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

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

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     

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.     

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.     

Nerve-independence of limb regeneration in larval Xenopus laevis is correlated to the level of fgf-2 mRNA expression in limb tissues

In both larval and adult urodele amphibians, limb blastema formation requires the presence of an adequate nerve supply. In previous research, we demonstrated that the hindlimb of early Xenopus laevis larvae formed a regeneration blastema even when denervated, while the denervated limb of late larvae did not. We hypothesized that the nerve-independence was due to the autonomous synthesis of a mitogenic neurotrophic-like factor by undifferentiated limb bud cells. In this paper, we demonstrate that fgf-2 mRNA is present in larval limb tissues and that its level is correlated to the extent of mesenchymal cells populating the limb: in early limbs, fgf-2 mRNA is present at high levels all over the limb, while, in late limbs, the fgf-2 expression is low and detectable only in the distal autopodium. After denervation, fgf-2 mRNA synthesis increases in amputated early limbs but not in amputated late limbs. The implantation of anti-FGF-2 beads into amputated early limbs hardly lowers the mitotic activity of blastema cells. However, FGF-2 beads implanted into the blastema of late limbs prevent the denervation-induced inhibition of mitosis and oppose blastema regression. Our data indicate that FGF-2 is a good candidate for the endogenous mitogenic factor responsible for blastema formation and growth in amputated and denervated early limbs. However, in amputated late limbs, the very limited fgf-2 expression is not sufficient to promote blastema formation in the absence of nerves.     

Hedgehog signaling controls dorsoventral patterning, blastema cell proliferation and cartilage induction during axolotl tail regeneration

Tail regeneration in urodeles requires the coordinated growth and patterning of the regenerating tissues types, including the spinal cord, cartilage and muscle. The dorsoventral (DV) orientation of the spinal cord at the amputation plane determines the DV patterning of the regenerating spinal cord as well as the patterning of surrounding tissues such as cartilage. We investigated this phenomenon on a molecular level. Both the mature and regenerating axolotl spinal cord express molecular markers of DV progenitor cell domains found during embryonic neural tube development, including Pax6, Pax7 and Msx1. Furthermore, the expression of Sonic hedgehog (Shh) is localized to the ventral floor plate domain in both mature and regenerating spinal cord. Patched1 receptor expression indicated that hedgehog signaling occurs not only within the spinal cord but is also transmitted to the surrounding blastema. Cyclopamine treatment revealed that hedgehog signaling is not only required for DV patterning of the regenerating spinal cord but also had profound effects on the regeneration of surrounding, mesodermal tissues. Proliferation of tail blastema cells was severely impaired, resulting in an overall cessation of tail regeneration, and blastema cells no longer expressed the early cartilage marker Sox9. Spinal cord removal experiments revealed that hedgehog signaling, while required for blastema growth is not sufficient for tail regeneration in the absence of the spinal cord. By contrast to the cyclopamine effect on tail regeneration, cyclopamine-treated regenerating limbs achieve a normal length and contain cartilage. This study represents the first molecular localization of DV patterning information in mature tissue that controls regeneration. Interestingly, although tail regeneration does not occur through the formation of somites, the Shh-dependent pathways that control embryonic somite patterning and proliferation may be utilized within the blastema, albeit with a different topography to mediate growth and patterning of tail tissues during regeneration.     

Cell interactions and regeneration control

This paper is a review of the main findings of our laboratory on the control of regeneration by cell interactions. These include results related to the role of both cell contact and local soluble factors in regeneration of the legs of insects and newts and of the parapodia and segments of nereis. The pattern of these structures is considered to be defined by positional information distributed as longitudinal and transverse positional value sequences carried by epidermal (insect) or mesenchymal (newt) cells. By associating tissues to create transverse and longitudinal discontinuities in these sequences, single or multiple regenerating structures were obtained. These structures are formed by the intercalation of cells characterized by intermediate positional values which fill the gap between the tissues in contact. Positional information may also be changed during regeneration by the nerve cord in nereis and retinoids in the newts. We describe additional cases where morphogenesis occurs without any overt discontinuity in positional information, such as from a locally injured or non-injured insect trochanter, or after deflection of nerves in nereis and newt. Regeneration following an amputation may be considered as a special case of intercalary regeneration, the first stage being the juxtaposition of normally non-contiguous cells resulting in a longitudinal or/and a transverse gap. We also report studies on local factors produced by nerves and the blastema during newt limb regeneration. The nerve factor is necessary for the division of blastemal cells. After denervation, mesenchyme differentiates in an abnormal way. The mitogenic signal from the nerves is mediated by the PKC pathway. Its production is enhanced by regeneration of cut nerve fibers. The blastema also produces growth factors. We show that the epidermal cap and mesenchyme contain acidic FGF-like factor, and that the proliferating mesenchyme stimulates nerve fibers to regrow into the blastema.     

