A novel glycine receptor alpha Z1 subunit variant in the zebrafish brain.

Alpha subunits of the inhibitory glycine receptor (GlyR) display genetic heterogeneity in mammals and zebrafish. This diversity is increased in mammals by the alternative splicing mechanism. We report here in zebrafish, the characterization of a new alphaZ1 subunit likely arising from alphaZ1 gene by an alternative splice process (alphaZ1L). This novel cDNA possesses 45 supplementary nucleotides at the putative exon2/exon3 boundary. The corresponding protein contains 15 additional amino acids in the NH2-terminal domain. Heterologous expression of homomeric GlyRalphaZ1L in human embryonic kidney-293 cells generates glycine-gated strychnine-sensitive chloride channels with no obvious discrepancy with pharmacological properties of GlyRalphaZ1. Moreover, zinc modulation of glycine-induced currents is identical in alphaZ1 and alphaZ1L glycine receptors. During ontogenesis, simultaneous alphaZ1 and alphaZ1L mRNA synthesis have been observed. Embryonic and adult alphaZ1 and alphaZ1L mRNA expressions are restricted to the CNS. Embryonic alphaZ1L mRNA anatomical pattern of expression is, however, highly restrained and strictly limited to the rostral part of the brain revealing a highly regionalized function of alphaZ1L in the CNS. This report contributes to the characterization of the diversity of glycine receptor isoforms in zebrafish and emphasizes the common mechanism used among vertebrates for creating GlyR variety and specificity.

Phylogenetic relationships and chromosomal location of five distinct glycine receptor subunit genes in the teleost Danio rerio.

Glycine receptors mediating synaptic inhibition are heteromeric proteins constituted of alpha and beta subunits. The mammalian GlyR subunits constitute a subgroup in the superfamily of ligand-gated ionic channels. To compare the evolutionary events in the mammalian and teleostean lineages for the receptor family, we first undertook systematic cloning of the constitutive subunits of the zebrafish glycine receptor. The isolation of two alpha subunits (alphaZ1 and alphaZ2) and one beta subunit (betaZ) has been reported previously and we report here the characterization of two novel alpha subunits, alphaZ3 and alphaZ4, increasing the known zebrafish subunits number to four alpha and one beta. Establishment of phylogenetic relationships reveals that alphaZ1, alphaZ3 and betaZeta are orthologous to mammalian alpha1, alpha3 and beta subunits. However, two zebrafish GlyRalpha subunit genes are orthologous to the unique avian and mammalian alpha4 subunit revealing a duplication of the alpha4 gene in zebrafish. Whole-mount in situ hybridization in 24-hours post fertilization (hpf) and 52-hpf embryos of the daughter gene products display very different expression patterns indicating distinct functions of the duplicated genes. Gene mapping reveals that the two duplicated genes are localized on two different linkage groups (LG5 and LG22) as would be daughter genes resulting from a large-scale duplication of the ancestral genome. Finally, we report that a linked pair of genes on human chromosome 4 (alpha3 and beta) is also linked on linkage group 1 in zebrafish (alphaZ3 and betaZ) as a consequence of a mosaic conserved syntheny.

Isolation and characterization of an alpha 2-type zebrafish glycine receptor subunit.

The complementary DNA for a novel alpha subunit of the glycine receptor, alphaZ2, was isolated from a zebrafish adult brain library. The molecular characteristics, phylogenetic relationships and messenger RNA length of this alphaZ2 subunit show it to be an alpha2-type glycine receptor subunit isoform. The leader peptide however, diverges from those of known glycine receptor alpha isoforms. Recombinantly expressed in Xenopus oocytes, alphaZ2 formed functional glycine receptor channels. These homomeric channels were activated by glycine and taurine, with apparent affinities similar to those reported for zebrafish alphaZ1 glycine receptor, and were also effectively antagonized by nanomolar concentrations of strychnine. However, during prolonged applications of agonists, ionic currents of alphaZ2 receptor channels declined to a much lower steady-state level than those of alphaZ1, indicating different desensitization properties. Analysis of messenger RNA revealed that alphaZ2 is specifically expressed in adult brain tissue and present in both adult and embryonic zebrafish. This report contributes to the characterization of the diversity of glycine receptor isoforms in vertebrates.

Regional distribution of glycine receptor messenger RNA in the central nervous system of zebrafish.

