Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.     

Caenorhabditis elegans dna-2 is involved in DNA repair and is essential for germ-line development

Caenorhabditis elegans germ cell proliferation and development were severely damaged in second generation dna-2 homozygotes. Even in the first generation, a much higher incidence of aberrant chromosomes in oocytes and resultantly higher embryonic lethality were found vs. wild type, when DNA breaks were induced by gamma-rays or camptothecin. The deficiency of dna-2 in combination with RNA interference on mre-11 gene expression synergistically aggravated germ-line development, especially oocyte formation. These results suggest that C. elegans Dna-2 is involved in a DNA repair pathway paralleling homologous recombination or non-homologous end joining with mre-11 participation.     

Abundance,Distribution, and Mutation Rates of Homopolymeric Nucleotide Runs in the Genome of Caenorhabditis elegans

Homopolymeric nucleotide runs, also called mononucleotide microsatellites, are a ubiquitous, dominant, and mutagenic feature of eukaryotic genomes. A clear understanding of the forces that shape patterns of homopolymer evolution, however, is lacking. We provide a focused investigation of the abundance, chromosomal distribution, and mutation spectra of the four strand-specific homopolymer types (A, T, G, C) 8 bp in the genome of Caenorhabditis elegans. A and T homopolymers vastly outnumber G and C HPs, and the run-length distributions of A and T homopolymers differ significantly from G and C homopolymers. A scanning window analysis of homopolymer chromosomal distribution reveals distinct clusters of homopolymer density in autosome arms that are regions of high recombination in C. elegans. Dramatic biases are detected among closely spaced homopolymers; for instance, we observe 994 A homopolymers immediately followed by a T homopolymer (5 to 3) and only 8 instances of T homopolymers directly followed by an A homopolymer. Empirical homopolymer mutation assays in a set of C. elegans mutation-accumulation lines reveal an 20-fold higher mutation rate for G and C homopolymers compared to A and T homopolymers. Nuclear A and T homopolymers are also found to mutate 100-fold more slowly than mitochondrial A and T homopolymers. This integrative approach yields a total nuclear genome-wide homopolymer mutation rate estimate of 1.6 mutations per genome per generation.Novel sequences are deposited in GenBank under accession numbers AY219759–AY219789.     

PYP-1, inorganic pyrophosphatase, is required for larval development and intestinal function in C. elegans

Inorganic pyrophosphatase (PPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) into phosphate (Pi), which provides a thermodynamic driving force for important biosynthetic reactions. The nematode Caenorhabditis elegans gene C47E12.4 encodes a PPase (PYP-1) which shows 54% amino acid identity with human PPase. PYP-1 exhibits specific enzyme activity and is mainly expressed in the intestinal and nervous system. A null mutant of pyp-1 reveals a developmental arrest at early larval stages and exhibits gross defects in intestinal morphology and function. The larval arrest phenotype was successfully rescued by reintroduction of the pyp-1 gene, suggesting that PYP-1 is required for larval development and intestinal function in C. elegans.     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.     

Tcl transposition and mutator activity in a Bristol strain of Caenorhabditis elegans

Summary In most strains of Caenorhabditis elegans with a low copy number of Tc1 transposable elements, germline transposition is rare or undetectable. We have observed low-level Tel transposition in the genome of the C. elegans var. Bristol strain KR579 (unc-13[e51]) resulting in an increase in Tc1 copy number and subsequent mutator activity. Examination of genomic blots from KR579 and KR579derived strains revealed that more Tc1-hybridizing bands were present than in other Bristol strains. A novel Tc1-hybridizing fragment was cloned from a KR579-derived strain. Unique sequence DNA flanking the Tc1 element identified a 1.6 kb restriction fragment length difference between the KR579 and N2 strains consistent with a Tc1 insertion at a new genomic site. The site of insertion of this Tel was sequenced and is similar to the published Tel insertion site consensus sequence. Several isolates of KR579 were established and maintained on plates for a period of 3 years in order to determine if Tc1 copy number would continue to increase. In one isolate, KR1787, a further increase in Tc1 copy number was observed. Examination of the KR1787 strain has shown that it also exhibits mutator activity as assayed by the spontaneous mutation frequency at the unc-22 (twitcher) locus. The KR579 strain differs from most low copy number strains in that it exhibits low-level transposition which has developed into mutator activity.     

