the origin and potentiality of progenitor cells that take part during epimorphic regeneration are known to some extent, the metabolic changes experienced and their associated implications, remain unexplored. However, a circuit with a potential role as a modulator of cellular metabolism along regeneration is that formed by Lin28/let-7. In this study, we report two Lin28 paralogs and eight mature let-7 microRNAs encoded in the axolotl genome. Particularly, in the proliferative blastema stage amxLin28B is more abundant in the nuclei of blastemal cells, while the microRNAs amx-let-7c and amx-let-7a are most downregulated. Functional inhibition of Lin28 factors increase the levels of most mature let-7 microRNAs, consistent with an increment of intermediary metabolites of the Krebs cycle, and phenotypic alterations in the outgrowth of the blastema. In summary, we describe the primary components of the Lin28/let-7 circuit and their function during axolotl regeneration, acting upstream of metabolic reprogramming events.

Expression Patterns of Three Regulatory Genes in Caudal Fin Regeneration of the Euryhaline Killifish, Aphanius hormuzensis (Teleostei: Aphaniidae)

Recent studies indicated that regeneration speed in caudal fin and kidney of the genus Aphanius is faster than the other traditional model zebrafish. Therefore, non-traditional model organisms, such as members of the genus Aphanius, can probably be suitable candidate to study regeneration phenomenon. In order to advance our knowledge on the regeneration ability in the genus Aphanius, firstly we investigated the histology organization of caudal fin regeneration process in ten individuals of Aphanius hormuzensis with 3.5–4.0 cm in total length. In addition, transcription of three controlling genes, i.e., the ligand (Eda) and the receptor (Edar) from ectodysplasin signaling pathway and mmp9 (matrix metallopeptidase 9), were examined during the caudal fin regeneration stages in Aphanius hormuzensis. For the later experiment, 55–60 fish specimens were used to obtain 50 mg tissue for each regeneration stages. Thereafter, the expression patterns of these genes were examined by quantitative real-time PCR. Our histological observations showed three distinct stages in caudal fin regeneration including a wound healing in 24 h post-amputation (hpa), the blastema formation in 72 hpa and the outgrowth with fin ray formation in 5 days post-amputation (dpa). The results of quantitative real-time PCR showed that mmp9 has a strong upregulation during the stages of blastema and fin ray formation. The Eda transcript level was significantly increased, and it has upregulation during the fin ray formation stage. Slight down-regulation of Edar transcripts was observed at the blastema stage, but it was upregulated thereafter. It is documented that ectodysplasin signaling has critical role in morphogenesis of ectodermal appendages, and mmp9 involves in the extracellular matrix remodeling during regeneration. As conclusion, Eda signaling is present and involve in different stages of the caudal fin regeneration in A. hormuzensis. Eda gene upregulation might support the hypothesis that the regulation of Eda/Edar signaling was controlled by the ligand (Eda) expression during the caudal fin regeneration of the studied killifish. Moreover, the upregulation of mmp9 in different stages of regeneration is consistent with its role in the removal of damaged cartilage matrix.

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.

Inhibition of Cyclooxygenase-2 Alters Wnt/β-Catenin Signaling in the Regenerating Tail of Lizard Hemidactylus flaviviridis.

Epimorphic regeneration in vertebrates involves the restoration of lost tissue or organs through the formation of a regeneration blastema and occurs through a complex interaction of a number of molecular signaling pathways. Of the many effectors of successful tail regeneration in the lizard Hemidactylus flaviviridis, one crucial pathway is the cyclooxygenase-2 (COX-2) mediated PGE2 signaling pathway. The current study was aimed at understanding whether COX-2 signaling plays any role in the expression of Wnt/β-Catenin signaling components during regenerative outgrowth in H. flaviviridis. Etoricoxib-selective inhibitor of the inducible isoform of COX-2-was administered to lizards orally. We tested the expression of β-Catenin during wound epidermis and blastema stages in the regenerating tail and found a reduction in its expression in response to drug treatment. Further, it was observed that the expression of canonical Wnt ligands was greatly altered due to COX-2 inhibition. Our results provide evidence of a cross-talk between the COX-2 induced PGE2 pathway and Wnt/β-Catenin signaling in the regenerating lizard tail. An understanding of the interaction among various signaling pathways will help elucidate the mechanism underlying epimorphosis in lizards, the only amniotes capable of appendage regeneration.

