Formation of the extracellular matrix during the epimorphic anterior regeneration of Owenia fusiformis: autoradiographical and in situ hybridization studies.

During post-traumatic regeneration of the polychaete annelid Owenia fusiformis, the extracellular matrix (ECM) formation was studied by light and electron microscopy and by histoautoradiography after incorporation of tritiated proline as marker for collagenic proteins. Three days after amputation, a new basement membrane was reformed in the blastema between the ectoderm and the mesoderm. At the same time, the cytoskeleton and the anchoring structures (hemidesmosomes) were differentiated in the basal part of the ectodermal cells. Four days after amputation, collagen fibers appeared in the extracellular matrix newly reformed between the ectodermal and mesodermal layers. The existence of a proximo-distal gradient in the organization of the new extracellular matrix and the accumulation of molecules labeled by 3H-proline was shown. This accumulation started at the level of the injured segment of the stump. Differences in labeling intensity were seen in the regenerate. Within specific organogenetic zones, i.e. the epidermal gland analagen, the branchial buds and the stomodeal invagination, the labeling between the ectodermal and mesodermal layers was less intense than in other parts of the regenerate. In the mesodermal connective septa (dissepiments), located between consecutive segments, the labeling and the accumulation of extracellular material occurred later than the formation of the ectodermal basement membrane. In situ hybridization of a DNA molecular probe corresponding partially to the coding region of the collagen-like gene Ocg8, showed a spatio-temporal expression of this gene. Northern blot analysis showed a single transcript of 6.6 kb. Four days after amputation the accumulation of this transcript was exclusively localized at the level of the ectodermal layer during differentiation of the regenerate. The ectoderm was thus shown to play a dynamic role during the first stages of traumatic regeneration, although it did not seem to be directly involved in the early events of the metameric process.

Expression of polysialylated neural cell adhesion molecule (PSA-N-CAM) in developing, adult and regenerating caudal spinal cord of the urodele amphibians.

The patterns of expression of polysialylated ("embryonic") form of Neural Cell Adhesion Molecule (PSA/E-N-CAM) and of all N-CAM isoforms were investigated by indirect immunofluorescence and immunoblotting during the development of the Central Nervous System (CNS) and during the regeneration of the caudal Spinal Cord (SC) of the amphibian urodeles Pleurodeles waltl (Pw) and Notophthalmus viridescens (Nv). In this study, a monoclonal antibody to group B Meningococcus (anti-Men-B) which recognizes alpha-2,8-linked sialic units of PSA-N-CAM, and polyclonal anti-total N-CAM antibodies were used. Total-N-CAM immunoreactivities were consistently detected throughout the CNS of developing and adult newts. PSA-N-CAM expression predominated in "embryonic" developing CNS and was reduced to certain CNS areas in the adult urodeles. In the case of SC, the expression level of this isoform of N-CAM dramatically decreased to become low and nearly restricted to some ependymoglial cell surfaces. Interestingly, during newt tail regeneration, PSA-N-CAM was intensely reexpressed in regenerating SC, at the surface of ependymoglial cell processes and in axonal compartments. Expression was maximal at 4 to 6 weeks following amputation, and then gradually returned to a normal adult low level in well differentiated SC. These findings strongly supported the view that the expression of PSA-N-CAM was associated with the properties of plasticity shown by the SC ependymoglial tissue in newts, during tail regeneration. On the other hand, the high level of PSA-N-CAM expression in axonal compartments of regenerating as well as developing SC suggested that these isoforms of N-CAM could be implicated in axonal outgrowth within the "tunnels" defined by the radial ependymoglial processes. This transient PSA-N-CAM expression could therefore be considered both as a negative modulator of cell-cell and cell-substrate interactions and as a permissive factor for neuron differentiation.

Remodeling processes during neural retinal regeneration in adult urodeles: an immunohistochemical survey.

