Roy A. Tassava

Denervation effects on DNA replication and mitosis during the initiation of limb regeneration in adult newts

Abstract Histological and cellular events during the first 2 weeks after amputation of adult newt forelimbs were compared under conditions of innervation and denervation in order to ascertain the first nerve-dependent step in the regeneration process. Formation of a wound epithelium, histolysis, and dedifferentiation occurred, despite the absence of nerves, in a manner similar to that of the regenerating limbs. Autoradiography showed that cells in the denervated limb-stumps incorporate [3H]thymidine into DNA beginning on day 4 and that during the first week after amputation these limb stumps resembled the contralateral controls in overall labeling indices, in the distribution of the labeled nuclei, and intensity of labeling. Microspectrophotometric determinations of nuclear DNA contents in the dedifferentiated cells of denervated and innervated limbs revealed that in both cases replication of the entire chromosomal complement occurred. Cell division, which first began 6 days after amputation, and early blastema formation were observed only in the innervated limb stumps. A likely explanation of these results, consistent with the preponderance of the literature on regeneration, is that limb stumps can initiate the regeneration process in the absence of nerves but that the cells are blocked in the G2 phase of the first cell cycle and consequently do not proliferate to form a blastema.

Expression of fibroblast growth factors 4, 8, and 10 in limbs, flanks, and blastemas of Ambystoma

Members of the fibroblast growth factor (FGF) family of molecules are critical to limb outgrowth. Here, we examine the expression of Fgfs in three types of limbs—embryonic (developing), mature (differentiated), and regenerating—as well as in the surrounding non–limb tissues in the Mexican axolotl, Ambystoma mexicanum. We have previously cloned partial cDNAs of Fgf4, 8, and 10 from the axolotl (Christensen et al., 2001 ); the complete Fgf10 cDNA sequence is presented here. Axolotl Fgf10 showed deduced amino acid sequence identity with all other vertebrate Fgf10 coding sequences of >62%, and also included conserved 5′ and 3′ untranslated regions in nucleotide sequence comparisons. Semiquantitative reverse transcriptase‐polymerase chain reaction showed that fibroblast growth factors are differentially expressed in axolotl limbs. Only Fgf8 and 10 were highly expressed during axolotl limb development, although Fgf4, 8, and 10 are all highly expressed during limb development of other vertebrates. Fgf4 expression, however, was highly expressed in the differentiated salamander limb, whereas expression levels of Fgf8 and 10 decreased. Expression levels of Fgf8 and 10 then increased during limb regeneration, whereas Fgf4 expression was completely absent. In addition, axolotl limb regeneration contrasted to limb development of other vertebrates in that Fgf8 did not seem to be as highly expressed in the distal epithelium; rather, its highest expression was found in the blastema mesenchyme. Finally, we investigated the expression of these Fgfs in non–limb tissues. The Fgfs were clearly expressed in developing flank tissue and then severely downregulated in mature flank tissue. Differential Fgf expression levels in the limb and shoulder (limb field) versus in the flank (non–limb field) suggest that FGFs may be instrumental during limb field specification as well as instrumental in maintaining the salamander limb in a state of preparation for regeneration.

Apical Epithelial Cap Morphology and Fibronectin Gene Expression in Regenerating Axolotl Limbs

Urodele amphibians (salamanders) are unique among adult vertebrates in their ability to regenerate limbs. The regenerated structure is often indistinguishable from the developmentally produced original. Thus, the two processes by which the limb is produced — development and regeneration — are likely to use many conserved biochemical and developmental pathways. Some of these limb features are also likely to be conserved across vertebrate families. The apical ectodermal ridge (AER) of the developing amniote limb and the larger apical epithelial cap (AEC) of the regenerating urodele limb are both found at the limbs distal‐most tip and have been suggested to be functionally similar even though their morphology is quite different. Both structures are necessary for limb outgrowth. However, the AEC is uniformly smooth and thickly covers the entire limb‐tip, unlike the AER, which is a protruding ridge covering only the dorsoventral boundary. Previous data from our laboratory suggest the multilayered AEC may be subdivided into separate functional compartments. We used hematoxylin and eosin (H+E) staining as well as in situ hybridization to examine the basal layer of the AEC, the layer that lies immediately over the distal limb mesenchyme. In late‐stage regenerates, this basal layer expresses fibronectin (FN) message very strongly in a stripe of cells along the dorsoventral boundary. H+E staining also reveals the unique shape of basal cells in this area. The stripe of cells in the basal AEC also contains the notch/groove structure previously seen in avian and reptilian AERs. In addition, AEC expression of FN message in the cells around the groove correlates with previous amniote AER localization of FN protein inside the groove. The structural and biochemical analyses presented here suggest that there is a specialized ridge‐like compartment in the basal AEC in late‐stage regenerates. The data also suggest that this compartment may be homologous to the AER of the developing amniote limb. Thus, the external differences between amniote limb development and urodele limb regeneration may be outweighed by internal similarities, which enable both processes to produce morphologically complete limbs. In addition, we propose that this basal layer of the AEC is uniquely responsible for AEC functions in regeneration, such as secreting molecules to promote mesenchymal cell cycling and dictating the direction of limb outgrowth. Finally, we include here a clarification of existing nomenclature to facilitate further discussion of the AEC and its basal layer. Dev Dyn;217:216–224.

