Description of International Caenorhabditis elegans Experiment first flight (ICE-FIRST).

Traveling, living and working in space is now a reality. The number of people and length of time in space is increasing. With new horizons for exploration it becomes more important to fully understand and provide countermeasures to the effects of the space environment on the human body. In addition, space provides a unique laboratory to study how life and physiologic functions adapt from the cellular level to that of the entire organism. Caenorhabditis elegans is a genetic model organism used to study physiology on Earth. Here we provide a description of the rationale, design, methods, and space culture validation of the ICE-FIRST payload, which engaged C. elegans researchers from four nations. Here we also show C. elegans growth and development proceeds essentially normally in a chemically defined liquid medium on board the International Space Station (10.9 day round trip). By setting flight constraints first and bringing together established C. elegans researchers second, we were able to use minimal stowage space to successfully return a total of 53 independent samples, each containing more than a hundred individual animals, to investigators within one year of experiment concept. We believe that in the future, bringing together individuals with knowledge of flight experiment operations, flight hardware, space biology, and genetic model organisms should yield similarly successful payloads.

Muscular degeneration in the absence of dystrophin is a calcium-dependent process.

Duchenne muscular dystrophy (DMD) is a progressive degenerative muscular disease that is due to mutations in the dystrophin gene. Neither the function of dystrophin nor the physiopathology of the disease have been clearly established yet. Several groups have reported elevated calcium concentrations in the mdx mouse model of DMD, but the effect of calcium levels on the progression of the disease continues to be a matter of debate. Here, we show that, in Caenorhabditis elegans, a gain-of-function mutation in the egl-19 calcium channel gene dramatically increases muscle degeneration in dystrophin mutants. Conversely, RNAi-mediated inhibition of egl-19 function reduces muscle degeneration by half. Therefore, our results demonstrate that calcium channel activity is a critical factor in the progression of dystrophin-dependent muscle degeneration.

Molecular, genetic and physiological characterisation of dystrobrevin-like (dyb-1) mutants of Caenorhabditis elegans.

Dystrobrevins are protein components of the dystrophin complex, whose disruption leads to Duchenne muscular dystrophy and related diseases. The Caenorhabditis elegans dystrobrevin gene (dyb-1) encodes a protein 38 % identical with its mammalian counterparts. The C. elegans dystrobrevin is expressed in muscles and neurons. We characterised C. elegans dyb-1 mutants and showed that: (1) their behavioural phenotype resembles that of dystrophin (dys-1) mutants; (2) the phenotype of dyb-1 dys-1 double mutants is not different from the single ones; (3) dyb-1 mutants are more sensitive than wild-type animals to reductions of acetylcholinesterase levels and have an increased response to acetylcholine; (4) dyb-1 mutations alone do not lead to muscle degeneration, but synergistically produce a progressive myopathy when combined with a mild MyoD/hlh-1 mutation. All together, these findings further substantiate the role of dystrobrevins in cholinergic transmission and as functional partners of dystrophin.

Genetic suppression of phenotypes arising from mutations in dystrophin-related genes in Caenorhabditis elegans.

BACKGROUND: Dystrophin is the product of the gene that is mutated in Duchenne muscular dystrophy (DMD), a progressive neuromuscular disease for which no treatment is available. Mice carrying a mutation in the gene for dystrophin (mdx mice) display only a mild phenotype, but it is aggravated when combined with a mutation in the MyoD gene. The nematode worm Caenorhabditis elegans has a dystrophin homologue (dys-1), but null mutations in dys-1 do not result in muscle degeneration. RESULTS: We generated worms carrying both the dys-1 null mutation cx18, and a weak mutation, cc561ts, of the C. elegans MyoD homologue hlh-1. The double mutants displayed a time-dependent impairment of locomotion and egg laying, a phenotype not seen in the single mutants, and extensive muscle degeneration. This result allowed us to look for genes that, when misexpressed, could suppress the dys-1; hlh-1 phenotype. When overexpressed, the dyc-1 gene - whose loss-of-function phenotype resembles that of dys-1 - partially suppressed the dys-1; hlh-1 phenotype. The dyc-1 gene encodes a novel protein sharing similarities with the mammalian neural nitric oxide synthase (nNOS)-binding protein CAPON, and is expressed in the muscles of the worm. CONCLUSIONS: As a C. elegans model for dystrophin-dependent myopathy, the dys-1; hlh-1 worms should permit the identification of genes, and ultimately drugs, that would reverse the muscle degeneration in this model.

Mutations in the dystrophin-like dys-1 gene of Caenorhabditis elegans result in reduced acetylcholinesterase activity.

