A new approach of urodele amphibian limb regeneration: study of myosin isoforms and their control by thyroid hormone.

In P. waltlii, an urodele amphibian species which undergoes spontaneous metamorphosis, study of native myosin in pyrophosphate gels at various stages of normal development demonstrates a complete larval to fast myosin isoforms transition, which occurs more precociously in forelimb muscles than in the dorsal and ventral muscles. In the neotenic species A. mexicanum, forelimb muscles development also presents a complete myosin isoforms transition which is in contrast with the partial myosin isoforms transition observed in the dorsal muscle. In metamorphosed or neotenic animals of both species aged 1 year, forelimb regeneration is characterized by a complete transition from larval to fast myosin isoforms, that occurs earlier and more rapidly than in normal forelimb development. When forelimb regeneration is studied in P. waltlii aged 4 years, the adult fast and slow isomyosins are expressed very early in the regeneration process. In experimental hypothyroidian P. waltlii, the larval to fast isoforms transition in regenerating forelimb muscles is slightly delayed. Experimental hyperthyroidism accelerates the disappearance of larval isomyosins in regenerating forelimb muscles, both in P. waltlii and A. mexicanum aged 1 year. This work demonstrates that changes in myosin isoform pattern during forelimb regeneration in adult urodele amphibians are different from changes occurring in the normal forelimb development. They take place without any thyroid hormone influence, as opposed to normal development, and appear to be age-dependent.

Expression of the proteoglycan SPOCK during mouse embryo development.

SPOCK is a modular proteoglycan, with homology with proteins involved in cell adhesion processes and neurogenesis. We have previously shown that SPOCK transcripts predominate in the adult mouse brain. Here, we report its expression during mouse embryonic development by in situ hybridization, and immunocytochemistry. SPOCK is actively expressed at the onset of neurogenesis during periods of neuron migration and axonal outgrowth. At a later developmental stage, its expression is particularly prevalent within developing synaptic fields. In the peripheral nervous system, SPOCK expression is also developmentally regulated particularly in dorsal root ganglion neurons.

Morphological and biochemical analysis of regeneration after cardiotoxin injury of Xenopus laevis fast muscles.

The anterior brachial muscle of Xenopus laevis forelimb was characterized as a fast-type muscle composed of type II fibers exclusively. Larval and adult muscles showed three distinct isomyosins composed by two different heavy chains, HCl and HCf, respectively, associated with the same fast light chains. Muscle regeneration was examined after degeneration of the myofibers by injection of cardiotoxin, a snake toxin. 24 h after the injury no myofibers and no myosin were detected. New myosins of larval and adult fast types started to be synthesized two weeks after the injury, during a stage of proliferation of mononucleated cells. 1 month after the injury, the regenerated muscles which showed structural differences with the normal muscle contained only fast isomyosins. The precocious larval to fast heavy chain transition observed in regenerating muscles of the adult X. laevis without any thyroid hormone influence shows that the myogenic program in adult muscle regeneration is regulated by factors that are different from those regulating normal development.

Pituitary-thyroid axis controls the final differentiation of the dorsal skeletal muscle in urodelan amphibians.

A histoenzymological study of the ATPase activity of myosin in the dorsal axis muscle (dorsalis trunci) was carried out on two species of urodelan amphibians: Pleurodeles waltlii, a euthyroid species with spontaneous metamorphosis and Ambystoma mexicanum, a neotenic hypothyroid species. P. waltlii and A. mexicanum underwent an operation after which cytological analysis of the remaining pituitary were carried out in parallel. The muscle phenotype of urodelan amphibians varies according to the thyroid status of the species. In euthyroid adults, IIA fibers are dominant whereas in hypothyroid adults, IIC fibers are dominant. The number of type IIB (fast) and type I fibers (slow) are similar in both species. Physiological or experimental modulation of the concentration of circulating thyroid hormones results in a modification of the muscle fiber type profile pertaining to the considered species. We found that pituitary (TSH) plays a dominant role in the maturation of type IIC fibers in both species. Moreover, it seems to modulate the development of IIA fibers in P. waltlii and that of IIB fibers in A. mexicanum. Its action is thus species specific. Through partial or total hypophysectomy experiments, we have been able to demonstrate the influence of the hypophysothyroidian axis on the appearance of the adult muscle phenotype during metamorphosis.

The emergence of an adult muscle phenotype in urodelan amphibians: an immunohistochemical study.