Dynamic features of neural retina regeneration in the adult newt Pleurodeles waltl were analyzed using immunohistochemical studies. Antibody to Glial Fibrillary Acidic Protein (GFAP) was used as a marker of the retinal glial supportive system in order to obtain an overview of the retinal reorganization pattern. Unexpectedly, retinal progenitor cells displayed GFAP staining, as did later Müller glial processes and astrocytes supporting ganglional axons. To study changes of plasticity during retinal restoration, the expression patterns of highly- (PSA) and weakly-sialylated N-CAM were examined by double staining. In the retina of adult newts, a sustained expression of total-N-CAM and PSA-N-CAM was detected. However, while an intense distribution of N-CAM was observed throughout the retina, PSA labeling was especially seen in the outer retinal layers. During retinal regeneration, similar widespread staining patterns were observed with the two antibodies, but labeling appeared higher with anti-total-N-CAM antibody than with anti-PSA-N-CAM antibody. On the other hand, tenascin (Tn) expression was analyzed for the first time during retinal regeneration. At the early stages, brightly stained matrix fibers of abundant Tn accumulating in the eye cavity were seen close to the retinal rudiment cells, which suggested that Tn was secreted from these cells. Tn expression was seen nearly throughout the retinal regenerate during neurite migration and then became restricted to the plexiform layers. In the light of the functions attributed to N-CAM and Tn in histogenetic events, the putative roles played by these morphoregulatory molecules in adult newt retinal regeneration were discussed.

Patterns of dystrophin expression in developing, adult and regenerating tail skeletal muscle of Amphibian urodeles.

The patterns of expression of dystrophin were investigated by indirect immunofluorescence and by immunoblotting in developing, adult and regenerating tail skeletal muscle of newts Pleurodeles waltl and Notophthalmus viridescens. In this study, a monoclonal antibody H-5A3 directed against the C-terminal region (residues 3357-3660) and a polyclonal antibody raised to the central domain (residues 1173-1738) of the chicken skeletal muscle dystrophin were used. Western blot analysis showed that these antibodies recognized a 400 kDa band of dystrophin (and may be of dystrophin-related protein) in the adult muscle tissues and in newt tail regenerates. During skeletal muscle differentiation or epimorphic regeneration (blastema), anti-dystrophin immunoreactivity gradually accumulated over the periphery of the myofibers. Dystrophin and laminin were first and concomitantly observed at the ends of the newly formed myotubes where they were anchored on connective tissue septa or bone processes by dystrophin-rich myotendinous structures. It is noteworthy that neuromuscular junctions, which most probably also contain dystrophin, are established in urodeles near the ends of the myofibers as shown by histochemical localization of AChE activity or fluorescent bungarotoxin detection of AChRs. In the stump transition zone close to the tail amputation level where tissue regeneration of injured muscle fibers took place, dystrophin staining located on the cytoplasmic surface of myofibers progressively disappeared during the dedifferentiation process which seemed to occur during muscle regeneration as suggested by electron microscopy. Furthermore, double labeling experiments using anti-dystrophin and anti-laminin antibodies showed a good correlation between the remodeling processes of the muscle fiber basal lamina and the loss of dystrophin along the sarcolemma of damaged and presumably dedifferentiating muscle cells.

Expression patterns of dystrophin products, especially of apodystrophin-1/Dp71, in the neural retina of Amphibian urodele Pleurodeles waltl.

