An immunofluorescent study of the distribution of fibronectin and laminin during limb regeneration in the adult newt

Fibronectin and laminin are two extracellular glycoproteins which are involved in various processes of cellular development and differentiation. The present investigation describes changes in their distribution during regeneration of the newt forelimb, as determined by indirect immunofluorescence. The distribution of fibronectin and laminin was similar in normal limb tissue components. These glycoproteins were localized in the pericellular region of the myofibers corresponding to its basement membrane; the perineurium and endoneurium of the nerves; and the basement membranes of blood vessels, skin epithelium, and dermal glands. The cytoplasm of myofibers, axons, skin epithelium, and bone matrix lacked fluorescence for both glycoproteins. After limb amputation in the regenerating blastema, extensive presence of fibronectin, but not laminin, was seen in and around the undifferentiated blastemal cells. Increased fluorescence for fibronectin was also seen during blastema growth, blastemal cell aggregation, and early stages of redifferentiation. As redifferentiation continued, staining for fibronectin slowly disappeared from the cartilage matrix and the myoblast fusion zone. Laminin was first observed around the regenerated myotubes; this was followed by the appearance of fibronectin suggesting a sequential formation of these two components of the new myotube basement membrane. In the regenerated limb, the distribution of laminin and fibronectin was similar to that seen in normal limb. Based on the distribution pattern of these glycoproteins, it is concluded that fibronectin may play an important role in blastemal cell aggregation, cell alignment, and initiation of redifferentiation. After redifferentiation, both laminin and fibronectin may be important in the determination of the architecture of the regenerated limb.     

Changes in the Distribution of Fibronectin During Limb Regeneration in Newts Using Immunocytochemistry

The distribution of fibronectin in regenerating newt limbs was studied using immunocytochemistry. At appropriate intervals after the initial amputation at the elbow (10–30 days), animals were reamputated at the shoulder and processed for light microscopy. The peroxidase-antiperoxidase technique was used to localize affinity-purified antibodies to fibronectin in limb tissues. At the amputation site, fibronectin was associated with basal laminae and connective tissues adjacent to dedifferentiating limb tissues destined to form the regeneration blastema. Accumulation and growth of the blastema was accompanied by the apparent de novo synthesis of fibronectin, where it appeared randomly in the interstitium between blastemal cells. The onset of chondrogenesis was characterized by a central condensation of prechondroblasts that formed the cartilage anlagen. Fibronectin formed an amorphous network between presumptive chondroblasts. As the mature cartilage phenotype was expressed and chondrocytes became isolated in lacunae, fibronectin was greatly reduced and then disappeared. The extracellular matrix surrounding undifferentiated blastemal cells still contained fibronectin. Fibronectin was also found in high concentrations between differentiating myoblasts. A condensation of fibronectin was also observed beneath the epidermis at the distal limb tip at the onset of digit formation. These observations are consistent with the hypothesis that fibronectin may play a key role in the morphogenetic events that result in the spatial organization and subsequent differentiation of cells during pattern formation in the regenerating limb.     

Nvbeta-actin and NvGAPDH as normalization factors for gene expression analysis in limb regenerates and cultured blastema cells of the adult newt, Notophthalmus viridescens

The red-spotted newt has the ability to fully regenerate complex structures by creating a pool of dedifferentiated cells that arise in response to tissue injury. An understanding of the mechanisms involved in the regenerative ability of the newt is limited by a lack of characterized assays. This deficiency includes the cloning and validation of housekeeping genes for normalizing gene expression data. We describe the cloning, characterization and real-time quantitative PCR evaluation of the normalization potential of the newt homologues of cytoplasmic beta-actin and GAPDH during newt limb regeneration and within the blastemal B1H1 cell line. Nvbeta-actin demonstrates a heterogeneous expression during limb regeneration and may be associated with differentiation state. The level of Nvbeta-actin expression in B1H1 cultures under conditions of myogenesis and serum resupplementation varies with the treatment. NvGAPDH is ubiquitously expressed during limb regeneration and within B1H1 cultures and does not demonstrate overall variations in expression levels. Thus, NvGAPDH is a more appropriate normalization factor in gene expression analyses during limb regeneration and treatments of B1H1 cultures.     

