Expression of the third component of complement, C3, in regenerating limb blastema cells of urodeles

In this study we have shown that complement component C3 is expressed in the regenerating tissue during urodele limb regeneration. C3 was expressed in the dedifferentiated regeneration blastema and in the redifferentiated limb tissues in the axolotl, Amblystoma mexicanum, and in Notophthalmus viridescens. This expression was verified by immunofluorescent staining using an Ab against axolotl C3 and by in situ hybridization with an axolotl C3 cDNA probe. In the early stages of regeneration C3 appeared to be equally present in all mesenchymal cells and in the wound epithelium, whereas in the later stages it was mainly expressed in the differentiating muscle cells. Since no expression was seen in the developing limb, it appears that the C3 expression was specific to the regeneration process. We then demonstrated by hybridization experiments that a blastema cell line of myogenic origin expresses C3. All these findings implicate C3 in the dedifferentiation process and may indicate a new role for this molecule in muscle differentiation.     

Expression of Msx genes in regenerating and developing limbs of axolotl

Msx genes, homeobox-containing genes, have been isolated as homologues of the Drosophila msh gene and are thought to play important roles in the development of chick or mouse limb buds. We isolated two Msx genes, Msx1 and Msx2, from regenerating blastemas of axolotl limbs and examined their expression patterns using Northern blot and whole mount in situ hybridization during regeneration and development. Northern blot analysis revealed that the expression level of both Msx genes increased during limb regeneration. The Msx2 expression level increased in the blastema at the early bud stage, and Msx1 expression level increased at the late bud stage. Whole mount in situ hybridization revealed that Msx2 was expressed in the distal mesenchyme and Msx1 in the entire mesenchyme of the blastema at the late bud stage. In the developing limb bud, Msx1 was expressed in the entire mesenchyme, while Msx2 was expressed in the distal and peripheral mesenchyme. The expression patterns of Msx genes in the blastemas and limb buds of the axolotl were different from those reported for chick or mouse limb buds. These expression patterns of axolotl Msx genes are discussed in relation to the blastema or limb bud morphology and their possible roles in limb patterning.     

Isoform-specific immunological detection of newt retinoic acid receptor delta 1 in normal and regenerating limbs

Retinoic acid (RA) exerts a variety of effects on the regenerating urodele limb including positional respecification of the blastema. The major RA receptor expressed in the newt limb and blastema is the delta 1 isoform and, in order to detect delta 1 in this context, we have made five affinity-purified antibodies against fusion proteins and peptides from non-overlapping regions of the molecule. These antibodies have been evaluated by reaction with transfected COS-7 cells, newt limb cells in culture and newt limb tissue sections. The most informative antibodies were RP6, directed against N-terminal region A sequence, and RP8, directed against C-terminal sequence. In western blots of blastemal extracts, delta 1 protein was detected as two major bands of immunoreactivity at positions consistent with the employment of two candidate methionine initiators identified by cDNA sequencing. Staining of adult limb sections with RP6 and RP8 showed reactivity in half of the nuclei in epidermal and mesenchymal tissues, a heterogeneity that was observed with adjacent nuclei in muscle fibres. In the regenerating limb, nuclei in the blastemal mesenchyme and wound epidermis were strongly reactive, although no axial variation in expression was detected.     

Expression of regeneration-associated cytoskeletal proteins reveals differences and similarities between regenerating organs