The expression patterns of the DMD (Duchenne Muscular Dystrophy) gene products, especially of Dp71 (apodystrophin-1) were investigated by immunofluorescence and immunoblotting in the retina of the Amphibian urodele Pleurodeles waltl. H-5A3 monoclonal antibody (mAb), directed against the C-terminal region of dystrophin/utrophin, and 5F3 mAb, directed against the last 31 amino acids of dystrophin and specific of Dp71, were used. Western blot analyses with H-5A3 mAb revealed distinct dystrophin-family isoforms in adult newt retinal extracts: a doublet 400-420 kDa, Dp260 isoform, a protein at about 120 kDa, and a diffuse zone at 70-80 kDa, which might correspond to Dp71. Reactivity with H-5A3 mAb appeared nearly restricted to the outer plexiform synaptic layer. On the other hand, Dp71-specific 5F3 mAb recognized trhee polypeptide bands at 70-80, 60-65 and 50-55 kDa in adult newt retina corresponding most probably to alternative spliced isoforms of Dp71. In immunohistochemistry by conventional epifluorescence microscopy, 5F3 labeling was mainly observed in the plexiform layers, the outer nuclear layer, and the photoreceptor inner segments, especially at the myoid regions. Analysis by confocal scanning laser microscopy (CSLM) revealed that 5F3 labeling was, in addition, present in the pigmented epithelium and the inner nuclear layer. Furthermore, CSLM showed that 5F3 staining at the myoids was concentrated at discrete domains underneath the plasma membrane. Our findings raised the question concerning the functional significance of Dp71 isoforms, especially at the myoid where Dp71 was detected for the first time, although it occurred here highly expressed. Putative role(s) played in this retinal compartment and other ones by Dp71 and/or other dystrophin isoforms were discussed.

Tenascin expression in developing, adult and regenerating caudal spinal cord in the urodele amphibians.

Tenascin (Tn) protein and transcripts were analyzed in developing, adult and regenerating caudal spinal cord (SC) of Pleurodeles waltl. A polyclonal antibody (PAb) against Xenopus Tn and a newt Tn cDNA probe were used. In Western blots, anti-Tn PAb recognized Tn polypeptides of 200-220 kDa in tail regenerate extracts, but also the homolog of Tn/Cytotactin/J1 in brain and SC of adult newt. Immunofluorescence studies showed some reactivity around ependymoglial cells and strong labeling in the nervous tracts, in the developing as well as in the regenerating SC or adult SC. Immunogold electron microscopy revealed the presence of Tn throughout the ependymoglial cells, particularly near and along the plasma membrane of radial processes surrounding axons, especially growth cones. Tn could be more precisely found within rough endoplasmic reticulum and Golgi structures, or again in the surrounding extracellular space. This suggested that Tn was at least produced by radial glial profiles forming axonal compartments in which axons grew. Using the DNA probe for Tn, expression of Tn mRNA was also examined by Northern blot and RNAase protection analyses and by in situ hybridization, respectively. The levels of transcripts, barely detectable in adult tail, increased in regenerates from 3 days through 4-8 weeks post-amputation. In situ Tn mRNA were mainly localized in the mesenchyme, especially at the epithelial-mesenchymal interface, and in the developing cartilage, at the early regeneration stages, whereas high amounts of transcripts were seen not only at these stages, but also later, in the regenerating SC. Our main results supported the view that, in the caudal SC of newts, Tn, synthesized by radial ependymoglial cells, was similarly expressed during regeneration as well as larval development, and exhibited a sustained high accumulation level in the adult SC. On the basis of the multifunctional properties of Tn, the putative roles played by Tn as a substrate for neuronal pathfinding and boundary shaping were discussed.

Pattern of RNA synthesis during the regeneration by Owenia fusiformis (polychaete annelid).

The binding of [3H] Uridine labelled RNAs and [32P] RNAs to poly U filters using the Sheldon et al. technique (1972) shows an increase in the poly A+ RNA populations from total RNAs and cytoplasmic RNAs in the prereplicative phase of the regenerating process in Owenia fusiformis. This increase confirms that the first stage of this process consists in an activation of the transcription of the genetic material concerning mainly mRNAs synthesis activation.

[Expression of glial, neurospecific, and extracellular antigens during retinal regeneration in adult newts]. / Ekspressia glial'nykh, neirospetsificheskikh i vnekletochnykh antigenov v protsesse regeneratsii setchatki u vzroslykh tritonov.