The influence of hypo‐osmotic stress on the regenerative capacity of the invasive polychaete Marenzelleria viridis from its native range

AbstractThe euryhaline polychaete Marenzelleria viridis is a notorious invader of the Baltic region and while many studies have focused on the effects of osmotic stress on larval development of the species, few have assessed its effect on adult specimens. This study investigated the effects of low salinity on the physiology of M. viridis by using its regenerative capacity as a proxy for physiologic performance. Specimens were collected in spring 2015 on sandy flats in Hempstead Bay (Long Island, New York), a site that falls within its native range. Worms were ablated between the 14th and 20th chaetigers and cultured at five different salinity treatments (24psu, 15psu, 10psu, 5psu and 0psu) that reflected the broad salinity regimes of its distributional range. Data for anterior regeneration were analysed and presented. Morphogenesis during anterior regeneration was characterized by the formation of a blastema from which the major anatomic structures emerged. All specimens cultured at 5–24psu successfully completed regeneration whereas 75% of specimens cultured at 0psu died and the survivors were unable to proceed beyond the blastema stage. Salinity did not have an effect on regeneration time (14.5–15.1 days) but did have an effect on the percentage of chaetigers regenerated (lowest – 77% at 5psu, highest – 97.5% at 15‰). The post‐regeneration phase was characterized by variability in pigmentation patterns in the regenerated anterior structures, which appear to be independent of salinity treatments. In conclusion, adult M. viridis appears to exhibit high tolerance to low salinity environments, which may have been inherited from its larval stages. In addition, the different pigmentation patterns observed in regenerated structures may allude to the variability of this feature in spionids.

Observing the development of the temporomandibular joint in embryonic and post-natal mice using various staining methods.

The temporomandibular joint (TMJ) is a specialized synovial joint that is essential for the movement and function of the mammalian jaw. The TMJ develops from two mesenchymal condensations, and is composed of the glenoid fossa that originates from the otic capsule by intramembranous ossification, the mandibular condyle of the temporal bone and a fibrocartilagenous articular disc derived from a secondary cartilaginous joint by endochondral ossification. However, the development of the TMJ remains unclear. In the present study, the formation and development of the mouse TMJ was investigated between embryonic day 13.5 and post-natal day 180 in order to elucidate the morphological and molecular alterations that occur during this period. TMJ formation appeared to proceed in three stages: Initiation or blastema stage; growth and cavitation stage; and the maturation or completion stage. In order to investigate the activity of certain transcription factors on TMJ formation and development, the expression of extracellular matrix (ECM), sex determining region Y-box 9, runt-related transcription factor 2, Indian hedgehog homolog, Osterix, collagen I, collagen II, aggrecan, total matrix metalloproteinase (MMP), MMP-9 and MMP-13 were detected in the TMJ using in situ and/or immunohistochemistry. The results indicate that the transcription factors, ECM and MMP serve critical functions in the formation and development of the mouse TMJ. In summary, the development of the mouse TMJ was investigated, and the molecular regulation of mouse TMJ formation was partially characterized. The results of the present study may aid the systematic understanding of the physiological processes underlying TMJ formation and development in mice.

Dynamic gene expression profiles during arm regeneration in the brittle star Amphiura filiformis

Echinoderms and in particular brittle stars display a remarkable ability to regenerate lost or damaged tissues. They offer an excellent model in which to study regeneration displaying extensive regenerative ability and close relationship to vertebrates providing the opportunity for comparative studies. Previous studies of gene expression during arm regeneration in brittle stars have focused on single genes commonly associated with the regenerative process. In this study we present the first microarray investigation of gene expression during arm regeneration in the brittle star Amphiura filiformis. We show the large-scale gene expression changes associated with the complex process of regeneration with over 50% of the clones measured showing a significant change at some point during the process when compared to non-regenerating arms. Particular attention is paid to genes associated with Hox gene expression regulation, neuronal development and the bone morphogenic protein BMP-1. Our data give an insight into the molecular control required during the various stages of regeneration from the stem cell rich blastema stage through to the highly differentiated regenerate. This work also forms an important basis for future gene expression investigations in this emerging model of limb regeneration.

Regulation of dermal fibroblast dedifferentiation and redifferentiation during wound healing and limb regeneration in the Axolotl

Adult urodeles (salamanders) are unique in their ability to regenerate complex organs perfectly. The Accessory Limb Model (ALM) in the axolotl allows for the identification of signals from the wound epidermis, nerves and dermal fibroblasts that interact to regenerate a limb. In the present study, we have used the ALM to identity the axolotl (Ambystoma mexicanum) orthologue of Twist (AmTwist), a basic helix-loop-helix transcription factor that is involved in the regeneration of the dermis during limb regeneration. AmTwist is expressed during the blastema stages in regeneration, but is inhibited by signals from the nerve during the early stages when dermal fibroblasts dedifferentiate to form blastema cells. As the dermis regenerates, AmTwist is expressed in association with the synthesis of type I collagen in the proximal region of the blastema. Exogenous bone morphogenetic protein-2 leads to an increase in AmTwist expression, and therefore may function as an endogenous regulator of AmTwist expression and dermis regeneration. The nerve appears to have a dual function in regeneration by coordinately regulating dedifferentiation and redifferentiation of dermal fibroblasts.