The unique events which allow regeneration of an entire organ to occur are formation of a specialized wound epidermis and accumulation of progenitor cells (blastemal cells) at the amputated surface to form a blastema. In order to identify some of the molecular events underlying the early stages of the regenerative process which are either common to different systems or specific to one of them, we have investigated whether molecules which are induced in limb blastemas are also expressed in skin repair and during regeneration of other complex body structures (lower jaws, upper jaws, and tails). In addition, we have addressed the issue of the identity of progenitor cells during jaw development and regeneration by analyzing the expression of limb blastemal markers in the developing head and face. We have focused on cytoskeletal components, and particularly on the epidermal keratin NvKII, the simple epithelial keratins 8 and 18 and 22/18, because they are among the few molecules which have been shown to be associated with regeneration in the limb and may play significant roles in various developmental processes. Some important findings emerge from this study: 1) Expression of the epidermal keratin NvKII, unlike that of its mammalian homologue K6, is not simply induced in response to wounding, but is associated with regeneration of specific organs. In fact, NvKII is expressed in regenerating limbs and tails, but not in upper or in lower jaw regenerates, demonstrating the existence of molecular differences in the composition of the wound epidermis in these systems. This, together with the fact that NvKII mRNA is regulated by retinoic acid, which differentially affects patterning of limbs and jaws, argues for distinct inductive abilities of the wound epidermis in different organs. 2) In contrast to the differential expression of the epidermal keratin NvKII, the regeneration-associated cytoskeletal molecules identified in limb blastemal cells are expressed in a similar fashion in jaw and tail blastemas. Therefore, it appears that similar cellular events lead to the establishment of an actively proliferating population of progenitor cells from the stump of different organs. Finally, the mesenchyme of the facial rudiments, unlike that of developing limb buds, expresses simple epithelial keratins. Thus, it appears that mesenchymal progenitor cells of developing and regenerating jaws are alike in regard to their intermediate filament content, and this may be related to nerve-dependent growth control of progenitor cells in different developing and regenerating systems.     

Cell sources, regulatory factors and gene expression in the regeneration of the crystalline lens and retina in vertebrate animals

Over the past century extensive experimental materials have been accumulated concerning cell sources of lens and retina regeneration, successive transformations of the cells, regulatory factors, and gene expression during restitution of these eye structures. The use of nuclear and cytoplasmic markers provided convincing evidence that the removed lens is restituted from the dorsal iris cells in vivo or from embryonic cells of the pigment epithelium and retina in vitro. The removed or destroyed retina is restituted as a result of transdifferentiation of the pigment epithelium cells in amphibians, fish, birds, and mammals during embryogenesis, in larvae of some anuran amphibians, and in adult newts. Cell precursors of rods are a cell source of retina regeneration in adult fish. A subpopulation of randomly distributed cells, which are a cell source of rod formation during the normal development of the eye was found in the external nuclear layer with the use of electron microscopy and nuclear and cytoplasmic markers. These cells are not only a source of regeneration of rods, but also of cones and cells of the internal nuclear layer after destruction of the corresponding retina layers. There is a peripheral growth area in the retina of vertebrates, where multi- and unipolar cells are localized, which provide for the retina growth during ontogenesis. A paradox of retina regeneration consists in that these little differentiated cells are not a source of complete restitution of the removed or destroyed retina. They make only a small contribution to its regeneration corresponding to the growth potential of cells of this eye region, while restitution of the retina proceeds only at the expense of cells of another type of differentiation. A factor controlling the differentiated state of the cell was found in the dorsal iris during studies of lens regeneration. Removal of this factor in the early stages of cell transformations leads to the initiation of lens regeneration. The factor is not specific and was identified in many cells of vertebrates, including the pigment epithelium and limb tissues, which, as is known, may be fully restituted. Studies of gene expression during lens and retina regeneration are now at the initial stage. The greatest advances were achieved on the model of transdifferentiation of the pigment epithelium cells of chick embryos into lentoids. Expression of genes MMP115 and pP344 was established in the pigment epithelium cells, which characterize the pigmented phenotype of the initial cells. Expression of the alpha-, beta-, and delta-crystallin genes was found in the lentoids, which characterize the phenotype of regenerating structures. The gene activity appears to be switched at an intermediate stage during cell dedifferentiation. Expression of the gamma-crystallin genes during lens regeneration in adult newts is initiated after completion of dedifferentiation and cell proliferation in the dorsal iris. The genes specifically expressed in the dorsal and ventral iris and in the retina rudiment have been identified by the method of gene subtraction. Expression of homeobox-containing genes from the family of PAX genes was found during lens regeneration in adult newts and retina regeneration in adult fish. The role of growth factors (FGF) as morphogenetic factors was proved, which are involved in a yet unknown way of altering the differentiation pathway of the initial cells during formation of the neuroepithelium rudiment in chick embryos, adult newts, and fish.     