Astrocyte activation by fibroblast growth factor-1 and motor neuron apoptosis: implications for amyotrophic lateral sclerosis

Fibroblast growth factor-1 (FGF1 or acidic FGF) is highly expressed in motor neurons. FGF-1 is released from cells by oxidative stress, which might occur from SOD-1 aberrant function in amyotrophic lateral sclerosis (ALS). Although FGF-1 is known to be neuroprotective after spinal cord injury or axotomy, we found that FGF-1 could activate spinal cord astrocytes in a manner that decreased motor neuron survival in co-cultures. FGF-1 induced accumulation of the FGF receptor 1 (FGFR1) in astrocyte nuclei and potently stimulated nerve growth factor (NGF) expression and secretion. The FGFR1 tyrosine kinase inhibitor PD166866 prevented these effects. Previously, we have shown that NGF secretion by reactive astrocytes induces motor neuron apoptosis through a p75(NTR)-dependent mechanism. Embryonic motor neurons co-cultured on the top of astrocytes exhibiting activated FGFR1 underwent apoptosis, which was prevented by PD166866 or by adding either anti-NGF or anti-p75(NTR) neutralizing antibodies. In the degenerating spinal cord of mice carrying the ALS mutation G93A of Cu, Zn superoxide dismutase, FGF-1 was no longer localized only in the cytosol of motor neurons, while FGFR1 accumulated in the nuclei of reactive astrocytes. These results suggest that FGF-1 released by oxidative stress from motor neurons might have a role in activating astrocytes, which could in turn initiate motor neuron apoptosis in ALS through a p75(NTR)-dependent mechanism.     

Matrix metalloproteinases (MMPs) are required for wound closure and healing during larval leg regeneration in the flour beetle,Tribolium castaneum

Regenerative abilities are found ubiquitously among many metazoan taxa. To compare mechanisms underlying the initial stages of limb regeneration between insects and vertebrates, the roles of matrix metalloproteinases (MMPs) and fibroblast growth factor (FGF) signaling were investigated in the red flour beetle, Tribolium castaneum. RNA interference-mediated knockdown of MMP2 expression delayed wound healing and subsequent leg regeneration. Additionally, pairwise knockdown of MMP1/2 and MMP2/3, but not MMP1/3, resulted in inhibition of wound closure. Wound healing on the dorsal epidermis after injury was also delayed when MMPs were silenced. Our findings show that functionally redundant MMPs play key roles during limb regeneration and wound healing in Tribolium. This MMP-mediated wound healing is necessary for the subsequent formation of a blastema. In contrast, silencing of FGF receptor did not interfere with the initial stages of leg regeneration despite the alterations in tanning of the cuticle. Thus, insects and vertebrates appear to employ similar developmental processes for the initial stages of wound closure during limb regeneration, while the role of FGF in limb regeneration appears to be unique to vertebrates.     

Partial purification of a low-molecular-weight growth factor from chicken brain

The regenerating amphibian limb serves as a useful model for studying factors influencing cell proliferation and differentiation. In particular, peripheral nerves are thought to provide a stimulus for growth of the blastema, presumably via the elaboration of an as yet unidentified neurotrophic factor. In the present study, pressure ultrafiltration coupled with chromatofocusing have proven to be effective methods of partially purifying a neurotrophic factor from adult chicken brains. This chick brain growth factor (CBGF) appears to be a heat-stable, basic peptide of low molecular weight (less than 6,000). It is a potent mitogen in vitro, at nanomolar concentrations, for both blastema cells and Swiss mouse 3T3 fibroblasts. CBGF is apparently distinct from other peptide mitogens and/or neuromodulators that have been reported to stimulate blastema growth in vivo and in vitro. These include substance P, FGF from bovine brain and pituitary, EGF, transferrin (sciatin), and spinal cord growth factor (SCGF). The possible relationship of CBGF to other neural regulatory molecules is discussed.     