Mutations of the Caenorhabditis elegans dystrophin/utrophin-like dys-1 gene lead to hyperactivity and hypercontraction of the animals. In addition dys-1 mutants are hypersensitive to acetylcholine and acetylcholinesterase inhibitors. We investigated this phenotype further by assaying acetylcholinesterase activity. Total extracts from three different dys-1 alleles showed significantly less acetylcholinesterase-specific activity than wild-type controls. In addition, double mutants carrying a mutation in the dys-1 gene plus a mutation in either of the two major acetylcholinesterase genes (ace-1 and ace-2) display locomotor defects consistent with a strong reduction of acetylcholinesterases, whereas none of the single mutants does. Therefore, in C. elegans, disruption of the dystrophin/utrophin-like dys-1 gene affects acetylcholinesterase activity.

In vitro interactions of Caenorhabditis elegans dystrophin with dystrobrevin and syntrophin.

Dystrophin, the product of the gene mutated in Duchenne muscular dystrophy (DMD) is bound by its C-terminus to a protein complex including the related protein dystrobrevin. Both proteins contain a putative coiled-coil domain consisting of two alpha-helices. It has been reported that the two proteins bind to each other by the first one of the two alpha-helices. We have revisited this question using the Caenorhabditis elegans homologs of dystrophin and dystrobrevin. In vitro interaction occurs through the more conserved second helix. We propose a new model of dystrophin interactions with associated proteins.

Dystrobrevin- and dystrophin-like mutants display similar phenotypes in the nematode Caenorhabditis elegans.

Dystrophin, the protein disrupted in Duchenne muscular dystrophy, forms a transmembrane complex with dystrophin-associated proteins. Dystrobrevins, proteins showing homology to the C-terminal end of dystrophin, and whose function is unknown, are part of the dystrophin complex. We report here that, in the nematode Caenorhabditis elegans, animals carrying mutations in either the dystrophin-like gene dys-1 or the dystrobrevin-like gene dyb-1 display similar behavioral and pharmacological phenotypes consistent with an alteration of cholinergic signalling. These findings suggest that: (1) dystrobrevin and dystrophin are functionally related and (2) their disruption impairs cholinergic signalling.

Serotonin inhibition of synaptic transmission: Galpha(0) decreases the abundance of UNC-13 at release sites.

We show that serotonin inhibits synaptic transmission at C. elegans neuromuscular junctions, and we describe a signaling pathway that mediates this effect. Release of acetylcholine from motor neurons was assayed by measuring the sensitivity of intact animals to the acetylcholinesterase inhibitor aldicarb. By this assay, exogenous serotonin inhibited acetylcholine release, whereas serotonin antagonists stimulated release. The effects of serotonin on synaptic transmission were mediated by GOA-1 (a Galpha0 subunit) and DGK-1 (a diacylglycerol [DAG] kinase), both of which act in the ventral cord motor neurons. Mutants lacking goa-1 G(alpha)0 accumulated abnormally high levels of the DAG-binding protein UNC-13 at motor neuron nerve terminals, suggesting that serotonin inhibits synaptic transmission by decreasing the abundance of UNC-13 at release sites.

Mutations in the Caenorhabditis elegans dystrophin-like gene dys-1 lead to hyperactivity and suggest a link with cholinergic transmission.

Mutations in the human dystrophin gene cause Duchenne muscular dystrophy, a common neuromuscular disease leading to a progressive necrosis of muscle cells. The etiology of this necrosis has not been clearly established, and the cellular function of the dystrophin protein is still unknown. We report here the identification of a dystrophin-like gene (named dys-1) in the nematode Caenorhabditis elegans. Loss-of-function mutations of the dys-1 gene make animals hyperactive and slightly hypercontracted. Surprisingly, the dys-1 mutants have apparently normal muscle cells. Based on reporter gene analysis and heterologous promoter expression, the site of action of the dys-1 gene seems to be in muscles. A chimeric transgene in which the C-terminal end of the protein has been replaced by the human dystrophin sequence is able to partly suppress the phenotype of the dys-1 mutants, showing that both proteins share some functional similarity. Finally, the dys-1 mutants are hypersensitive to acetylcholine and to the acetylcholinesterase inhibitor aldicarb, suggesting that dys-1 mutations affect cholinergic transmission. This study provides the first functional link between the dystrophin family of proteins and cholinergic transmission.

Modulation of serotonin-controlled behaviors by Go in Caenorhabditis elegans.

Seven transmembrane receptors and their associated heterotrimeric guanine nucleotide-binding proteins (G proteins) have been proposed to play a key role in modulating the activities of neurons and muscles. The physiological function of the Caenorhabditis elegans G protein Go has been genetically characterized. Mutations in the goa-1 gene, which encodes an alpha subunit of Go (G alpha o), cause behavioral defects similar to those observed in mutants that lack the neurotransmitter serotonin (5-HT), and goa-1 mutants are partially resistant to exogenous 5-HT. Mutant animals that lack G alpha o and transgenic animals that overexpress G alpha o [goa-1(xs) animals] have reciprocal defects in locomotion, feeding, and egg laying behaviors. In normal animals, all of these behaviors are regulated by 5-HT. These results demonstrate that the level of Go activity is a critical determinant of several C. elegans behaviors and suggest that Go mediates many of the behavioral effects of 5-HT.