Spatial localization and time of expression of glial, neurospecific, and extracellular antigens were studied during retina regeneration in adult newts using antibodies against the glial acidic fibrillar protein (GFAP), neurospecific molecules (N-CAM and PSA-N-CAM), and tenascin (Tn). At the early stages of regeneration (5th day after the operation), slightly differentiated cells in the retina growth area, which are not cellular sources of regeneration, were brightly stained by the antibodies. The one-two-layered retina rudiment formed from the pigment epithelium layer a week later was also intensely stained by the antibodies to GFAP. The retina rudiment cells belong to slightly differentiated precursors of the regenerating retina. It was first shown that the antibodies to GFAP were not only markers of the glial cells, but also markers of slightly differentiated precursors of the regenerating retina. Expression of neurospecific antigens was found in depigmented cells of the retina rudiment. It appeared to have been initiated by cell interactions in the regenerate. An expression of tenascin was found in the cells migrating in the eye cavity and contacting with the retina rudiment cells. The role of tenascin in interaction with the retina rudiment is unknown.

DNA synthesis during the first stages of anterior regeneration in the polychaete annelid Owenia fusiformis (dedifferentiation and early phases of differentiation).

We have analysed DNA synthesis in early phases of regeneration in a marine Polychaete Annelid, Owenia fusiformis. The length and efficiency of the prereplicative phase was found to vary with the diurnal rhythm of activity of the animal; that is, it depends on the initial state of the cell population at the moment of the onset of proliferative stimulatin. When animals were operated on at 12 a.m., the duration of the prereplicative phase of the first cells stimulated to proliferate was found to be 12 h. The remaining cells entered the S-phase progressively in waves until the 3rd day following amputation when nearly 100% of the blastema cells were stimulated. At that time the cell-cycles of these dividing cells were found to be highly synchronized. Blastema differentiation takes place on the 4th day and is initiated by stomodeum formation. During the differentiation phase, DNA synthesis is restricted to small areas of the regenerating part. The system described is viewed as a new instrument for investigating the control of the cell cycle in synchronized and subsequently differentiating tissue cells.

[Synthesis of nuclear proteins incorporated into chromatin during anterior traumatic regeneration of an annelid (Owenia fusiformis)]. / Synthèse des protéines nucléaires incorporées dans la chromatine durant la régémération traumatique antérieure d'une Annélide (Owenia fusiformis).

3H Trp and 14C Lys incorporation in proteins from chromatin of normal and regenerating animals, purified on metrizamide, show that the synthesis of these proteins is different in the two cases.

[Daily variations of the level of neural secretions in Owenia fusiformis. Persistance of a daily rhythm in cultured tissues]. / Variations journalières du taux des sécrétions nerveuses chez Owenia fusiformis. Persistance d'un rhythme journalier dans les tissus en culture

A quantitative study of elementary granules in neuron axons of the nervous chain of Owenia fusiformis (Annelid polychaete) shows that daily variations of the number of secretory granules occur in unamputated animals and in ventral tissue cultures. These variations are correlated with the thythm of the cell cycle.

Expression of muscle actin genes in early differentiation stages of tail regeneration of the urodele amphibian Pleurodeles waltlii.

Amputation of the tail of the amphibian Pleurodeles waltlii leads to the formation of a blastema composed of undifferentiated cells during the first stages of regeneration. Between the 3rd and 4th week following amputation, the first fully differentiated striated muscle cells appear, and in the 6th week myogenic differentiation extends throughout the regenerate. Myoblasts appear in cell patches where cells fuse as differentiation proceeds. Using a cloned cDNA probe to a striated muscle actin gene specifically expressed in adult skeletal muscle, we show that no corresponding mRNA remains during dedifferentiation. A new actin transcript of the same length as cardiac actin transcript appears in the 4th week after amputation. We conclude that for the myogenic cell line, differentiation in the regenerate is accompanied by an actin isoform transition similar to that seen in embryonic terminal differentiation of higher vertebrates.