The monoclonal antibody 22/18 recognizes a conformational change in an intermediate filament of the newt,Notophthalmus viridescens,during limb regeneration

Summary Previous work has shown that the monoclonal antibody 22/18 identifies progenitor cells (blastemal cells) which depend on the nerve for their division in the early stages of limb regeneration in the newt,Notophthalmus viridescens. This antibody also reacts with cultured cells derived from the newt limb, and the intensity of immunoreactivity appears related to cell density and differentiation into myotubes. We report here that the monoclonal antibody 22/18 recognizes a polypeptide (22/18 antigen) which is intracellular and filamentous. Double staining of cells with 22/18 monoclonal antibody and antibodies against various cytoskeletal components indicates that the epitope is expressed on an intermediate filament component. Although this antibody is specific for blastemal cells in cryostat sections of the regenerating limb, its reactivity on immunoblots is not confined to this tissue. The 22/18 antigen is differentially affected by aldehyde fixatives distinguished by the spacing of their reactive groups. While formaldehyde fixation impairs detection of the antigen, ethylene glycol-bis[succinic acid n-hydroxysuccinimide ester] reveals the antigen in sections of normal and regenerating limbs in a distribution that is consistent with the one obtained from immunoblots. We suggest that the 22/18 monoclonal antibody detects a change in protein conformation, probably related to changes in the physiological state of the cell, that occurs transiently during regeneration and possibly during development.     

Glial growth factor and nerve-dependent proliferation in the regeneration blastema of Urodele amphibians

After amputation of a limb from Urodele amphibians, division of the blastemal cells (the progenitor cells of the regenerate) depends on one or more unidentified growth factors provided by the nerve supply. Here we show that glial growth factor (GGF), a mitogenic protein previously purified from the bovine pituitary, is present in newt nervous system extracts. It is also detectable in extracts of the forelimb regeneration blastema, and its level there decreases after denervation. We have previously shown that blastemal cells dependent on the nerve for division are marked by a monoclonal antibody called 22/18. When denervated blastemas are cultured in the presence of partially purified GGF from newt brain, or pure GGF from the bovine pituitary, the thymidine labeling index of blastemal cells that are 22/18-positive is increased as much as sevenfold. These data indicate that GGF plays a role in nerve-dependent proliferation in the blastema.     

Transient expression of simple epithelial keratins by mesenchymal cells of regenerating newt limb

Structural proteins of the intermediate filament family are an early indicator of differentiation before organogenesis becomes apparent. Keratin intermediate filaments are characteristically expressed only by epithelial and not by mesenchymal cells. Here we show, using monoclonal antibodies, a transient expression of the keratin pair 8 and 18 in a population of mesenchymal cells in the regenerating newt limb, specifically in the undifferentiated progenitor cells (blastemal cells) which give rise to the new tissues. These keratins are also expressed in cultured limb cells that can differentiate into muscle. In contrast no reactivity with anti-keratin 8 and 18 antibodies was observed in the newt limb bud at an early stage of development, indicating a molecular difference between the developing and regenerating limb. The molecular weights of the newt proteins detected by these antibodies are very similar to those of human keratins 8 and 18, further supporting the immunocytochemical evidence that the newt homologs of these keratins are expressed in blastemal cells. This is the first demonstration of keratin expression in mesenchymal progenitor cells in an adult animal.     

Mechanism of Action of Secreted Newt Anterior Gradient Protein

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

Image analysis of blastema cell proliferation in denervated limb regenerates of the newt, Pleurodeles waltlii M.

When denervated at the medium bud stage, limb blastemas of the newt, Pleurodeles waltlii Michah, stop growing. In order to better understand the role of nerves in the cell cycle in blastemas, we studied the distribution of mesenchymal cells in the G0-1, G1, S, G2 and M phases 48 and 96 h after denervation. The cell-cycle phases were determined by examining Feulgen-stained nuclei using a SAMBA 200 (System for Analytical Microscopy in Biological Applications) cell image processor. The cell nuclei were automatically analyzed by calculating 18 parameters related to the densitometry and texture of chromatin, and the shape of each nucleus. Cell-cycle phases were classified according to the unsupervised recognition method using a SAMBA 200 system as proposed by Moustafa and Brugal for cell-kinetics analysis. The classification obtained was tested against the results of stepwise linear discriminant analysis performed according to the method of Giroud. Our results show that, in blastemas 96 h after denervation, the percentage of cells in the S, G2, and M phases decreases significantly, while the percentage of G1 and G0-1 cells increases (+ 51% for G1 cells; + 30% for G0-1 cells). Thus, it appears that denervation of medium-bud-stage limb blastemas promotes the lengthening of G1 and premature exiting of cells from the cycle into the G0-1 phase. These results show that nerves (i.e., neurotrophic factor) regulate cell kinetics during newt limb regeneration by maintaining blastema mesenchymal cells in the cell-cycle.     