Electrophoretic techniques adapted for the analysis of muscles of lower invertebrates reveal four myosin heavy chain isoforms in the dorsalis trunci of Pleurodeles waltlii: two fast (MHC-IIA, MHC-IIB), and one slow (MHC-I) in the adult and one isoform (MHC-La) in the larvae. Polyclonal antibodies were prepared against the larval (anti-MHC-La) and one of the fast myosin (MHC-IIA) isoforms and their specificity was confirmed by western blot analysis. An immunohistochemical analysis was then carried out on frozen sections of the dorsalis trunci of P. waltlii at different stages of development. From stage 44 it was possible to demonstrate the presence of MHC-IIA in the small diameter fibers at the periphery of the muscle; the number and diameter of these fibers increased from stage 44 to stage 56 when anatomical metamorphosis had finished. By stage 56 these fibers could also be readily identified using standard histochemical techniques as type IIA fibers. We conclude that fast IIA myosin is expressed well before the final adult muscle phenotype has been established and its expression is therefore independent of thyroid hormone.

Cloning and expression of a thyroid hormone receptor alpha 1 in the perennibranchiate amphibian Proteus anguinus.

We demonstrated the presence of thyroid hormone receptor alpha mRNAs in tissues of the perennibranchiate amphibian Proteus anguinus, which is insensitive to thyroid hormone. From P. anguinus muscle we cloned and sequenced the 3' coding and untranslated region of a cDNA corresponding to a thyroid hormone receptor alpha 1. Using cDNA-PCR and in situ hybridization, we showed a tissue-specific expression of thyroid hormone receptor alpha genes, which was not upregulated by thyroid hormone as opposed to that observed in the TH-sensitive species, Xenopus laevis.

Myosin structure and thyroidian control of myosin synthesis in urodelan amphibian skeletal muscle.

Electrophoretic analysis in non-dissociating conditions reveals three types of myosin in adult urodelan amphibian skeletal muscles: 3 isoforms of fast myosin (FM), one isoform of intermediate myosin (IM) and one or two isoforms of slow myosin (SM). Each type is characterized by a specific heavy chain HCf (FM), HCi (IM) and HCs (SM), respectively. In all urodelan species, as in mammals, fast isomyosins associate HCf and the three fast light chains LC1f, LC2f, and LC3f. In most urodelan species the intermediate myosin contains LC1f and LC2f and can be considered as an homodimer of the alkali LC1f. However, in Euproctus asper, IM is characterized by the association of both slow and fast LC with HCi. Slow myosin is a hybrid molecule associating HCs with slow and fast LC. During metamorphosis, a myosin isoenzymic transition occurs consisting in the replacement of three larval myosins (LM) characterized by a specific heavy chain (HCI), by the adult isomyosins with lower electrophoretic mobilities. At the same time there is a change in the ATPase myofibrillar pattern, with the larval fiber types being replaced by adult fibers of types I, IIA and IIB. In the neotenic and perennibranchiate species, which do not undergo spontaneous metamorphosis, sexually mature larval animals present a change in the myosin isoenzymic profile, but no complete transition. The coexistence of larval and adult isomyosins and the persistence of transitional fibers of type IIC in the skeletal muscle are demonstrated. Experimental hypo- and hyperthyroidism indicate that thyroid hormone stimulates the regression of the larval isomyosins, possibly through indirect pathways. In contrast, the appearance and the persistence of the adult isomyosins seem to be independent of thyroid hormone. Thus, the control of the isoenzymic transition in the skeletal muscle of urodelan amphibians appears to imply indirect mechanisms, operating differently on each of the two phases of the complete transition.

Myosin structure in the eel (Anguilla anguilla L.). Demonstration of three heavy chains in adult lateral muscle.

Myosin extracts from central white fibers and peripheral red fibers of the lateral muscle of eel (Anguilla anguilla) were analysed by electrophoresis under non-dissociating conditions, which demonstrated a polymorphism of myosin isoforms. The light and heavy subunit content of the isomyosins was established using SDS-PAGE and two-dimensional electrophoresis. In the central white muscle, 3 myosin isoforms FM3, FM2, FM1, were characterized by 3 types of fast light chain and one fast heavy chain HCf; the existence of a fourth isomyosin is discussed. In the peripheral red muscle, two myosin isoforms were found, SM1 and SM2, each characterized by a specific heavy chain, HCs1 or HCs2, and containing the same slow light chain content. This work demonstrates for the first time the existence of 3 heavy chains in the skeletal muscle of a fish.