Functional constraint and divergence in the G protein family in <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> and <Emphasis Type="Italic">Caenorhabditis briggsae</Emphasis>

Part of the challenge of the post-genomic world is to identify functional elements within the wide array of information generated by genome sequencing. Although cross-species comparisons and investigation of rates of sequence divergence are an efficient approach, the relationship between sequence divergence and functional conservation is not clear. Here, we use a comparative approach to examine questions of evolutionary rates and conserved function within the guanine nucleotide-binding protein (G protein) gene family in nematodes of the genus Caenorhabditis. In particular, we show that, in cases where the Caenorhabditis elegans ortholog shows a loss-of-function phenotype, G protein genes of C. elegans and Caenorhabditis briggsae diverge on average three times more slowly than G protein genes that do not exhibit any phenotype when mutated in C. elegans, suggesting that genes with loss of function phenotypes are subject to stronger selective constraints in relation to their function in both species. Our results also indicate that selection is as strong on G proteins involved in environmental perception as it is on those controlling other important processes. Finally, using phylogenetic footprinting, we identify a conserved non-coding motif present in multiple copies in the genomes of four species of Caenorhabditis. The presence of this motif in the same intron in the gpa-1 genes of C. elegans, C. briggsae and Caenorhabditis remanei suggests that it plays a role in the regulation of gpa-1, as well as other loci.Electronic Supplementary Material Supplementary material is available for this article at     

The thioredoxin TRX-1 regulates adult lifespan extension induced by dietary restriction in Caenorhabditis elegans

Dietary restriction (DR) is the only environmental intervention known to extend adult lifespan in a wide variety of animal models. However, the genetic and cellular events that mediate the anti-aging programs induced by DR remain elusive. Here, we used the nematode Caenorhabditis elegans to provide the first in vivo evidence that a thioredoxin (TRX-1) regulates adult lifespan extension induced by DR. We found that deletion of the gene trx-1 completely suppressed the lifespan extension caused by mutation of eat-2, a genetic surrogate of DR in the worm. However, trx-1 deletion only partially suppressed the long lifespan caused by mutation of the insulin-like receptor gene daf-2 or by mutation of the sensory cilia gene osm-5. A trx-1::GFP translational fusion expressed from its own promoter in ASJ neurons (Ptrx-1::trx-1::GFP) rescued the trx-1 deletion-mediated suppression of the lifespan extension caused by mutation of eat-2. This rescue was not observed when trx-1::GFP was expressed from the ges-1 promoter in the intestine. In addition, overexpression of Ptrx-1::trx-1::GFP extended lifespan in wild type, but not in eat-2 mutants. trx-1 deletion almost completely suppressed the lifespan extension induced by dietary deprivation (DD), a non-genetic, nutrient-based model of DR in the worm. Moreover, DD upregulated the expression of a trx-1 promoter-driven GFP reporter gene (Ptrx-1::GFP) in ASJ neurons of aging adults, but not that of control Pgpa-9::GFP (which is also expressed in ASJ neurons). We propose that DR activates TRX-1 in ASJ neurons during aging, which in turn triggers TRX-1-dependent mechanisms to extend adult lifespan in the worm.     

SER-1, a Caenorhabditis elegans 5-HT2-like receptor, and a multi-PDZ domain containing protein (MPZ-1) interact in vulval muscle to facilitate serotonin-stimulated egg-laying

Serotonin (5-HT) stimulation of egg-laying in Caenorhabditis elegans is abolished in ser-1 (ok345) animals and is rescued by ser-1 expression in vulval muscle. A PDZ binding motif (ETFL) at the SER-1 C-terminus is not essential for rescue, but facilitates SER-1 signaling. SER-1 binds specifically to PDZ domain 10 of the multi-PDZ domain protein, MPZ-1, based on GST pulldown and co-immunoprecipitation. mpz-1 is expressed in about 60 neurons and body wall and vulval muscles. In neurons, GFP-tagged MPZ-1 is punctate and colocalizes with the synaptic marker, synaptobrevin. The expression patterns of ser-1 and mpz-1 overlap in 3 pairs of neurons and vulval muscle. In addition, MPZ-1 also interacts with other GPCRs with acidic amino acids in the -3 position of their PDZ binding motifs. mpz-1 RNAi reduces 5-HT stimulated egg-laying in wild type animals and in ser-1 mutants rescued by muscle expression of SER-1. In contrast, mpz-1 RNAi has no effect on 5-HT stimulated egg-laying in ser-1 mutants rescued by expression of a truncated SER-1 that lacks the C-terminal PDZ binding motif. The overexpression of MPZ-1 PDZ domain 10 also inhibits 5-HT stimulated egg-laying. These studies suggest that the SER-1/MPZ-1 interaction facilitates SER-1 mediated signaling.     

Caenorhabditis elegans DAZ-1 is expressed in proliferating germ cells and directs proper nuclear organization and cytoplasmic core formation during oogenesis

The deleted in azoospermia (DAZ) family genes encode potential RNA-binding proteins that are expressed exclusively in germ cells in a wide range of metazoans. We have previously shown that mutations in daz-1, the only DAZ family gene in Caenorhabditis elegans, cause pachytene stage arrest of female germ cells but do not affect spermatogenesis. In this study, we report that DAZ-1 protein is most abundantly expressed in proliferating female germ cells, in a manner independent of the GLP-1 signaling pathway. DAZ-1 is dispensable in males but it is expressed also in male mitotic germ cells. Detailed phenotypic analyses with fluorescence microscopy and transmission electron microscopy have revealed that loss of daz-1 function causes multiple abnormalities as early as the onset of meiotic prophase, which include aberrant chromatin structure, small nucleoli, absence of the cytoplasmic core, and precocious cellularization. Although the reduced size of nucleoli is indicative of a low translational activity in these cells, artificial repression of general translation in the germline does not phenocopy the daz-1 mutant. Thus, we propose that DAZ-1 in C. elegans plays essential roles in female premeiotic and early meiotic germ cells, probably via regulating the translational activity of specific target genes required for the progression of oogenesis.     