Neural crest-like cells originate from the spinal cord during tail regeneration in adult amphibian urodeles.

Using in vitro cell-marking experiments and transplantation in tail regenerates, we have recently shown (Benraiss et al., 1996) that clonal cells derived from adult newt spinal cord (SC) cultures could find suitable cues in blastemal mesenchyme to enable them to differentiate into melanocytes or Schwann cells. This led to the question of whether neural crest-like cell derivatives might emerge from the ependymal tube as tail regeneration proceeded. To address this question we used the biolistic method for in situ transfection of caudal SC cells. These cells were transfected with an alkaline phosphatase marker gene. The potentialities of transfected cell derivatives in tail regenerates were analyzed using histochemistry or immunohistochemistry. As early as eight days after transfection, labeled cells were detected in the regenerating SC and around its "terminal vesicle" (TV). Two to four weeks following transfection, most of the labeled cell derivatives could be identified either by dark granules as melanocytes or by galactocerebroside staining as Schwann cells. Electron microscopic investigations revealed the incompletely organized cytoarchitecture of the TV, suggesting that an exit of cells was possible at this level, at least from its "open" dorsal part. Furthermore, the localization of ciliated cells in the blastemal mesenchyme, especially around the TV, supported this view by suggesting that they might be ependymal cells detached from it. Our findings therefore led us to believe that in the newt, during tail regeneration, neural crest-like cells emerging from the TV could participate in the formation of the peripheral nervous system, especially by providing Schwann cells and melanocytes.

Variations in accessibility of DNA during traumatic regeneration by Owenia fusiformis (polychaete annelid).

One of the earliest events observed in the 'dedifferentiation phase' of traumatic regeneration is an in vivo increase in transcription. In order to see whether that increase corresponds to a variation at the level of genetic material, we prepared chromatin from normal and regenerating animals (12 h after amputation) for transcription in vitro by E. coli RNA polymerase. The template activity of regenerating chromatin was 2.1-fold higher than normal under standard conditions and showed an altered response to variation of the ionic strength of the medium. The accessibility of DNA in the chromatin was tested using mild digestion by DNAase II. Almost twice as much DNA was solubilised from regenerating chromatin as from normal chromatin. These data were correlated with morphological changes (decondensation) observed by electron microscopy.

[Experimental study of the synthesis of lysosomal enzymes by the synovial membrane: acid phosphatases]. / Etude expérimentale de la synthèse des enzymes lysosomiales par la synoviale: phosphatases acides

In a system consisting of synovial samples taken surgically and maintained in culture, the authors studied the regulation of enzyme synthesis by taking acid phosphatase as the test enzyme. By means of double labelling and the use of protein synthesis inhibitors, they were able to demonstrate that this synthesis was stimulated by the addition of rheumatoid factor to the culture medium and that it depended on the transcription activity of the genome and on the translation of the information molecules.

Neurogenesis during caudal spinal cord regeneration in adult newts.

After tail amputation in urodele amphibians, dramatic changes appear in the spinal cord rostral to the amputation level. Transection induces a proliferation response in cells lining the ependymal canal, giving rise to an ependymal tube in which neurogenesis occurs. Using the thymidine analog bromodeoxyuridine (BrdU) in short- and long-term labeling of cells undergoing DNA synthesis (S phase of the cell cycle), specific cell markers, and cell cultures, we show that neurons derive from the proliferative ependymal layer of the ependymal tube.

Muscle dedifferentiation and contractile protein synthesis during post-traumatic regeneration by Owenia fusiformis (polychaeta annelid).