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.

Monoclonal Antibody MT2 Identifies the Urodele α1 Chain of Type XII Collagen, a Developmentally Regulated Extracellular Matrix Protein in Regenerating Newt Limbs

We previously described the upregulation of the MT2 antigen during urodele limb regeneration and characterized the MT2 antigen as a 310- to 325-kDa chondroitin-sulfated glycoprotein with a core protein of 285-300 kDa. In this study, we screened a newt blastema cDNA library using monoclonal antibody (mAb) MT2 and obtained a 1-kb cDNA fragment, designated Isolate (IS)-1. Subsequent screening of the same library using IS-1 cDNA as a probe provided IS-2, a 2.8-kb cDNA. IS-2 overlaps IS-1 at its 5′ end, is highly homologous to a portion of the α1 chain of the chicken type XII collagen cDNA (α1[XII]), and spans a third of the chicken α1[XII] cDNA, from the last 62 amino acids of the second A domain of von Willebrand factor to the first two repeats of the fourth fibronectin type III domain. The peptide sequence deduced from cDNA IS-2 demonstrates invariable tryptophan, leucine, threonine, and tyrosine residues that are highly conserved among all the fibronectin type III domains within IS-2 and between corresponding sequences of IS-2 and chicken α1[XII]. A Northern blot showed a 10-kb band that corresponds to the size of the chicken α1[XII] mRNA. A fusion gene was constructed by inserting the IS-2 cDNA downstream from the malE gene of Escherichia coli, which encodes maltose-binding protein (MBP). The isopropyl β-d-thiogalactoside-induced fusion protein had the expected molecular weight and reacted to both mAb MT2 and rabbit anti-MBP serum. We conclude that mAb MT2 identifies the urodele α1[XII].The expression pattern of the type XII collagen gene in newt limb regenerates was examined by in situ hybridization. Type XII collagen transcripts first appeared at 3 days after amputation in cells of the basal layer of the wound epithelium. At Day 10, both the basal wound epithelial cells and the distal mesenchyme cells were highly transcriptionally active. At mid-bud and late-bud blastema stages, wound epithelium expression had decreased, whereas the mesenchyme remained strongly active in transcription and showed a tendency toward distal regionalization. Condensing cartilage showed no signal. Finally, at the late digit stage, hybridization became largely restricted to the perichondrium. The in situ results suggest a developmental role for type XII collagen in regeneration.

Re‐programming of expression of the KGFR and bek variants of fibroblast growth factor receptor 2 during limb regeneration in newts (Notophthalmus viridescens)

We have previously shown, by in situ hybridization, that fibroblast growth factor receptor 2 (FGFR2) is present in the basal layer of wound epithelium during limb regeneration in newts (Notophthalmus viridescens). In contrast, FGFR1 expression is observed throughout the blastema mesenchyme but is distinctly absent from the wound epithelium (Poulin et al. [1993] Development 119:353-361). Sequence analysis revealed that we have isolated both the KGFR and bek variants of FGFR2. These two variants differ only in the second half of the last of their three (or two) Ig-like domains. In this report, we show the expression patterns of FGFR2 variants during limb regeneration by in situ hybridization. During the pre-blastema stages of regeneration, FGFR2 expression was observed in the basal layer of the wound epithelium and in the cells of the periosteum. The wound epithelial hybridization was observed when the KGFR-specific probe was used while the bek-specific probe hybridized to mRNA in the cells of the periosteum. As regeneration progresses to the blastema stages, KGFR expression continued to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. The bek-specific hybridization pattern observed at this stage corresponds specifically to the mesenchymal hybridization. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. Interestingly, there appears to be a dorsoventral gradient of the expression of both KGFR and bek variants of FGFR2, which are opposite each other at the later stages of regeneration. Thus, re-programming of expression of the two FGFR2 variants is required during the initial wound closure of limb regeneration. Remarkably, the expression patterns of KGFR and bek mimic those observed in the mouse limb bud during early embryonic development (Orr-Urtreger et al. [1993] Dev. Biol. 18:475-486). Moreover, our results suggest that the two FGFR2 variants have distinct roles in limb regeneration. Further investigation regarding the potential sources of the FGF ligands will help establish the roles that FGFs and FGFRs play in limb regeneration.

Examination of fibronectin distribution and its sources in the regenerating newt limb by immunocytochemistry and in situ hybridization.