Antigenic variation of the Epstein-Barr virus nuclear antigen EBNA1 as revealed by monoclonal antibodies

To obtain specific immunological probes for studying molecular mechanisms involved in cell renewal, cell differentiation, and pattern formation in intact and regenerating planarians, we have produced a hybridoma library specific for the asexual race of the fresh-water planarian Dugesia (Girardia) tigrina. Among the 276 monoclonal antibodies showing tissue-, cell-, cell subtype-, subcellular- and position-specific staining, we have found monoclonal antibodies against all tissues and cell types with the exception of neoblasts, the undifferentiated totipotent stem-cells in planarians. We have also detected position-specific antigens that label anterior, central, and posterior regions. Patterns of expression uncovered an unexpected heterogeneity among previously thought single cell types, as well as interesting cross-reactivities that deserve further study. Characterization of some of these monoclonal antibodies suggests they may be extremely useful as molecular markers for studying cell renewal and cell differentiation in the intact and regenerating organism, tracing the origin, lineage, and differentiation of blastema cells, and characterizing the stages and mechanisms of early pattern formation. Moreover, two position-specific monoclonals, the first ones isolated in planarians, will be instrumental in describing in molecular terms how the new pattern unfolds during regeneration and in devising the pattern formation model that best fits classical data on regeneration in planarians.     

Retinoids as endogenous components of the regenerating limb and tail

The role of retinoids (vitamin A derivatives) in limb and tail regeneration is the focus of this review. The dramatic effects of excess retinoids on regeneration are considered, which consist of the respecification of each of the cardinal axes of the limb and the transformation of tail regenerates into limbs. The evidence that retinoids are endogenous components of the regeneration blastema and are required for normal regeneration is then addressed. This evidence consists of the fact that various techniques can detect endogenous retinoic acid in the blastema and that retinoic acid receptors are present, and an individual function in regeneration has been established for each of three receptors. Finally, experiments are described in which retinoic synthesis has been inhibited, and this results in the inhibition of both limb development and limb and tail regeneration.     

Inhibition of chondrogenesis by Wnt gene expression in vivo and in vitro

The Wnt family of secreted signaling proteins are implicated in regulating morphogenesis and tissue patterning in a wide variety of organ systems. Several Wnt genes are expressed in the developing limbs and head, implying roles in skeletal development. To explore these functions, we have used retroviral gene transfer to express Wnt-1 ectopically in the limb buds and craniofacial region of chick embryos. Infection of wing buds at stage 17 and tissues in the head at stage 10 resulted in skeletal abnormalities whose most consistent defects suggested a localized failure of cartilage formation. To test this hypothesis, we infected micromass cultures of prechondrogenic mesenchyme in vitro and found that expression of Wnt-1 caused a severe block in chondrogenesis. Wnt-7a, a gene endogenously expressed in the limb and facial ectoderm, had a similar inhibitory effect. Further analysis of this phenomenon in vitro showed that Wnt-1 and Wnt-7a had mitogenic effects only in early prechondrogenic mesenchyme, that cell aggregation and formation of the prechondrogenic blastema occurred normally, and that the block to differentiation was at the late-blastema/early-chondroblast stage. These results indicate that Wnt signals can have specific inhibitory effects on cytodifferentiation and suggest that one function of endogenous Wnt proteins in the limbs and face may be to influence skeletal morphology by localized inhibition of chondrogenesis.     