Characterization of a newt tenascin cDNA and localization of tenascin mRNA during newt limb regeneration by in situ hybridization

We previously showed that tenascin, a large, extracellular matrix glycoprotein, exhibits a temporally and spatially restricted distribution during urodele limb regeneration. To further investigate the role of tenascin in regeneration, we cloned a newt tenascin cDNA, NvTN.1, that has 70% homology to the chicken tenascin sequence. A deduced amino acid sequence of NvTN.1 showed a modular structure unique to tenascin characterized by epidermal growth factor-like and fibronectin type III repeats. To determine the cellular origin of tenascin protein during limb regeneration, we localized tenascin transcripts by in situ hybridization using a riboprobe synthesized from NvTN.1. Transcripts could not be detected in normal limb tissues but first became detectable in the wound epithelium at 2 days and in the distal mesoderm at 5 days after amputation. These wound epithelial cells are probably the source of tenascin protein found within and immediately underneath the wound epithelium. At preblastema stages, hybridization was seen in cells associated with most of the distal mesodermal tissues but not in dermis. At blastema stages, essentially every mesenchymal cell contained tenascin transcripts. Thus, regardless of origin, blastemal mesenchymal cells may share a common regulatory mechanism that results in tenascin gene transcription. Finally, during redifferentiation stages of regeneration, tenascin gene transcription was associated with both differentiation and growth. The results show that initiation of tenascin gene expression is an early event in regeneration and continued tenascin gene transcription is associated with some of the important processes of regeneration, namely wound epithelial-mesenchymal interactions, dedifferentiation, initiation of cell cycling, blastema outgrowth, and cellular differentiation.

Cloning and neuronal expression of a type III newt neuregulin and rescue of denervated, nerve‐dependent newt limb blastemas by rhGGF2

Urodele amphibians are the only vertebrates that can regenerate their limbs throughout their life. The critical feature of limb regeneration is the formation of a blastema, a process that requires an intact nerve supply. Nerves appear to provide an unidentified factor, known as the neurotrophic factor (NTF), which stimulates cycling of blastema cells. One candidate NTF is glial growth factor (GGF), a member of the neuregulin (NRG) growth factor family. NRGs are both survival factors and mitogens to glial cells, including Schwann cells. All forms of NRGs contain an EGF-like domain that is sufficient to activate NRG receptors erbB2, erbB3, and erbB4. To investigate the involvement of neuregulin in newt limb regeneration, we cloned and characterized one neuregulin isoform, a neuregulin with a cysteine-rich domain (CRD-NRG), from newt (Notophthalmus viridescens) spinal cord. Results of in situ hybridization showed that the newt CRD-NRG is highly expressed in dorsal root ganglia and spinal cord neurons that innervate the limbs. We also demonstrated the biological activity of recombinant human GGF2 (rhGGF2) in urodele limb regeneration. When rhGGF2 was injected into denervated, nerve-dependent axolotl blastemas, the labeling index (LI) of blastema cells was maintained at a level near to that of control, innervated blastemas, whereas without rhGGF2 the LI decreased significantly. In another experiment, rhGGF2 was delivered into denervated, nerve-dependent blastemas either by direct infusion into blastemas or by injection into the intraperitoneal cavity. The denervated blastemas were rescued into a regeneration response.

Extracellular matrix protein turnover during salamander limb regeneration

After amputation of a salamander limb, the extracellular matrix undergoes remodeling. The extracellular matrix that maintains the differentiated state of limb tissues is broken down and replaced by an extracellular matrix essential for dedifferentiation and blastema formation. We used monoclonal antibodies in immunohistochemistry methods and riboprobes in in situ hybridization to evaluate the upregulation of tenascin, type XII collagen, fibronectin, and the MT5 antigen. The Stump 1 antigen, an extracellular matrix protein that is abundant in the normal limb, is downregulated during regeneration and reappears late in regeneration as differentiation occurs. In the embryo, the Stump 1 antigen is also absent from the early limb bud and first appears during differentiation stages. Tenascin and fibronectin are also upregulated in the limb bud of the embryo, and these two extracellular matrix proteins appear to function during limb regeneration in adults and limb development in embryos. However, type XII collagen and the MT5 antigen are not found in the limb bud, indicating that type XII collagen and the MT5 antigen have roles in the regenerating limb but not in the embryo limb bud.