Total lactate dehydrogenase activity of tail muscle is not cold-adapted in nocturnal lizards from cool-temperate habitats

The dependence of metabolic processes on temperature constrains the behavior, physiology and ecology of many ectothermic animals. The evolution of nocturnality in lizards, especially in temperate regions, requires adaptations for activity at low temperatures when optimal body temperatures are unlikely to be obtained. We examined whether nocturnal lizards have cold-adapted lactate dehydrogenase (LDH). LDH was chosen as a representative metabolic enzyme. We measured LDH activity of tail muscle in six lizard species (n = 123: three nocturnal, two diurnal and one crepuscular) between 5 and 35 °C and found no differences in LDH-specific activity or thermal sensitivity among the species. Similarly, the specific activity and thermal sensitivity of LDH were similar between skinks and geckos. Similar enzyme activities among nocturnal and diurnal lizards indicate that there is no selection of temperature specific LDH enzyme activity at any temperature. As many nocturnal lizards actively thermoregulate during the day, LDH may be adapted for a broad range of temperatures rather than adapted specifically for the low temperatures encountered when the animals are active. The total activity of LDH in tropical and temperate lizards is not cold-adapted. More data are required on biochemical adaptations and whole animal thermal preferences before trends can be established.     

Requirements of Kettin, a giant muscle protein highly conserved in overall structure in evolution, for normal muscle function, viability, and flight activity of Drosophila

Kettin is a giant muscle protein originally identified in insect flight muscle Z-discs. Here, we determined the entire nucleotide sequence of Drosophila melanogaster kettin, deduced the amino acid sequence of its protein product (540 kD) along with that of the Caenorhabditis elegans counterpart, and found that the overall primary structure of Kettin has been highly conserved in evolution. The main body of Drosophila Kettin consists of 35 immunoglobulin C2 domains separated by spacers. The central two thirds of spacers are constant in length and share in common two conserved motifs, putative actin binding sites. Neither fibronectin type III nor kinase domains were found. Kettin is present at the Z-disc in several muscle types. Genetic analysis showed that kettin is essential for the formation and maintenance of normal sarcomere structure of muscles and muscle tendons. Accordingly, embryos lacking kettin activity cannot hatch nor can adult flies heterozygous for the kettin mutation fly.     

A novel zinc-carboxypeptidase SURO-1 regulates cuticle formation and body morphogenesis in Caenorhabditis elegans

Cuticle formation and molting are critical for the development of Caenorhabditis elegans. To understand cuticle formation more clearly, we screened for suppressors in transgenic worms that expressed dominant ROL-6 collagen proteins. The suro-1 mutant, which is mild dumpy, exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in three suro-1 mutants, and found that suro-1 (ORF R11A5.7) encodes a putative zinc-carboxypeptidase homologue. The expression of this enzyme in the hypodermis and the genetic interactions between this enzyme and other collagen-modifying enzyme mutants suggest a regulatory role in collagen processing and cuticle organization for this novel carboxypeptidase. These findings aid our understanding of cuticle formation during worm development.     

The hedgehog-related gene wrt-5 is essential for hypodermal development in Caenorhabditis elegans

The Caenorhabditis elegans genome encodes a series of hedgehog-related genes, which are thought to have evolved and diverged from an ancestral Hh gene. They are classified into several families based on their N-terminal domains. Here, we analyze the expression and function of a member of the warthog gene family, wrt-5, that lacks the Hint/Hog domain. wrt-5 is expressed in seam cells, the pharynx, pharyngeal-intestinal valve cells, neurons, neuronal support cells, the excretory cell, and the reproductive system. WRT-5 protein is secreted into the extracellular space during embryogenesis. Furthermore, during larval development, WRT-5 protein is secreted into the pharyngeal lumen and the pharyngeal expression changes in a cyclical manner in phase with the molting cycle. Deletion mutations in wrt-5 cause embryonic lethality, which are temperature sensitive and more severe at 15 degrees C than at 25 degrees C. Animals that hatch exhibit variable abnormal morphology, for example, bagging worms, blistering, molting defects, or Roller phenotypes. We examined hypodermal cell junctions using the AJM-1Colon, two colonsGFP marker in the wrt-5 mutant background and observed cell boundary abnormalities in the arrested embryos. AJM-1Colon, two colonsGFP protein is also misplaced in pharyngeal muscle cells in the absence of WRT-5. In conclusion, we show that wrt-5 is an essential gene that - despite its lack of a Hint domain - has multiple functions in C. elegans and is implicated in cell shape integrity.