Expression and neural control of myogenic regulatory factor genes during regeneration of mouse soleus.

Given the importance of the myogenic regulatory factors (MRFs) for myoblast differentiation during development, the aims of this work were to clarify the spatial and temporal expression pattern of the four MRF mRNAs during soleus regeneration in mouse after cardiotoxin injury, using in situ hybridization, and to investigate the influence of innervation on the expression of each MRF during a complete degeneration/regeneration process. For this, we performed cardiotoxin injury-induced regeneration experiments on denervated soleus muscle. Myf-5, MyoD, and MRF4 mRNAs were detected in satellite cell-derived myoblasts in the first stages of muscle regeneration analyzed (2--3 days P-I). The Myf-5 transcript level dramatically decreased in young multinucleated myotubes, whereas MyoD and MRF4 transcripts were expressed persistently throughout the regeneration process. Myogenin mRNA was transiently expressed in forming myotubes. These results are discussed with regard to the potential relationships between MyoD and MRF4 in the satellite cell differentiation pathway. Muscle denervation precociously (at 8 days P-I) upregulated both the Myf-5 and the MRF4 mRNA levels, whereas the increase of both MyoD and myogenin mRNA levels was observed later, in the late stages of regeneration (30 days P-I). This significant accumulation of each differentially upregulated MRF during soleus regeneration after denervation suggests that each myogenic factor might have a distinct role in the regulatory control of muscle gene expression. This role is discussed in relation to the expression of the nerve-regulated genes, such as the nAChR subunit gene family. (J Histochem Cytochem 49:887-899, 2001)

Long-term denervation modulates differentially the accumulation of myogenin and MRF4 mRNA in adult Xenopus muscle.

In adult Xenopus laevis, we analyzed, using reverse transcriptase-polymerase chain reaction (RT-PCR) and in situ hybridization, the influence of long-term muscle denervation on the accumulation of MRF4 and myogenin transcripts. The brachial muscle was denervated by cutting the brachial nerve and was examined after 4 months. MRF4 mRNA levels decreased about two-fold in denervated muscle as compared with contralateral muscle. Myogenin mRNA levels, by contrast, were induced about five-fold by denervation. This report shows that muscle denervation persistently reduces the levels of MRF4 transcripts suggesting that MRF4 expression may be induced by innervation and hence may be involved in mediating transcriptional responses to innervation. The up-regulation of myogenin by denervation suggests that myogenin expression may compensate for the down-regulation of MRF4 gene.

Myosin isoforms and their light chains from the ventricular muscle of the urodelan amphibian Pleurodeles waltlii: comparison with myosin from skeletal muscles.

Myosin extracted from ventricular muscle of the urodelan amphibian Pleurodeles waltlii was analyzed in comparison with myosin extracted from skeletal muscles by native, one-dimensional SDS gel electrophoresis and two-dimensional gel electrophoresis. Two myosin isoforms were detected in ventricular muscle using pyrophosphate gel electrophoresis. These isomyosins contained two types of light chain subunits, LC1v and LC2v. Two-dimensional gel electrophoresis showed that LC1v comigrated with the slow light chain LC1s, whereas LC2v was characterized by a specific mobility, distinct from LC2s and LC2f. Diaphragm muscle was characterized by the coexistence of larval and adult myosin isoforms.

[High-performance society and doping]. / Société de la performance et dopage.

Doping is not limited to high-level athletes. Likewise it is not limited to the field of sports activities. The doping phenomenon observed in sports actually reveals an underlying question concerning the notion of sports itself, and more widely, the society's conception of sports. In a high-performance society, which is also a high-risk society, doping behavior is observed in a large number of persons who may or may not participate in sports activities. The motivation is the search for individual success or profit. The fight against doping must therefore focus on individual responsibility and prevention in order to preserve athlete's health and maintain the ethical and educational value of sports activities.

Myosin isoenzymes and their subunits in urodelan amphibian fast skeletal muscle. Coexistence of larval and adult heavy chains in neotenic individuals.