Two modes of microtubule-associated protein 1B phosphorylation are differentially regulated during peripheral nerve regeneration

Two major modes of MAP1B phosphorylation (I and II), respectively recognized by monoclonal antibodies 150 and 125, have been related to remodeling and formation of processes in the mature nervous system. To gain insight into the cytoskeletal modifications underlying peripheral nerve regeneration, the pattern of expression of both MAP1B phosphorylated modes was studied during this process. Sciatic nerves from adult Wistar rats were crushed and animals allowed to survive for 5, 7, 10 or 14 days. After those survival periods, damaged and undamaged sciatic nerves, dorsal root ganglia (DRG), and spinal cords, were subjected to immunohistochemistry and Western blot, using antibodies 150 and 125. At all survival periods analysed, MAP1B phosphorylated at mode I was concentrated at the distal region of regenerating nerves whereas mode II phosphorylation underwent an overall decrease in regenerating axons that was less evident in more proximal nerve regions. Very high levels of MAP1B phosphorylated at mode II were detected in the bodies of DRG neurons and in bodies and dendrites of spinal motor neurons. This phosphorylation mode was also encountered in some Schwann cells and oligodendroglia associated with more proximal regions of regenerating axons. In this study we conclude that MAP1B was differentially phosphorylated depending on the cell type, subcellular compartment and stage of the regenerative process and discuss the possible functional implications that differential expression of each MAP1B phosphorylation mode might have during nerve regeneration.     

Lipoxygenase products and expression of 5-lipoxygenase and 5-lipoxygenase-activating protein in human cultured synovial cells

Limb regeneration is a phenomenon occurring only in some urodeles. The process seems to be initiated by the dedifferentiation of the terminally differentiated cells. These cells differentiate, subsequently, to the tissues that comprise the limb, thus reconstructing the pattern of the missing limb part. In this paper we review and present evidence that certain cell types of the limb have the capacity to differentiate to different cell types than their original one by cellular metaplasia. This switch is called transdifferentiation. The focus of this review is the process of dedifferentiation which is the necessary prerequisite for differentiation, and the possible mechanisms involved.     

Transdifferentiation of muscle to electric organ: regulation of muscle-specific proteins is independent of patterned nerve activity

Transdifferentiation is the conversion of one differentiated cell type into another. The electric organ of fishes transdifferentiates from muscle but little is known about how this occurs. To begin to address this question, we studied the expression of muscle- and electrocyte-specific proteins with immunohistochemistry during regeneration of the electric organ. In the early stages of regeneration, a blastema forms. Blastemal cells cluster, express desmin, fuse into myotubes, and then express alpha-actinin, tropomyosin, and myosin. Myotubes in the periphery of the blastema continue to differentiate as muscle; those in the center grow in size, probably by fusing with each other, and lose their sarcomeres as they become electrocytes. Tropomyosin is rapidly down-regulated while desmin, alpha-actinin, and myosin continue to be diffusely expressed in newly formed electrocytes despite the absence of organized sarcomeres. During this time an isoform of keratin that is a marker for mature electrocytes is expressed. One week later, the immunoreactivities of myosin disappears and alpha-actinin weakens, while that of desmin and keratin remain strong. Since nerve fibers grow into the blastema preceding the appearance of any differentiated cells, we tested whether the highly rhythmic nerve activity associated with electromotor input plays a role in transdifferentiation and found that electrocytes develop normally in the absence of electromotor neuron activity.     

Xefiltin, a Xenopus laevis neuronal intermediate filament protein, is expressed in actively growing optic axons during development and regeneration