Injury requirement for initiation of regeneration of newt limbs which have whole skin grafts.

THERE are three known requirements for salamander limb regeneration—injury, nerves, and wound epidermis1,2. During the past few years we have examined cellular events in amputated, regenerating limbs and compared these with non-regenerating limbs after either denervation3,4 or after whole skin grafts over the amputation surface5. Results of these experiments led to the formulation of an hypothesis6 which suggests unique roles for injury, nerves, and the wound epidermis during the initiation of regeneration : injury is important to cause dedifferentiation of limb stump tissue cells, entry of these cells into G1 of the cell cycle and DNA replication; nerves are important for one or more G2 events and thus indirectly for mitosis; and the wound epidermis maintains these cells in the cell cycle so that significant outgrowth (blastema formation) can occur. In the present study we have examined the extent and kinds of injury necessary to initiate regeneration in newt limbs which have had skin grafts for 5 weeks and externally show no regeneration. The results suggest that non-cycling dedifferentiated cells are present in 5-week skin graft limbs which immediately begin cycling when a wound epidermis forms after re-injury.

A developmentally regulated wound epithelial antigen of the newt limb regenerate is also present in a variety of secretory/transport cell types☆

The role of the wound epithelium in amphibian limb regeneration is not understood. We showed previously that monoclonal antibody (mAb) WE3 stains the wound epithelium but not skin epidermis, suggesting that the WE3 antigen may be a marker for, or be important in, the function of the wound epithelium. In the present study, we conducted an extensive immunohistochemical survey of adult newt tissues to define the distribution of the WE3 antigen. The results show that the antigen is most commonly found in tissues specialized in macromolecular secretion and/or ion transport. Since the enzyme, carbonic anhydrase, serves as a useful marker for a variety of specialized transporting cell types, we examined whether this enzyme was present in WE3-reactive cells. Of the tissues examined, a striking degree of colocalization of carbonic anhydrase and the WE3 antigen was observed, further strengthening the view that the WE3 antigen is an important constituent of specialized transporting cells. A preliminary biochemical characterization suggests that the antigen is probably a glycoprotein, which elutes during gel filtration as a species of over 660 kDa. Possible implications for the function of the wound epithelium are discussed.

Effects of peripheral nerve implants on the regeneration of partially and fully innervated urodele forelimbs

This study addresses the cellular mechanism of the nerve requirement for regeneration of the urodele forelimb. Others have suggested that only the Schwann cell lineage of the blastema requires nerves for regeneration and that upon limb denervation, Schwann cells arrest in the cell cycle and produce a factor that inhibits the cycling of the remaining blastema cells. Our objective was to test this Schwann cell inhibitor model. First, pieces of peripheral nerve were implanted into partially denervated (one third of the nerve supply cut) axolotl forelimbs in an attempt to provide sufficient additional Schwann cells to increase the threshold nerve requirement above that provided by the remaining nerves. These limbs showed delayed regeneration in 68% of the cases and mild deformities, as seen by Victoria Blue staining, in 10% of the cases, as compared with control, partially denervated contralateral limbs that received grafts of muscle or frozen/thawed nerve. Second, when pieces of peripheral nerve were implanted into fully innervated newt limbs, blastema formation was limited, and regeneration was delayed in 80% of experimental cases when compared with control, contralateral newt limbs with muscle or frozen/thawed nerve implants. The results support the inhibition model and further link the need for nerves in regeneration to a possible specific requirement by Schwann cells.

Rescue of blocked cells by reinnervation in denervated forelimb stumps of larval Ambystoma

Cells of amputated, denervated larval Ambystoma forelimbs dedifferentiate and enter the cell cycle but do not subsequently proliferate sufficiently to form a blastema. The denervated limb stump resorbs slowly until reinnervation stimulates regeneration. We used this system to investigate the fate of cells in denervated limbs which undergo early but limited cycling in response to amputation. In Experiment 1, cells were labeled with [3H]thymidine (3H-T) on Day 4 postamputation (PA)/Day 3 postdenervation (PD). Labeled cells were still present on Day 7 PA, but were less frequently observed on Day 13 PA when the limbs were reinnervated and beginning to regenerate. In Experiment 2 we denervated 1 day preamputation to obtain earlier reinnervation and prevent loss of Day 4 PA labeled cells. Cells labeled with 3H-T on Day 4 PA/Day 5 PD were present throughout the denervation period and most were still present on Day 13 PA. Little or no mitotic activity was found among the labeled cells after the initial round of cycling. The apparent cell cycle block was released upon reinnervation on Days 12 and 13 PA when cycling resumed. Labeled mitotic figures were present on Day 13 PA, and the mitotic index of the labeled population increased as a result of reinnervation. These results demonstrate that blocked cells are rescued by nerves, re-enter the cell cycle, and thus contribute to the reinnervation blastema.