The distributions of native myosin isoforms were examined by electrophoresis under non-dissociating conditions, in the fast twitch dorsal skeletal muscle of young larvae, neotenic adults and metamorphosed adults of urodelan amphibians. Both heavy and light chains of myosin isoenzymes were analysed. In pyrophosphate acrylamide gel electrophoresis three isoenzymes were demonstrated in larval myosin; other isoforms of lower electrophoretic mobility were observed in metamorphosed adults myosin. Larval and adult isoenzymes were shown to coexist in myosin from neotenic adults. Analysis of heavy chains in denaturing conditions and proteolytic digestion revealed the sequential occurrence during development of two types of heavy chains, one larval and one adult, that coexist in the myosin of neotenic adults only. Analysis of light chain patterns under denaturing conditions revealed the existence of three fast light chains which displayed no modification during the course of development. The neotenic urodelan amphibian species model represents actually the only model in which the coexistence of larval (or neonatal) and adult heavy chains is maintained throughout life in adults.

[Transplantation into the blastocoel of Pleurodeles waltlii Michah., of blastomers isolated from blocked blastulae obtained from interspecific nuclear transplants between Ambystoma mexicanum Shaw. and Ambystoma dumerilii Dugès (urodele amphibians)]. / Transplantation intrablastocoelienne chez Pleurodeles waltlii Michah., de blastomères isolés de blastulas bloquées, obtenues par greffe nucléaire interspécifique entre Ambystoma mexicanum Shaw. et Ambystoma dumerilii Dugès (amphibiens urodèles)

Ectodermic blastomeres from arrested nucleocytoplasmic blastulae obtained by nuclear graft between Ambystoma mexicanum (A. m.) and Ambystoma dumerilii (A. d.), are transplanted in the blastocoele of recipient Pleurodeles waltlii blastulae. Histo-autoradiographic analysis shows that hybrid cells have cellular affinities and multiplication capacities very different from normal Ambystoma mexicanum blastulae isolated cells. The possibility that a revitalisation of lethal blastomeres can occur should not be retained.

Analysis of MyoD, myogenin, and muscle-specific gene mRNAs in regenerating Xenopus skeletal muscle.

We have analyzed in adult Xenopus laevis, using in situ hybridization, the spatial and temporal expression patterns of MyoD, myogenin, and alpha-skeletal actin and fast myosin heavy chain mRNAs during muscle regeneration following cardiotoxin injury. MyoD transcripts could be detected in the satellite cells as early as the first stage of regeneration and were expressed persistently throughout the regeneration process. Myogenin mRNAs were transiently expressed in forming myotubes. alpha-Skeletal actin and fast myosin heavy chain mRNAs were detected precociously, before the young myotube stage. This work has shown, for the first time, the presence of myogenin transcripts during Xenopus myogenesis.

[Quantitative and qualitative analysis of the effects of various doses of beta-irradiation (50-5000 rads) on epidermal cells of embryos and larvae of Pleurodeles waltlii Mich. (Amphibia, Urodela)]. / Analyse qualitative et quantitative des effets de differentes doses d'irradiation beta (50-5 000 rad) sur les cellules epidermioues d'embryons et de larves de Pleurodeles waltlii Mich. (amphibiens-urodeles).

Embryos of Pleurodeles waltlii at the hatching stage were irradiated with doses of 50 to 5 000 rad. From 70 to 500 rad chromosomal aberrations appear; they are studied respectively 24,48 hours and 3 weeks after the treatment. Breakages are observed, that may be followed by rearrangements, i.e. acentric, telocentric and dicentric fragments, chromatid translocations and chromosome translocations. With time, the cells showing the most severe abnormalities are eliminated by the developing larvae. From 1 000 rad cytoplasmic structures (membrane systems and mitochondria) are alterated .

Expression of myogenic regulatory factors during muscle development of Xenopus: myogenin mRNA accumulation is limited strictly to secondary myogenesis.

To clarify the acquisition of the adult muscle pattern in Xenopus laevis, in situ hybridization and reverse transcriptase-polymerase chain reaction were used to correlate the time course of gene expression for myogenic regulatory factors (Myf-5, MyoD, and myogenin) with the expression of contractile protein (myosin heavy chain; MHC) genes during hindlimb formation compared with their expression in dorsal body muscles. After the precocious expression of Myf-5 and MyoD mRNA in limb bud (stage 50), myogenin mRNA strongly accumulated later at paddle stages (stages 52/53) concomitantly with the accumulation of both the larval and the adult MHC mRNAs. In dorsal body muscles, as early as stage 52, myogenin transcripts accumulated in a few small, secondary myofibers expressing the adult MHC mRNA that were located along the dorsomedial edge, but they were never detected in the large, primary myofibers of the body expressing the larval MHC mRNA. During metamorphosis, the areas expressing both the adult MHC and the myogenin transcripts gradually expanded from the dorsomedial edge to the ventral side of the dorsal body muscles, accounting for the progression of the secondary "adult" myogenesis described previously (Nishikawa and Hayashi [1994] Dev. Biol. 165:86-94). This work shows that, in Xenopus, the accumulation of myogenin mRNA is restricted to secondary myogenesis, including the formation of new muscles in developing limbs as well as in dorsal muscles during body remodeling. This shows that myogenin is not required for primary myogenesis, and it suggests a crucial role for myogenin in the terminal differentiation program, including myoblast fusion and the activation of adult-type muscle genes.