Neurofilaments are an important structural component of the axonal cytoskeleton and are made of neuronal intermediate filament (nIF) proteins. During axonal development, neurofilaments undergo progressive changes in molecular composition. In mammals, for example, highly phosphorylated forms of the middle- and high-molecular-weight neurofilament proteins (NF-M and NF-H, respectively) are characteristic of mature axons, whereas nIF proteins such as alpha-internexin are typical of young axons. Such changes have been proposed to help growing axons accommodate varying demands for plasticity and stability by modulating the structure of the axonal cytoskeleton. Xefiltin is a recently discovered nIF protein of the frog Xenopus laevis, whose nervous system has a large capacity for regeneration and plasticity. By amino acid identity, xefiltin is closely related to two other nIF proteins, alpha-internexin and gefiltin. alpha-Internexin is found principally in embryonic axons of the mammalian brain, and gefiltin is expressed primarily in goldfish retinal ganglion cells and has been associated with the ability of the goldfish optic nerve to regenerate. Like gefiltin in goldfish, xefiltin in Xenopus is the most abundantly expressed nIF protein of mature retinal ganglion cells. In the present study, we used immunocytochemistry to study the distribution of xefiltin during optic nerve development and regeneration. During development, xefiltin was found in optic axons at stage 35/36, before they reach the tectum at stage 37/38. Similarly, after an orbital crush injury, xefiltin first reemerged in optic axons after the front of regeneration reached the optic chiasm, but before it reached the tectum. Thus, during both development and regeneration, xefiltin was present within actively growing optic axons. In addition, aberrantly projecting retinoretinal axons expressed less xefiltin than those entering the optic tract, suggesting that xefiltin expression is influenced by interactions between regenerating axons and cells encountered along the visual pathway. These results support the idea that changes in xefiltin expression, along with those of other nIF proteins, modulate the structure and stability of actively growing optic axons and that this stability is under the control of the pathway which growing axons follow.     

Passive cutaneous anaphylaxis in the domestic fowl

Histochemical demonstration of beta-glucuronidase was carried out in the normal and regenerating tail of the house lizard, Hemidactylus flaviviridis. Greater enzyme activity was particularly discernible in the stratum germinativum of the skin of the normal and fully regenerated tail. During regeneration most of the cellular elements of the blastema and the differentiating regenerate exhibited enzyme activity. Distribution of the enzyme beta-glucuronidase is correlated with the high degree of cellular proliferation. The role of the enzyme in the catabolism of mucopolysaccharides is also implicated during the wound-healing phase. In the full-grown regenerate the intensity and distribution of the enzyme activity were found to be the same as those observed in the normal tail.     

Epidemiology of violence and theft in the workplace

Using sequence information derived from the Drosophila melanogaster (Dm) ecdysteroid receptor (EcR)- and retinoid X receptor (RXR)-encoding gene homologs, we have isolated cDNA clones corresponding to the DNA-binding domains (DBD) for these two nuclear receptors from the fiddler crab, Uca pugilator (Up). Both genes appear to be represented in 1-2 copies in the Up genome, and unlike Dm, contain an intron within the DBD-encoding region. Sequence comparisons to the Dm EcR and RXR homologs indicate 76 and 82% nucleotide identity, respectively, corresponding to 6 and 4 single-amino acid substitutions which primarily cluster in the region of the molecule involved in dimerization. RT-PCR analysis indicates that both the EcR and RXR homologs are expressed during the initial stages of limb regeneration, temporally concomitant with early blastema formation and the secretion of a flexible sac cuticle at the site of limb loss.     

Involvement of the sonic hedgehog, patched 1 and bmp2 genes in patterning of the zebrafish dermal fin rays

The signaling molecule encoded by Sonic hedgehog (shh) participates in the patterning of several embryonic structures including limbs. During early fin development in zebrafish, a subset of cells in the posterior margin of pectoral fin buds express shh. We have shown that regulation of shh in pectoral fin buds is consistent with a role in mediating the activity of a structure analogous to the zone of polarizing activity (ZPA) (Akimenko and Ekker (1995) Dev. Biol. 170, 243-247). During growth of the bony rays of both paired and unpaired fins, and during fin regeneration, there does not seem to be a region equivalent to the ZPA and one would predict that shh would play a different role, if any, during these processes specific to fish fins. We have examined the expression of shh in the developing fins of 4-week old larvae and in regenerating fins of adults. A subset of cells in the basal layer of the epidermis in close proximity to the newly formed dermal bone structures of the fin rays, the lepidotrichia, express shh, and ptc1 which is thought to encode the receptor of the SHH signal. The expression domain of ptc1 is broader than that of shh and adjacent blastemal cells releasing the dermal bone matrix also express ptc1. Further observations indicate that the bmp2 gene, in addition to being expressed in the same cells of the basal layer of the epidermis as shh, is also expressed in a subset of the ptc1-expressing cells of the blastema. Amputations of caudal fins immediately after the first branching point of the lepidotrichia, and global administration of all-trans-retinoic acid, two procedures known to cause fusion of adjacent rays, result in a transient decrease in the expression of shh, ptc1 and bmp2. The effects of retinoic acid on shh expression occur within minutes after the onset of treatment suggesting direct regulation of shh by retinoic acid. These observations suggest a role for shh, ptc1 and bmp2 in patterning of the dermoskeleton of developing and regenerating teleost fins.     