Bone marrow-derived fibroblast growth factor-2 induces glial cell proliferation in the regenerating peripheral nervous system

ABSTRACT: BACKGROUND: Among the essential biological roles of bone marrow-derived cells, secretion of many soluble factors is included and these small molecules can act upon specific receptors present in many tissues including the nervous system. Some of the released molecules can induce proliferation of Schwann cells (SC), satellite cells and lumbar spinal cord astrocytes during early steps of regeneration in a rat model of sciatic nerve transection. These are the major glial cell types that support neuronal survival and axonal growth following peripheral nerve injury. Fibroblast growth factor-2 (FGF-2) is the main mitogenic factor for SCs and is released in large amounts by bone marrow-derived cells, as well as by growing axons and endoneurial fibroblasts during development and regeneration of the peripheral nervous system (PNS). RESULTS: Here we show that bone marrow-derived cell treatment induce an increase in the expression of FGF-2 in the sciatic nerve, dorsal root ganglia and the dorsolateral (DL) region of the lumbar spinal cord (LSC) in a model of sciatic nerve transection and connection into a hollow tube. SCs in culture in the presence of bone marrow derived conditioned media (CM) resulted in increased proliferation and migration. This effect was reduced when FGF-2 was neutralized by pretreating BMMC or CM with a specific antibody. The increased expression of FGF-2 was validated by RT-PCR and immunocytochemistry in co-cultures of bone marrow derived cells with sciatic nerve explants and regenerating nerve tissue respectivelly. CONCLUSION: We conclude that FGF-2 secreted by BMMC strongly increases early glial proliferation, which can potentially improve PNS regeneration.     

Effect of transferrin on amphibian limb regeneration: a blastema cell culture study

Summary In order to study mitogenic control during axolotl limb regeneration, we have developed a primary blastema cell culture as a very sensitive bioassay for blastema mitogens. Transferrin, an iron-binding glycoprotein which has been shown to be the neurotrophic factor for muscle cells, is the mitogen which has been analysed in the present report. Addition of approximately 2 g human transferrin/ ml of serum-free culture medium enhances blastema cell proliferation 11-fold over control levels and 2-fold over that produced by the addition of nerve extracts or purified growth factors extracted from nerve tissues (basic and acidic fetal growth factor, FGF). At a higher concentration (20 g/ml), transferrin alone has no mitogenic effect unless the medium is also supplemented with FeCl₃ (100 M). The results are discussed with regard to the sensitivity of the blastema cell culture bioassay and in the context of the neurotrophic theory of urodele limb regeneration.     

Fibroblast growth factor-1 induces heme oxygenase-1 via nuclear factor erythroid 2-related factor 2 (Nrf2) in spinal cord astrocytes: consequences for motor neuron survival

Fibroblast growth factor-1 (FGF-1) is highly expressed in motor neurons and can be released in response to sublethal cell injury. Because FGF-1 potently activates astroglia and exerts a direct neuroprotection after spinal cord injury or axotomy, we examined whether it regulated the expression of inducible and cytoprotective heme oxygenase-1 (HO-1) enzyme in astrocytes. FGF-1 induced the expression of HO-1 in cultured rat spinal cord astrocytes, which was dependent on FGF receptor activation and prevented by cycloheximide. FGF-1 also induced Nrf2 mRNA and protein levels and prompted its nuclear translocation. HO-1 induction was abolished by transfection of astrocytes with a dominant-negative mutant Nrf2, indicating that FGF-1 regulates HO-1 expression through Nrf2. FGF-1 also modified the expression of other antioxidant genes regulated by Nrf2. Both Nrf2 and HO-1 levels were increased and co-localized with reactive astrocytes in the degenerating lumbar spinal cord of rats expressing the amyotrophic lateral sclerosis-linked SOD1 G93A mutation. Overexpression of Nrf2 in astrocytes increased survival of co-cultured embryonic motor neurons and prevented motor neuron apoptosis mediated by nerve growth factor through p75 neurotrophin receptor. Taken together, these results emphasize the key role of astrocytes in determining motor neuron fate in amyotrophic lateral sclerosis.     