Using monoclonal antibodies (mAbs) reactive to newt limb regenerates, we hope to gain insight into the identity and function of regeneration significant molecules. mAb MT4 (matrix 4) identifies an extracellular matrix (ECM) protein that is strongly up-regulated first in the distal stump and then in the blastema during regeneration. Within the first 24 hr after amputation the MT4 antigen is localized to an acellular space beneath the wound epithelium, and first appears in the basal cells of the wound epithelium between days 5 and 7. At mid-bud blastema stages, the MT4 antigen is homogeneously distributed as thin fibers in the blastema ECM, and is later largely restricted to the distal tip of the blastema and the areas of cartilage condensation. After extraction and immunoblotting, the MT4 antigen was observed as three reduced species of M(r) 225, 250, and 260. Taken together, the immunoblot and immunocytochemistry results suggested that mAb MT4 recognized newt fibronectin (FN). Sequence from a cDNA (NvFN.10) obtained by screening a newt blastema cDNA expression library with mAb MT4 conclusively identified the MT4 antigen as FN. To further investigate the expression of FN in regeneration, cDNA NvFN.10 was used to construct a riboprobe and in situ hybridization was done. In the unamputated limb only a few scattered cells expressed the FN gene. Within the first 3 days after amputation strong hybridization signal was observed in the basal cells of the wound epithelium. Most of the stump cells that dedifferentiated and accumulated beneath the wound epithelium at 7 days expressed the FN gene, while the basal cells of the wound epithelium maintained their expression. At mid- and late-bud blastema stages the vast majority of the blastema cells were strongly expressing the FN gene, but the wound epithelial cells now showed only weak FN transcription. Thus initially FN comes from the plasma. Then FN is synthesized by both the wound epithelium and mesenchyme. Finally, at blastema stages FN is produced primarily by the mesenchyme. The expression pattern of FN throughout regeneration suggests that this glycoprotein has roles in wound epithelial and mesenchymal cell migration and mesenchymal cell proliferation and differentiation.

Developmental Regulation of a Matrix Metalloproteinase during Regeneration of Axolotl Appendages

Removal of specific extracellular matrix (ECM) components has been implicated in the initiation of salamander limb regeneration. Remodeling of the ECM at the distal stump is necessary for the release of cells that eventually contribute to the blastema. Several matrix metalloproteinases (MMPs) have been well characterized as important to various physiological and pathological processes, such as bone remodeling and tumor invasion. The goal of this study is to identify and characterize MMPs that modulate the ECM during appendage regeneration in the axolotl Ambystoma mexicanum. By analyzing axolotl tissue extracts using gelatin-substrate gels, we have identified a 90-kDa gelatinase/collagenase that is upregulated within hours after limb amputation. This gelatinase shows a dramatic elevation in activity during the dedifferentiation and blastema stages. Its activity declines by the palette stage and returns to its basal level by the digit stages. The increase in activity of the 90-kDa gelatinase in response to amputation is independent of the nerve supply and the wound epithelium but these factors affect its subsequent downregulation. In addition to the blastema, the 90-kDa gelatinase can be detected in the stump at least 4 mm proximal to the regenerate. Similar regulation of the 90-kDa gelatinolytic activity is observed during tell regeneration and flank wound healing. We suggest that this 90-kDa gelatinase/collagenase may play a role in the initiation and rapid growth phase of axolotl regeneration and wound healing.

Heterogeneity in the expression of fibroblast growth factor receptors during limb regeneration in newts (Notophthalmus viridescens).

Two closely related fibroblast growth factor receptors, FGFR1 and FGFR2, have been cloned from a newt (Notophthalmus viridescens) limb blastema cDNA library. Sequence analysis revealed that we have isolated both the bek and KGFR variants of FGFR2. These two variants differ only in the second half of the last of their three Ig-like domains. The expression patterns of FGFR1 and FGFR2 during limb regeneration have been determined by in situ hybridization. During the preblastema stages of regeneration, FGFR2 expression is observed in the basal layer of the wound epithelium and in the cells of the periosteum. As regeneration progresses to the blastema stages, FGFR2 expression continues to be observed in the basal layer of the wound epithelium with additional hybridization seen in the blastema mesenchyme closely associated with the bisected bones. From the early bud to the mid-bud blastema stage, FGFR1 expression is observed throughout the blastema mesenchyme but, unlike FGFR2, is distinctly absent from the wound epithelium. In the differentiation stages of regeneration, the mesenchymal expression of FGFR2 becomes restricted to the cells of the condensing cartilage and later to the perichondrium. During these later stages of regeneration, the wound epithelium hybridization to the FGFR2 probe is no longer observed. The expression patterns of these receptors suggest that FGFR1 and FGFR2 have distinct roles in limb regeneration, despite their sharing a number of the FGF ligands. Further investigation regarding the potential sources of the FGF ligands will help establish the role that FGFs and FGFRs play in limb regeneration.