[Expression of hepatitis B virus (HBV) genome in the oocytes of two species of amphibians]. / Expression du génome du virus de l'hépatite B (HBV) dans les ovocytes de deux espèces d'amphibiens.

Xenopus laevis and Pleurodeles waltlii oocyte nuclei are injected with a recombinant plasmid pCP 10, carrying two hepatitis B virus (HBV) genomes in a tandem arrangement. Transcription occurs in oocytes of both species and production of viral RNAs is demonstrated; better quantitative results are obtained with P. waltlii oocytes. Two viral proteins are detected: Hepatitis B surface antigen (Ag HBs) and Hepatitis B core antigen (Ag HBc). These proteins can be shown only in oocyte homogenates, and not in the incubation media. Another viral marker (Ag HBe) was not detected.

Changes in calmodulin level after fertilization and during first cleavage in the egg of the urodelan amphibian Pleurodeles waltlii.

We report three significant calmodulin rises related to Pleurodeles waltlii egg fertilization and following developmental events. These elevations are correlated to the major obvious Ca2+-dependent events: Na+-H+ exchange, activation of NAD kinase, triggering of cortical reaction, resumption of meiotic division II, initiation of DNA synthesis and regulation of cell division. Therefore, it is suggested that alterations in calmodulin level in fertilized egg may be part of the Ca2+-dependent regulatory mechanisms which turn on metabolisms, initiate development and govern cell cleavages.

Partial purification of the maturation-promoting factor MPF from unfertilized eggs of Xenopus laevis.

A 200-fold purification of the maturation-promoting factor or MPF from unfertilized eggs of Xenopus laevis is reported for the first time. Purification was achieved by three successive column chromatographies on hydroxyapatite, trisacryl blue and L-arginine-agarose. The presence of MPF was assessed by the usual maturation criteria after injections of test material into immature stage VI unstimulated X. laevis oocytes: the precocious appearance of the maturation spot (within 45-120 min), the germinal vesicle breakdown, the presence of the first polar body and the second metaphase spindle. Purification was monitored by the decrease of the minimal amount of protein injected in a constant volume (50 nl) required to induce 50% frequency of germinal vesicle breakdown. This amount decreased from 500 ng in the crude extract to 2.5 ng in the 200-fold purified material. Analysis by SDS-PAGE of the crude extract showed about 40 Coomassie-blue-stained polypeptides with molecular masses ranging from 300 kDa to 20 kDa, whereas in the 200-fold purified MPF only 5 stained polypeptides were revealed, with molecular masses of 62, 53, 49, 39 and 37 kDa. In vitro phosphorylations for the detection of kinase activities for endogenous and exogenous substrates were monitored by analysis of autoradiograms of SDS-PAGE, after treatment of fractions with [gamma-32P]ATP. Only inactive fractions eluted from columns ahead of MPF, and fractions containing MPF activity were tested. Phosphorylation of numerous stained polypeptides was demonstrated in the crude MPF extract and exogenous substrates such as phosvitin, casein and histone type II-AS were also strongly phosphorylated. In the MPF fraction, purified on hydroxyapatite, a polypeptide of 53 kDa was more highly and specifically phosphorylated and the presence of kinase activities was observed for the above three exogenous substrates. In the 100-fold and 200-fold purified MPF, phosphorylation of endogenous substrates could not be shown and kinase activities for the above three substrates were drastically decreased as compared with the crude and purified MPF obtained after hydroxyapatite column chromatography. However, neither endogenous phosphorylations nor kinase activities with the above exogenous substrates could be shown in inactive fractions eluted ahead of MPF at the different purification steps. Some characteristics of the purified material are also described in this paper.