During anterior traumatic regeneration of the polychaete annelid Owenia fusiformis, we have observed by electron microscopy the dedifferentiation processes of muscle cells. The dedifferentiated cells are reminiscent of undifferentiated myoblasts. They form the blastema and redifferentiate in muscle cells in the regenerate. The in vivo and in vitro biochemical studies of the biosynthesis of contractile proteins, as markers of the terminal differentiation programme of the muscle cells, showed that gene expression correlated with the terminal differentiation programme (at last for muscle cells) seemed unaffected. It seems in fact that contractile protein synthesis is regulated partly at the translational level during traumatic regeneration in the invertebrate.

Early innervation of skeletal muscle during tail regeneration in urodele amphibians.

The innervation pattern of skeletal muscles was studied in the normal and regenerating tail of Notophthalmus viridescens. Silver staining for nerve endings and histochemical localization of acetylcholinesterase (AChE) were used for light microscopy. In In normal musculature, AChE positive reactions were localized at the ends of the muscle fibers where they are anchored on connective tissue septa by myotendinous junctions. At this level, silver staining shows nerve terminals forming endplates. During regeneration, positive reactions for AChE appear de novo as dense plates localized at the ends of the newly formed myotubes. The mechanisms involved in the localization of AChE on this surface seem to operate before previous local contacts by nerve terminals. From the ultrastructural data and immunohistochemical results with anti-laminin antibody, these observations suggest that regenerating muscle fibers determine a region of post-synaptic specialization in close relation with the organization of myotendinous regions and basement membrane formation. Nerve-muscle contacts appear at these levels at stage IV (15-20 days after amputation) in the stump and in the rostral part of the regenerate (transition zone). These nerve terminals are provided by the disorganized peripheral nervous system of the injured segment. In the regenerate a similar pattern of AChE reaction can be seen in every myotube, differentiating according to a rostro-caudal gradient. Innervation at the ends of the muscle fibers is in spatiotemporal relation with the exists of the ventral roots from the regenerating nerve cord as the regenerate continues to grow in length.

The wound epithelium of regenerating limbs of Pleurodeles waltl and Notophthalmus viridescens: studies with mAbs WE3 and WE4, phalloidin, and DNase 1.

The wound epithelium of regenerating limbs of the American newt, Notophthalmus viridescens (Nv), up-regulates a number of antigens, including those recognized by mAbs WE3 and WE4. In the present study, we show that the WE3 antigen is up-regulated in a similar fashion in the wound epithelium of the European newt, Pleurodeles waltl (Pw). mAb WE3 and WE4 reactivities to secretory/transport body cell types, including integumentary glands, perineurium, endothelium, and conjunctiva, are also similar in these two species of newt. However, mAb WE4 reacts to both the epidermis and wound epithelium in Pw, whereas in Nv, mAb WE4 reacts only to the wound epithelium. Because the WE3 antigen is cytoskeleton-associated and Western blots reveal a 43 kDa species, we compared mAb WE3 reactivity with that of rhodamine-labeled phalloidin, a known actin-binding compound. Phalloidin did not react preferentially to the wound epithelium, conjunctiva, or other cell types strongly reactive to mAb WE3. Pretreatment of sections and tissue extracts with DNAse 1, a protein known to bind to actin, nearly abolished mAb WE3 reactivity in tissue sections and both WE3 and WE4 reactivity in ELISA assays, respectively. The results lead to the hypothesis that the WE3 and WE4 antigens are actin-binding proteins unique to the wound epithelium and other secretory/transport cell types.

Presence in invertebrate genomes of sequences characterized by the repetition of the triplet CCPurine.

In Drosophila melanogaster (Dm), polypeptidic domains have been found in different morphogenetic genes. Two types of them are characterized by the repetition of nucleotidic triplets: the M repeat (CAX) and the paired repeat (CAXCCX). In this paper we described a third type of repeat isolated from the genome of a Polychaete annelid: Owenia fusiformis. This repeat is characterized by the repetition of the triplet CCPurine. Phylogenetic studies showed the presence of this repeat in all the invertebrate genomes tested (eight copies in Dm genome) while we failed to detect it in vertebrate genomes.