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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.

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.

The urodele amphibians are nearly the only adult vertebrates able to regener- ate their missing or amputated tail. The most strik- ing feature of this model lies in the ability of the spinal cord (SC) to differentiate, within the regen- erating tail, a new ependymal tube from which the SC and the peripheral nervous system originate. A fundamental question is whether, in response to tail excision, the ependymoglia of the old SC stump behaves as an embryonic neuroepithelium. To evaluate this possibility, cell lines from primary cell cultures of adult SC were established for the first time in newts, and two cell clones, immuno- chemically characterized as ependymoglial cell populations, could be obtained. To analyze the po- tentialities of these clonal cells, after transplanta- tion in tail regenerates, cell-marking experiments, using either in vitro transfection with lac2 gene or the lineage tracer lysinated rhodamine dextran (LRD), were performed. One to 2 weeks postim- plantation, most of labeled derivatives were iden- tified as melanocytes. Interestingly, labeled cells were also seen integrated in the ependymoglia of the regenerating SC. Two to 6 weeks after implan- tation in young regenerates, we also observed LRD-labeled elongated cells close to nerves or myofibers which were unambiguously identified as Schwann cells by galactocerebroside staining. Taken together, these findings showed that clonal cells derived from adult newt SC cultures could largely find, in regenerate mesenchyme, suitable environmental conditions to differentiate into mel- anocytes or Schwann cells. Because these two cell types arise from neural crest cells during embryo- genesis, this supports the interesting view that multipotent cells are still present in the SC of adult urodeles.

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. © 1993 Wiley-Liss, Inc.

In the regenerating newt tail, epimorphic regeneration—which recapitulates morphologically normal embryonic development—proceeds along a rostrocaudal differentiation gradient. Innervation of the new myomeres results from the spinal roots of segments rostral to the amputation plane and from ventral roots emerging from the lateroventral region of the regenerating spinal cord, in which motor neurons are differentiating. Electron microscopy and an indirect immunofluorescence study with anti-glial fibrillary acid protein (GFAP) confirm that the ventrolateral part of the regenerated ependymal tube gives rise to cells of the ventral root sheath and the spinal ganglia. Anti-GFAP and anti-neurofilament antibodies showed that ependymoglial cells and Schwann cells may play a role in neuronal pathfinding by helping guide and stabilize pioneering axons as they extend toward the myomeres. The carbohydrate epitope NC-1 is expressed in the spinal cord, in sheath cells of the spinal ganglia and in the non-myelin-forming Schwann cells of the peripheral nervous system. L1, a Ca+ + independent neural cell adhesion molecule, was detected in the axonal compartments of the regenerating spinal cord, on immature and/or non-myelin-forming Schwann cells within the peripheral nervous system (PNS), and on nerve fibers within the regenerate. These immunohistochemical observations collectively support the hypothesis that Schwann cells already present in the blastema could be involved in organizing neural pathways. © 1992 Wiley-Liss, Inc.

The innervation pattern of skeletal muscles was studied in the normal and regen- erating tail of Notophthalmus viridescens. Silver staining for nerve endings and histochemical localization of acetylcholinesterase (AChE) were used for light microscopy. 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 termi- nals. 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 mem- brane formation. Nerve-muscle contacts appear at these levels at stage IV (15-20 days after amputation) in the stump and in the rostra1 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 spatio- temporal relation with the exits of the ventral roots from the regenerating nerve cord as the regenerate continues to grow in length. An adult urodele amphibian can regenerate its tail after amputation by forming a blastema at the tip of the stump. This blastema gives rise to a regenerate in which cell differentiation occurs. Epidermal structures, skeleton, central (CNS) and peripheral (PNS) nervous systems, and skele- tal muscles are regenerated in a normal struc- tural pattern through five recognizable stages of which the main histological characteristics are summarized in Table 1, from the data of Iten and Bryant ('76a) and from our observations in re- spect to the shape and the days post-amputation of the regenerate. The number of segmented structures (myomeres and vertebrae) that form in the regenerate is directly proportional to the number removed by amputation (Iten and Bry- ant, '76a,b). The evidence that two distinct modes of skele- tal muscle regeneration exist was proposed by Cox ('69) studying muscle regeneration in the liz- ard tail and generalized by Carlson ('70) to the amphibian regeneration: 1) a tissue mode that re

We have determined the patterns of expression of tenascin, an extracellular matrix (ECM) glycoprotein, by indirect immunohistofluorescence and immunoblots during the post-traumatic regeneration of the tail distal part of the amphibian urodele Pleurodeles waltl. In normal tails of adult Pleurodeles, tenascin expression is mostly restricted to few connective tissues including the periosteum, the ligaments of vertebral articulation, myotendinous structures, the perimysium and the pia mater; the extracellular matrix is virtually negative. In certain areas of the adult skin the molecule is expressed around cells of the inner epidermal layer, apparently associated with the cell surface. In the first 4-6 days following tail amputation, tenascin expression increases in the stump region in areas surrounding dedifferentiating tendons; the early blastemic epithelium does not express the molecule. The local increase of tenascin in areas where cells dedifferentiate and start to migrate, precedes the increase in fibronectin that occurs later in the mesenchyme of the blastema. From the 8th day of regeneration, there is a sharp increase of the level of expression of the molecule in the extracellular matrix of the loose mesenchyme underlying the epithelium of the blastema which remains negative. The maximal expression in the matrix is reached in 4-to 6-week-old regenerates and then gradually decreased. High levels of tenascin are present in sites of muscle condensation as fibrils oriented parallel to the direction of alignment of myogenic cells and in sites of chondrogenesis particularly in regions of precartilage formation. After the second week of regeneration, tenascin is strongly expressed in the basal lamina of the regenerated skin and, after the fourth week, also at the level of epidermaldermal junctional areas. Like in normal tail ends, in regenerates older than 8 – 9 weeks, tenascin expression is nearly restricted to the muscle connective tissue and myotendinous structures. These results are discussed in view of the possible multiple morphogenetic roles of tenascin in tissue regeneration and repair.