We report the cloning of the zebrafish beta subunit of the glycine receptor and compare the anatomical distribution of three glycine receptor subunit constituents in adult zebrafish brain (alphaZ1, alphaZ2 and betaZ) to the expression pattern of homologous receptor subunits (alpha1, alpha2 and beta) in the mammalian adult CNS. Non-radioactive hybridization was used to map the distribution of the alphaZ1, alphaZ2 and betaZ glycine receptor subunit messenger RNAs in the adult zebrafish brain. The anterior-posterior expression gradient found in adult zebrafish brain was similar to that reported in mammalian CNS. However, the glycine receptor transcripts, notably the alphaZ1 subunit, were more widely distributed in the anterior regions of the zebrafish than in the adult mammalian brain. The isoform-specific distribution pattern was less regionalized in zebrafish than in the rat mammalian CNS. Nevertheless, there was some regionalization of alphaZ1, alphaZ2 and betaZ transcripts in the diencephalic and mesencephalic nuclei where different sensory and motor centers express either alphaZ1/betaZ or alphaZ2 subunits. In contrast to the widespread distribution of the beta subunit in adult mammalian brain, alphaZ2 messenger RNA presented the widest expression territory of all three glycine receptor subunits tested. alphaZ2 messenger RNA was expressed in the absence of alphaZ1 and betaZ messenger RNA in the outer nuclear layer of the retina, the inferior olive and the raphe of the medulla oblongata, as well as in the nucleus of Cajal of the medulla spinalis. In contrast, an identified central neuron of the reticular formation, the Mauthner cell, expresses all three glycine receptor subunits (alphaZ1, alphaZ2 and betaZ). This report extends the already described glycine receptor expression in the vertebrate CNS and confirms the importance of glycine-mediated inhibition in spinal cord and brainstem.

Cloning, expression and electrophysiological characterization of glycine receptor alpha subunit from zebrafish.

The glycine receptor is a ligand-gated anion channel protein, providing inhibitory drive within the nervous system. We report here the isolation and functional characterization of a novel alpha subunit (alphaZ1) of the glycine receptor from adult zebrafish (Danio rerio) brain. The predicted amino acid sequence is 86%, 81% and 77% identical to mammalian isoforms alpha1, alpha3 and alpha2, respectively. AlphaZ1 exhibits many of the molecular features of mammalian alpha1, but the sequence patterns in the M4 and C-terminal domains are more similar to alpha2/alpha3. Phylogenetic analysis indicates that alphaZ1 is more closely related to the mammalian alpha1 subunits, being positioned, however, on a distinct branch. The alphaZ1 messenger RNA is 9.5 kb, similar to that described previously for alpha1 messenger RNAs. When expressed in Xenopus oocytes or a human cell line (BOSC 23), alphaZ1 forms a homomeric receptor which is activated by glycine and antagonized by strychnine. This receptor demonstrates unexpectedly high sensitivity to taurine and can also be activated by GABA. These results are consistent with physiological findings in lamprey and goldfish, and they suggest that this teleost fish glycine receptor displays a lower selectivity to neurotransmitters than that reported for glycine mammalian receptors.

Cytokeratin 8 is a suitable epidermal marker during zebrafish development.

We have cloned and characterized the zebrafish (Danio rerio) homologous cytokeratin 8 (zf-K8) cDNA. This cytokeratin belongs to the gene family of intermediate filaments and it is a component of the cytoskeleton of epithelial cells. Gene expression analysis during embryonic development and at adult stages presented here revealed that zf-K8 mRNA is inherited maternally and that it is present in the oocyte, the zygote and in the cleavage stage embryo. After mid blastula transition this gene is expressed in all surface cells, notably in those of the enveloping layer (EVL) and of the periderm, as well as in a subpopulation of the deep cells (DEL) presumed to be intestinal progenitors. During later embryonic stage zf-K8 mRNA is strongly expressed in the developing pectoral fin. In adult zebrafish, the zf-K8 gene is not only expressed in simple epithelia such as the colorectal intestine, but also, in contrast to other vertebrates, it is present in stratified skin and differentiated fins. These observations suggest that the zf-K8 gene is an appropriate epidermal marker during zebrafish ontogenesis.

Expression of myosin heavy chain and of myogenic regulatory factor genes in fast or slow rabbit muscle satellite cell cultures.