A monoclonal antibody detects a difference in the cellular composition of developing and regenerating limbs of newts

We have previously described a monoclonal antibody (called 22/18) that reacts with the early blastemal cells of the regenerating limb of the newt (Notophthalmus viridescens). In embryos of two newt species the antibody reacts with the epidermis, glial cells in the neural tube, the lens and cells in a restricted region of the aorta. In the developing limb bud less than 1% of the mesenchymal cells were reactive with 22/18, although most cells stained brightly with an antibody to another cytoskeletal component. When limbs were amputated prior to the arrival of nerves (axons and Schwann cells) at the amputation plane there was no extra reactivity with 22/18 as compared to the contralateral unamputated control, even though the amputated buds regenerated satisfactorily. Limbs amputated after nerves are present at the plane of amputation respond by forming a 22/18-positive blastema. The appearance of the 22/18 responses is a function of the stage of limb development as shown by amputation of forelimb and hindlimb buds at a larval stage where development of the forelimb is greatly advanced relative to the hindlimb. The distribution of the 22/18-positive cells in larval blastemas showed them to be closely associated with axons as detected by double staining with an antiserum to a neurofilament subunit. The clear antigenic difference between development and regeneration may be related to the relationship between embryonic regulation and epimorphic regeneration, and also to the acquisition of nerve-dependent proliferation of blastemal cells.     

The role of cartilage and fibronectin during respecification of pattern induced in the regenerating amphibian limb by retinoic acid

When retinoic acid (RA) is applied to the regenerating limb the positional information of blastemal cells is respecified and extra limb segments develop. We are trying to elucidate the molecular basis of the action of RA and report here experiments focused on the role that fibronectin (FN) might play in the process. The FN distribution in stump tissues, regeneration blastemas and RA-treated blastemas was investigated by immunocytochemistry. Two effects of RA were observed. Firstly, excessive dedifferentiation of the severed cartilage at the amputation plane, resulting in lumps of FN-positive matrix being released into the blastema; secondly, blastemal cells tend to aggregate together into FN-positive accumulations. Excessive dedifferentiation of the cartilage plays no role in the RA-induced respecification of pattern, because we show that extra segments are still produced in RA-treated limbs from which all the cartilage has been removed. The effect on blastemal cell FN distribution was investigated in several ways. Axolotl plasma FN and cellular FN were characterised on immunoblots, and no obvious change was observed after RA treatment; neither were there changes in amounts of FN detected by ELISA. Levels of FN synthesis were measured by [35S]-methionine labelling and again no change observed after RA treatment. We conclude that the change in FN distribution observed by immunocytochemistry after RA treatment may be due to the retention of FN on the surface of the blastemal cells rather than to any effect on the levels of synthesis of this molecule.     

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.     

Regenerate epithelium and skin glands of the adult newt react to the same monoclonal antibody

A search for specific proteins involved in newt limb regeneration, using monoclonal antibodies against forelimb blastemas, led to the detection of an antigen in the regenerate epithelium. Fluorescent-antibody-labeled cells first appeared just prior to blastema outgrowth. From bud through early digit stages this antibody reacted with nearly all of the regenerate epithelial cells. Other tissues also reacted, including nerve, blood vessels, and gastrointestinal tract. The behavior of the reactive cells in the regenerate epithelium, and their close association with immediately adjacent skin glands, raises several new possibilities for the origin of the regenerate epithelium.     