Isozyme selective alterations of the expression of cytochrome P450 during regeneration of male rat liver following partial hepatectomy

1. During liver regeneration in the male rat, the metabolic activities of imipramine were differentially affected depending on the specific metabolic pathways. Imipramine N-demethylation was markedly reduced whereas 2-hydroxylation showed only a moderate reduction following partial hepatectomy. 2. A slight decline was observed in the hepatic microsomal content of CYP2D apoprotein, whereas a substantial decrease occurred in CYP2C11 content during liver regeneration. Since imipramine 2-hydroxylation and N-demethylation are mediated by CYP2D and 2C11 respectively, metabolic pathway-specific alterations in the activities of imipramine metabolism are explained by the isozyme selective alteration in the levels of CYPs in regenerating liver. 3. No significant effect of regeneration was observed on expression of CYP2B1 and 2E1 apoproteins. CYP3A2 apoprotein, one of the male-specific CYP isoforms, was significantly suppressed in regenerating liver showing a similar pattern of alteration to the levels of CYP2C11. The alteration pattern of the CYP1A1 level was different to the above with a moderate decline at the first day post-operation and a marked rebound thereafter. 4. In the partially hepatectomized male rate, no significant increase in androstenedione 5-alpha reductase activity, an activity predominant in the female rat, was detected. It is concluded that the pattern of alterations of hepatic oxidative metabolism during liver regeneration was not related to the functional feminization of the liver.     

Heterogeneity among muscle precursor cells in adult skeletal muscles with differing regenerative capacities

Skeletal muscle has a remarkable capacity to regenerate after injury, although studies of muscle regeneration have heretofore been limited almost exclusively to limb musculature. Muscle precursor cells in skeletal muscle are responsible for the repair of damaged muscle. Heterogeneity exists in the growth and differentiation properties of muscle precursor cell (myoblast) populations throughout limb development but whether the muscle precursor cells differ among adult skeletal muscles is unknown. Such heterogeneity among myoblasts in the adult may give rise to skeletal muscles with different regenerative capacities. Here we compare the regenerative response of a masticatory muscle, the masseter, to that of limb muscles. After exogenous trauma (freeze or crush injuries), masseter muscle regenerated much less effectively than limb muscle. In limb muscle, normal architecture was restored 12 days after injury, whereas in masseter muscle, minimal regeneration occurred during the same time period. Indeed, at late time points, masseter muscles exhibited increased fibrous connective tissue in the region of damage, evidence of ineffective muscle regeneration. Similarly, in response to endogenous muscle injury due to a muscular dystrophy, widespread evidence of impaired regeneration was present in masseter muscle but not in limb muscle. To explore the cellular basis of these different regenerative capacities, we analyzed the myoblast populations of limb and masseter muscles both in vivo and in vitro. From in vivo analyses, the number of myoblasts in regenerating muscle was less in masseter compared with limb muscle. Assessment of population growth in vitro indicated that masseter myoblasts grow more slowly than limb myoblasts under identical conditions. We conclude that the impaired regeneration in masseter muscles is due to differences in the intrinsic myoblast populations compared to limb muscles.