We investigated the myogenic properties of rabbit fast or slow muscle satellite cells during their differentiation in culture, with a particular attention to the expression of myosin heavy chain and myogenic regulatory factor genes. Satellite cells were isolated from Semimembranosus proprius (slow-twitch muscle; 100% type I fibres) and Semimembranosus accessorius (fast-twitch muscle; almost 100% type II fibres) muscles of 3-month-old rabbits. Satellite cells in culture possess different behaviours according to their origin. Cells isolated from slow muscle proliferate faster, fuse earlier into more numerous myotubes and mature more rapidly into striated contractile fibres than do cells isolated from fast muscle. This pattern of proliferation and differentiation is also seen in the expression of myogenic regulatory factor genes. Myf5 is detected in both fast or slow 6-day-old cell cultures, when satellite cells are in the exponential stage of proliferation. MyoD and myogenin are subsequently detected in slow satellite cell cultures, but their expression in fast cell cultures is delayed by 2 and 4 days respectively. MRF4 is detected in both types of cultures when they contain striated and contractile myofibres. Muscle-specific myosin heavy chains are expressed earlier in slow satellite cell cultures. No adult myosin heavy chain isoforms are detected in fast cell cultures for 13 days, whereas cultures from slow cells express neonatal, adult slow and adult fast myosin heavy chain isoforms at that time. In both fast and slow satellite cell cultures containing striated contractile fibres, neonatal and adult myosin heavy chain isoforms are coexpressed. However, cultures made from satellite cells derived from slow muscles express the slow myosin heavy chain isoform, in addition to the neonatal and the fast isoforms. These results are further supported by the expression of the mRNA encoding the adult myosin heavy chain isoforms. These data provide further evidence for the existence of satellite cell diversity between two rabbit muscles of different fibre-type composition, and also suggest the existence of differently preprogrammed satellite cells.

Modifications of gene expression in myotonic murine skeletal muscle are associated with abnormal expression of myogenic regulatory factors.

The mouse mutants ADR ("arrested development of righting") and the allelic CRP ("cramp") are characterized by a myotonic phenotype resulting from a dysfunction of the skeletal muscle chloride channel which leads to myotonic trains of actions potentials in response to stimuli. Compared to normal mouse muscle, numerous biochemical modifications have been found in the ADR muscle, and changes are observed in the expression of certain isoforms of contractile proteins. We have therefore measured the levels of the mRNA transcripts encoding the myosin heavy chain isoforms (MyHC) in both mutants. Transcripts for the myogenic regulatory factors were also studied since they are known to play a role in the induction of muscle-specific gene transcription, and their own expression is modified by different electrical activity patterns. In both mutants, the mRNA encoding the IIB MyHC was considerably decreased. In contrast, the mRNAs for the IIA, IIX, and beta/slow MyHCs were increased. The mRNA for the neonatal MyHC mRNA was not detectable, and therefore fiber regeneration does not appear to play a role in these phenomena. Among the myogenic regulatory factors, herculin is the most abundant in adult muscle; however, herculin mRNA undergoes a large decrease in myotonic muscle which does not seem to be related to the changing fiber type. The levels of MyoD and myogenin mRNAs are also modified with the former decreasing and the latter increasing. Qualitatively similar changes are seen in the ADR and CRP mutants; however, they are generally less pronounced in CRP. These observations suggest that specific myogenic factors may be linked to the expression of individual MyHC genes and that abnormal expression of some of the factors may be associated with myotonic muscle pathology.

Modulation of embryonic and muscle-specific enolase gene products in the developing mouse hindlimb.

During striated muscle development, the glycolytic enzyme enolase (EC 4.2.1.11) undergoes an isozymic transition, from the embryonic alpha alpha form towards the muscle-specific forms alpha beta and beta beta. The regulation of this transition was analyzed in mouse hindlimb muscles from embryonic day 15 (E15) to the adult stage. The quantitative modulations of the levels of the transcripts and subunits of alpha and beta enolase genes were determined. The absolute amounts of alpha and beta enolase mRNAs were estimated using in vitro synthesized transcripts as calibration standards, thus allowing an evaluation of their relative contribution at each stage examined. The muscle-specific beta enolase mRNA is already present at E15. Its level then increases and, from E17, this transcript becomes predominant. This accumulation is biphasic: a steep prenatal rise, corresponding to a net increase per fiber, accompanies the formation of secondary myofibers and the development of innervation; a second rise, beginning at postnatal day 5, is temporally correlated with the definitive specialization of the myofibers. Most of the decrease in alpha mRNA level occurs postnatally. No temporal or quantitative correlation between the up-regulation of beta mRNA and the down-regulation of alpha mRNA levels is observed throughout hindlimb muscle development. Quantitative immunoblotting analyses carried out in parallel show that the enolase isozymic transition is mainly controlled at the mRNA level.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium-induced and voltage-dependent inactivation of calcium channels in crab muscle fibres.