Transferrin is necessary and sufficient for the neural effect on growth in amphibian limb regeneration blastemas

Cell proliferation during the early phase of growth in regenerating amphibian limbs requires a permissive influence of nerves. Based on analyses of proliferative activity in denervated blastemas, it was proposed that nerves provide factors important for cells to complete the proliferative cycle rather than for mitogenesis itself. One such factor, the iron-transport protein transferrin (Tf), is abundant in regenerating peripheral nerves where it is axonally transported and released at growth cones. Using blastemas in organ culture, which have been widely used in previous investigations of the neural effect on growth, it was shown here that the growth-promoting activity of neural extract was completely removed by immuno-absorption with antiserum against Tf and restored by addition of Tf. Purified Tf or a low molecular weight ferric ionophore were as active as the neural extract in this assay, indicating that the trophic effect of Tf involves its capacity for iron delivery. Both Tf and ferric ionophore also maintained DNA synthesis in denervated blastemas in vivo . A dose-response assay indicated that purified axolotl Tf stimulates growth of cultured blastemal cells at concentrations as low as 100 ng/mL. The Tf mRNA in axolotl nervous tissue was shown by northern analysis to be similar in size to that of liver. These results are discussed together with those from previous in vitro studies of blastemal growth and support the hypothesis that cell division in the blastema depends on axonally released Tf during the early, nerve-dependent phase of limb regeneration.     

Blastema cell proliferation in vitro: effects of limb amputation on the mitogenic activity of spinal cord extracts

Primary cultures of mesenchymal cells of axolotl limb blastemas provide a very sensitive in vitro bioassay for studying nerve dependence of newt regeneration. These cells can be stimulated by crude spinal cord extracts of non-amputated animals in a dose-dependent manner up to 60 micrograms protein/ml of culture medium; at this concentration the mitotic index is increased 4-fold. Spinal cord extracts of axolotls 14 days after forelimb amputation (i.e., late bud stage) are more efficient in stimulating blastema cell proliferation (+50%) than extracts of axolotls 7 days after forelimb amputation (i.e., early bud stage) or of axolotls without amputation. In a similar manner, spinal cord extracts of young axolotls 14 days after forelimb amputation, are more stimulatory than older axolotls 14 d after forelimb amputation which regenerate only a very small blastema during the same time. It appears that spinal cord mitogenic activity is enhanced after limb amputation, probably in correlation with blastema cell requirements for limb regeneration.     

Transferrin and the trophic effect of neural tissue on amphibian limb regeneration blastemas

Nerves promote regeneration of amputated urodele limbs, but the chemical basis of the effect is not known. We have examined the possible involvement of the iron-transport factor transferrin, which is important for cell proliferation and is present in vertebrate nervous tissue. Newt brain extract stimulated incorporation of [3H]thymidine in cultured blastemas from regenerating newt forelimbs, showing a biphasic dose-response similar to that of heterologous transferrin. As shown previously for transferrin, the inhibitory effect of brain extract at high concentrations was relieved by the addition of iron. Activity of brain extract was reduced by treatment with an iron-chelating agent and fully restored by the readdition of iron. Double immunodiffusion of newt tissue extracts and antibodies against newt plasma transferrin demonstrated the presence of transferrin-like factors in brain, spinal cord, and peripheral nerve. These results indicate that activity of transferrin may be part of the trophic effect of brain extract on cultured blastemas.     

Ganglia implantation as a means of supplying neurotrophic stimulation to the newt regeneration blastema: cell-cycle effects in innervated and denervated limbs.

Regulation of blastema cell proliferation during amphibian limb regeneration is poorly understood. One unexplained phenomenon is the relatively low level of active cell cycling in the adult newt blastema compared to that of larval axolotls. In the present study, we used ganglia implantation as a means of "superinnervating" normally innervated adult newt blastemas to test whether blastema cell subpopulations are responsive to nerve augmentation. The effectiveness of implanted ganglia to provide neurotrophic stimulation was demonstrated in denervated blastemas. Blastemas implanted with 2 dorsal root ganglia and simultaneously denervated 14 days after amputation exhibited control levels of cell cycle activity 6 days later, as measured by 3H-thymidine pulse labeling. Denervated blastemas that were sham-operated or implanted with pituitary glands exhibited cell-cycle declines similar to those of denervated blastemas without implanted ganglia. Thus, 2 implanted ganglia provide neurotrophic stimulation equivalent to that of the normal nerve supply. Dorsal root ganglia implanted into normally innervated blastemas, which should effectively double neurotrophic activity to the blastema, had no effect on cell-cycle activity, innervated blastemas implanted with ganglia for 6 days exhibited pulse labeling indices similar to those of normally innervated blastemas. These data indicate that neurotrophic stimulation is not normally limiting in innervated limbs, and that some other factor, whether extrinsic or intrinsic to blastema cells, accounts for the relatively low level of active cell cycling in the adult newt blastema.     

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. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 150–158, 2000