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.

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.

The urodele amphibians are nearly the only adult vertebrates able to regenerate their missing or amputated tail. The most striking feature of this model lies in the ability of the spinal cord (SC) to differentiate, within the regenerating tail, a new ependymal tube from which the SC and the peripheral nervous system originate. A fundamental question is whether, in response to tail excision, the ependymoglia of the old SC stump behaves as an embryonic neuroepithelium. To evaluate this possibility, cell lines from primary cell cultures of adult SC were established for the first time in newts, and two cell clones, immunochemically characterized as ependymoglial cell populations, could be obtained. To analyze the potentialities of these clonal cells, after transplantation in tail regenerates, cell-marking experiments, using either in vitro transfection with lacZ gene or the lineage tracer lysinated rhodamine dextran (LRD), were performed. One to 2 weeks postimplantation, most of labeled derivatives were identified as melanocytes. Interestingly, labeled cells were also seen integrated in the ependymoglia of the regenerating SC. Two to 6 weeks after implantation in young regenerates, we also observed LRD-labeled elongated cells close to nerves or myofibers which were unambiguously identified as Schwann cells by galactocerebroside staining. Taken together, these findings showed that clonal cells derived from adult newt SC cultures could largely find, in regenerate mesenchyme, suitable environmental conditions to differentiate into melanocytes or Schwann cells. Because these two cells types arise from neural crest cells during embryo-genesis, this supports the interesting view that multipotent cells are still present in the SC of adult urodeles.

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.

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.

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.

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.

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.

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)n and the paired repeat (CAXCCX)n. 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.

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.

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.

Striated Ciliary Roots (SCRs), about 3 μm long, are attached to the basal bodies of branchial crown ciliated epithelial cells ofOwenia. These SCRs appear to consist of 5–7-nm diameter filaments organized in a cross-striation pattern with an apparent variable periodicity of 50 to 80 nm. The most exciting observation emerging from this study is the constant and conspicuous close spatial relationship between SCRs and fairly well developed Golgi apparatus. By enhancing contrast and preservation of cell components, the OsFeCN postfixation-staining of material prefixed in glutaraldehyde in the presence of calcium has revealed some fine-structural details within the SCR-Golgi Association. By means of the calcium precipitation method, with antimonate or oxalate in conjunction with X-ray microanalysis, we have identified calcium within SCR dark bands and SCR-associated Golgi bodies. The ability to bind calcium makes the Golgi apparatus a likely candidate for Ca2+ regulation of putative contraction of the SCRs and/or ciliary motility. The slight period variability measured in the SCRs and cytochemical localization of Mg2+, Ca2+-dependent ATPase activities associated with cross striations support the view that theOwenia SCRs may be contractile organelles.

Morphological and immunocytochemical studies were used to determine the correlations among cytoskeleton, junction complex, and basement membrane reorganization during anterior regeneration in Owenia fusiformis. Electron microscopical observations showed that, at the point of amputation, in conjunction with the disorganization of the extracellular matrix, alterations in the adhesive junctions caused dramatic changes in the distribution of intermediary filaments. Fluorescence microscopy investigations with fluorescein – phalloidin and antibodies to actin and Owenia tropomyosin visualized a basal actin network in epidermal cells, which was reorganized during regeneration. Two or three days after amputation, this cortical mat was not observed in blastema where the basal membrane was lacking. It was still incompletely reassembled after 4 or 5 days, i.e., when the thin reforming basal membrane, which stained with anti type IV collagen antibody, did not yet display collagen fibrils. In the epidermis of 6- to 7-day-old regenerates, the basal membrane, intercellular junctions, and actin and intermediary filament cytoskeletons exhibited nearly normal structural features. Use of antibody to Owenia paramyosin showed that the reorganization of the muscles during regeneration was concomitant with actin cytoskeletal reconstruction, as appeared after antitropomyosin labeling. Immunofluorescence findings were checked and (or) specified by immunogold staining.

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.

Experiments on 14C-valine uptake by Phoronis psammophila Cori revealed polyphasic kinetics of uptake at external concentrations higher than 1.5 μM. This uptake displays seasonal variations, with a maximum in summer. In winter, increases in uptake are induced by preincubation in high amounts of amino-acids in the external medium; such increases are, however, inhibited by the action of cycloheximide.

We previously demonstrated, during the prereplicative phase of traumatic regeneration, an increase of transcription of poly(A) + -RNA which was correlated to variations in the accessibility of DNA in the chromatin to different probes. In this paper, we study the variations at the chromatin-associated protein level. In chromatin extracted from normal and from 12 h regenerating animals, quantitative and qualitative changes (modifications of the electrophoretic pattern and phosphorylation) were found in non-histone proteins extractable by low ionic concentration (probably ribonucleoproteins).

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.

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.

Resume L'hybridation des RNA, marques par la [ 3 H]-uridine ou le [ 32 P], sur filtres poly U selon la technique de Sheldon et al. (1972). extraits au cours de la phase prereplicative des processus de regeneration, chez Owenia fusiformis, montre une augmentation de la population d'RNA poly A + dans la fraction totale et dans la fraction cyloplasmique. Ce resultat confirme qu'un des premiers evenements biochimiques de la regeneration consiste en une augmentation des facultes de transcription du materiel genetique concernant en partie la synthese de RNA messagers.

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.