Dissection of the Drosophila pourquoi-pas? promoter: complex ovarian expression is driven by distinct follicle cell- and germ line-specific enhancers.

The pourquoi-pas? (pqp) gene of Drosophila melanogaster encodes a zinc finger protein present in the oocyte nucleus, the nurse cells and, at a lower level, in the follicle cells. Null mutations of the pqp gene lead to female sterility. We have undertaken a functional dissection of the pqp promoter by following the expression of the lacZ reporter gene in the ovaries of transgenic flies. pqp sequences, necessary for expression of the lacZ gene in a pattern similar to that of the endogenous pqp gene, are located between positions -210 and +30, relative to the transcription start site. These sequences, subdivided in follicle cell- and germ line-specific regions, appear to function in a direction-independent and distance-sensitive manner. The -210/-40 region, sharing stretches of sequence similarity with 5' sequences of follicle cell-specific genes, promotes lacZ expression only in the follicle cells. The -80/+30 region is germ line-specific. The promoter limits, deduced from the deletion experiments presented here, are in accordance with the molecular analysis of pqp mutants.

The Drosophila pourquoi-pas?/wings-down zinc finger protein: oocyte nucleus localization and embryonic requirement.

The pourquoi-pas? (pqp) gene of Drosophila melanogaster encodes a Cys2/His2 zinc finger protein and is abundantly transcribed in adult ovaries. During oogenesis, we immunodetected the pqp protein in the nucleus of nurse cells at stages 1-6, in a spherical structure within the oocyte nucleus at stages 7-9, and uniformly distributed in the oocyte nucleus and in nurse cell nuclei at later stages. The pqp protein is also present at a lower level in the nuclei of follicle cells, embryos, and larvae. By means of a polymerase chain reaction (PCR) screen, we recovered three independent and phenotypeless P-element insertions at the pqp locus. In a second step, two excision-induced deletions of the pqp gene were isolated after mobilization of one of these P elements. The pqp mutants display zygotic (spread and drooping wings, cross-vein defects, extra bristles) and maternal (embryonic lethality) recessive phenotypes. The chromosomal position (98EF) of the pqp gene and the drooping wing phenotype of the pqp mutants agree with the hypothesis that the pqp gene is the wings down (wdn) gene for which T.H. Morgan isolated (and lost) mutants in the 1920s. This is the first reported occurrence of a zinc finger protein in the nucleus of the Drosophila oocyte.

Spatial distribution of the Sm antigen in Drosophila early embryos.

Anti-Sm antibodies recognize the major small nuclear RNA-protein particles (snRNPs) involved in pre-mRNA processing. The spatial distribution of the snRNPs has been investigated in Drosophila embryos up to the cellularization stage (cycle 14), using the Y12 anti-Sm antibody. Our results show that: 1) all or most of the Sm antigen is localized in the cytoplasm of the syncytial blastoderm until the 12th cycle of division, in both the nuclear and cytoplasmic compartments at cycle 13, and then in the nuclei at cycle 14 and later. This relocalization takes place when zygotic transcriptional activation occurs; 2) at the subcellular level, the Sm antigen localizes in a speckled pattern and in foci-like structures within the nucleus of Drosophila blastoderm embryos; 3) strikingly, some nuclei of embryos at the 14th cycle appear to contain more snRNPs than others. The position of these nuclei differs from one embryo to another, and their distribution does not resemble any known developmental pattern of Drosophila embryogenesis. We propose that random differences in snRNP concentration may serve as an epigenetic signal for stochastic events occurring during development.

sry h-1, a new Drosophila melanogaster multifingered protein gene showing maternal and zygotic expression.

Low-stringency hybridization of the Drosophila serendipity (sry) finger-coding sequences revealed copies of homologous DNA sequences in the genomes of members of the family Drosophilidae and higher vertebrates. sry h-1, a new Drosophila finger protein-coding gene isolated on the basis of this homology, encodes a 3.2-kilobase (kb) mRNA accumulating in eggs and abundant in early embryos. The predicted sry h-1 protein product, starting at an internal initiation site of translation, is a 868-amino-acid basic polypeptide containing eight TFIIIA-like fingers encoded by three separate exons. Links separating individual fingers in the sry h-1 protein are variable in length and sequence, in contrast with the invariant H/C link found in most multi-fingered proteins. The similarity of the developmental pattern of transcription of sry h-1 with that of several other Drosophila finger protein genes suggests the existence of a complex set of such genes encoding an information which is, at least partly, maternally provided to the embryo and required for activation of gene transcription in early embryos or maintenance of gene activity during subsequent development.