Msx1‐2 immunolocalization in the regenerating tail of a lizard but not in the scarring limb suggests its involvement in the process of regeneration

The immunolocalization of the muscle segmental homoeobox protein Msx1‐2 of 27–34 kDa in the regenerating tail blastema of a lizard shows prevalent localization in the apical ependyma of the regenerating spinal cord and less intense labelling in the wound epidermis, in the apical epidermal peg (AEP), and in the regenerating segmental muscles. The AEP is a micro‐region of the regenerating epidermis located at the tail tip of the blastema, likely corresponding to the AEC of the amphibian blastema. No immunolabelling is present in the wound epidermis and scarring blastema of the limb at 18–21 days of regeneration, except for sparse repairing muscles. The presence of a proximal–distal gradient of Msx1‐2 protein, generated from the apical ependyma, is suggested by the intensity of immunolabelling. The AEP and the ependyma are believed to induce and maintain tail regeneration, and this study suggests that Msx1‐2 proteins are components of the signalling system that maintains active growth of the tail blastema. The lack of activation and production of Msx1‐2 protein in the limb are likely due to the intense inflammatory reaction following amputation. This study confirms that, like during regeneration in fishes and amphibians, also the blastema of lizards utilizes common signalling pathways for maintaining regeneration.     

A stepwise model system for limb regeneration

The amphibian limb is a model that has provided numerous insights into the principles and mechanisms of tissue and organ regeneration. While later stages of limb regeneration share mechanisms of growth control and patterning with limb development, the formation of a regeneration blastema is controlled by early events that are unique to regeneration. In this study, we present a stepwise experimental system based on induction of limb regeneration from skin wounds that will allow the identification and functional analysis of the molecules controlling this early, critical stage of regeneration. If a nerve is deviated to a skin wound on the side of a limb, an ectopic blastema is induced. If a piece of skin is grafted from the contralateral side of the limb to the wound site concomitantly with nerve deviation, the ectopic blastema continues to grow and forms an ectopic limb. Our analysis of dermal cell migration, contribution, and proliferation indicates that ectopic blastemas are equivalent to blastemas that form in response to limb amputation. Signals from nerves are required to induce formation of both ectopic and normal blastemas, and the diversity of positional information provided by blastema cells derived from opposite sides of the limb induces outgrowth and pattern formation. Hence, this novel and convenient stepwise model allows for the discovery of necessary and sufficient signals and conditions that control blastema formation, growth, and pattern formation during limb regeneration.     

Nerve-dependent and -independent events in blastema formation during Xenopus froglet limb regeneration

Blastema formation, the initial stage of epimorphic limb regeneration in amphibians, is an essential process to produce regenerates. In our study on nerve dependency of blastema formation, we used forelimb of Xenopus laevis froglets as a system and applied some histological and molecular approaches in order to determine early events during blastema formation. We also investigated the lateral wound healing in comparison to blastema formation in limb regeneration. Our study confirmed at the molecular level that there are nerve-dependent and -independent events during blastema formation after limb amputation, Tbx5 and Prx1, reliable markers of initiation of limb regeneration, that start to be expressed independently of nerve supply, although their expressions cannot be maintained without nerve supply. We also found that cell proliferation activity, cell survival and expression of Fgf8, Fgf10 and Msx1 in the blastema were affected by denervation, suggesting that these events specific for blastema outgrowth are controlled by the nerve supply. Wound healing, which is thought to be categorized into tissue regeneration, shares some nerve-independent events with epimorphic limb regeneration, although the healing process results in simple restoration of wounded tissue. Overall, our results demonstrate that dedifferentiated blastemal cells formed at the initial phase of limb regeneration must enter the nerve-dependent epimorphic phase for further processes, including blastema outgrowth, and that failure of entry results in a simple redifferentiation as tissue regeneration.     

Stimulation in cell culture of mesenchymal cells of newt limb blastemas by EDGF I or II (basic or acidic FGF)

After amputation of a newt limb, a blastema forms on the amputation plane and later differentiates to regenerate all the missing parts of the limb. Proliferation of blastema cells is under the control of severed nerves which deliver a 'neurotrophic factor' (NTF) of unknown nature. In order to characterize this factor we use a primary culture of blastema mesenchymal cells; changes in mitotic index after 48-h colchicine treatment indicate mitogenic activity of potential growth substances. These cells, which are stimulated by nerve extracts (mitotic index X 6), were tested with two purified growth factors extracted from bovine retina or brain (EDGF I = basic FGF and EDGF II = acidic FGF). We show that these two growth factors stimulate proliferation of blastema cell cultures in a dose-dependent manner. Maximal stimulation was obtained at 3 pM for EDGF I (mitotic index X 5.7) or 300 pM for EDGF II (mitotic index X 4.9). So it appears that these two growth factors have a mitogenic activity on blastema mesenchymal cells similar to that obtained with nerve extracts. The fact that two different growth factors can stimulate these cells raises the question of whether both are present in NTF and/or whether there are receptors to both EDGF I and EDGF II on mesenchymal cell membranes.