Inactivation of Ca channels was examined in crab muscle fibres using the voltage-clamp method. A satisfactory suppression of outward currents was attempted by the use of K+ blocking agents: TEA, 4AP and Cs ions instead of K+ ions applied extracellularly. The inactivation of Ca current appeared as a bi-exponential process. The faster component had a mean value of the time constant of 50 ms while the second component inactivated at a tenfold slower rate. The extent of inactivation of the faster component increased as the Ca current itself increased in different experimental conditions. Inactivation decreased when ICa was reduced for large applied depolarizations. The time constant of the faster calcium component also depended on the calcium current. Thus the results suggested that Ca2+ entry leads to inactivation of one component of calcium current in crab muscle. Substitution of Ca2+ ions by Sr2+ or Ba2+ ruled out the hypothesis concerning an accumulation process which would explain the decrease of the inward current. The second slower component of Ca current was better described by a voltage-dependent mechanism and its rate was not modified in Ca2+ rich solution or when the inward current was carried by Sr2+ or Ba2+ ions. Thus in crab muscle fibres, inactivation is mediated by both calcium entry and a voltage-gated mechanism.

Activation of skinned muscle fibers by calcium and strontium ions.

Intact and mechanically skinned skeletal muscle fibers of the crab Carcinus maenas have been used. The aim of the experiments was to determine the origin of the mechanical activity recorded in intact crab muscle fibers exhibiting an inward strontium current in strontium solution without calcium. To do so, the effect of strontium ions in inducing activation of contractile proteins and calcium release from the sarcoplasmic reticulum has been studied. The properties of the sarcoplasmic reticulum membrane towards strontium ions, i.e., the efficiency of the calcium ATPase towards strontium ions and the capability to release strontium ions have been investigated. Results show that the contractile proteins have a lower affinity for strontium than for calcium ions. However, the maximum bound strontium is identical to the maximum bound calcium. As for the sarcoplasmic reticulum, strontium ions can induce a calcium release and also can be taken up by the calcium ATPase and be released. We concluded that the mechanical activity in intact fibers bathed in a strontium medium has two origins: first, a direct and partial activation of the contractile proteins by strontium ions flowing through the calcium channel; second, a contractile proteins activation of calcium ions released by the sarcoplasmic reticulum by a "strontium-induced calcium release" mechanism.

Role of the different calcium sources in the excitation-contraction coupling in crab muscle fibers.

Excitation-contraction coupling in crab muscle fibers was studied in voltage-clamp conditions. Extracellular calcium is essential for the mechanical activity. Two calcium influxes induced by membrane depolarization contribute to tension development: one is the inward calcium current responsible for the phasic tension, the other is a calcium influx dependent on extracellular sodium and calcium concentrations and is responsible for the tonic tension. These calcium influxes are not sufficient to activate contractile proteins. Experiments with procaine and caffeine show that a calcium release from the sarcoplasmic reticulum is required.

Calcium-induced calcium release mechanism from the sarcoplasmic reticulum in skinned crab muscle fibres.

Mechanically skinned skeletal muscle fibres of the crab Carcinus maenas have been used to investigate the mechanism of calcium release from the sarcoplasmic reticulum. Calcium release has been monitored by the amplitude and kinetics of the tension developed by the fibre. Results show that a very low calcium concentration, insufficient to directly activate contractile proteins, induces a release of calcium from the SR. This release is stimulated by low concentrations of caffeine and inhibited by small amounts of EGTA. Thus, a graded calcium-induced calcium release mechanism dependent on extrareticular calcium concentration has been demonstrated in skinned crab muscle fibre.

Intramuscular calcium movements: experiments from the Soviet biosatellite Biocosmos.

Experiments have been performed in skeletal muscle fibres from the lateral head of gastrocnemius muscle of female rats. Changes in intramuscular calcium movements due to microgravity conditions have been tested by tension measurements in chemically skinned muscle fibres. Our results show that microgravity induces i) a decrease in maximal muscle strength developed by contractile proteins ii) a decrease of intensity and rate of both Ca release and Ca uptake by the sarcoplasmic reticulum.

Effects of ryanodine on the ionic currents and the calcium conductance in crab muscle fibers.

The effects of ryanodine, a neutral alkaloid, on crab muscle fibers were investigated under voltage clamp conditions using the double sucrose-gap technique. In the presence of ryanodine, contracture develops without any membrane depolarization and the Ca-conductance variables are shifted in a hyperpolarizing direction. As a result, the Ca channels are activated at the resting potential. During the action potential, the Ca channels appear to open faster and for a longer period of time. The K currents are also modified. These different effects may be interpreted by a common mechanism related to a change of the membrane electrical field induced by increased Ca activity in the cytoplasm.