Expression of the homeotic gene mab-5 during Caenorhabditis elegans embryogenesis.

mab-5 is a member of a complex of homeobox-containing genes evolutionarily related to the Antennapedia and bithorax complexes of Drosophila melanogaster. Like the homeotic genes in Drosophila, mab-5 is required in a particular region along the anterior-posterior body axis, and acts during postembryonic development to give cells in this region their characteristic identities. We have used a mab-5-lacZ fusion integrated into the C. elegans genome to study the posterior-specific expression of mab-5 during embryogenesis. The mab-5-lacZ fusion was expressed in the posterior of the embryo by 180 minutes after the first cleavage, indicating that the mechanisms responsible for the position-specific expression of mab-5-lacZ act at a relatively early stage of embryogenesis. In embryos homozygous for mutations in the par genes, which disrupt segregation of factors during early cleavages, expression of mab-5-lacZ was no longer localized to the posterior. This suggests that posterior-specific expression of mab-5 depends on the appropriate segregation of developmental factors during early embryogenesis. After extrusion of any blastomere of the four-cell embryo, descendants of the remaining three cells could still express the mab-5-lacZ fusion. In these partial embryos, however, the fusion was often expressed in cells scattered throughout the embryo, suggesting that cell-cell interactions and/or proper positioning of early blastomeres are required for mab-5 expression to be localized to the posterior.     

Molecular characterization of two homologs of the Caenorhabditis elegans cadmium-responsive gene cdr-1: cdr-4 and cdr-6

A novel cadmium-inducible gene, cdr-1, was previously identified and characterized in the nematode Caenorhabditis elegans and found to mediate resistance to cadmium toxicity. Subsequently, six homologs of cdr-1 were identified in C. elegans. Here, we describe two homologs: cdr-4, which is metal inducible, and cdr-6, which is noninducible. Both cdr-4 and cdr-6 mRNAs contain open reading frames of 831 nt and encode predicted 32-kDa integral membrane proteins, which are similar to CDR-1. cdr-4 expression is induced by arsenic, cadmium, mercury, and zinc exposure as well as by hypotonic stress. In contrast, cdr-6 is constitutively expressed at a high level in C. elegans, and expression is not affected by these stressors. Both cdr-4 and cdr-6 are transcribed in postembryonic pharyngeal and intestinal cells in C. elegans. In addition, cdr-4 is transcribed in developing embryos. Like CDR-1, CDR-4 is targeted to intestinal cell lysosomes in vivo. Inhibition of CDR-4 and/or CDR-6 expression does not render C. elegans more susceptible to cadmium toxicity; however, there is a significant decrease in their lifespan in the absence of metal. Although nematodes in which CDR-4 and/or CDR-6 expression is knocked down accumulate fluid in the pseudocoelomic space, exposure to hypertonic conditions did not significantly affect growth or reproduction in these nematodes. These results suggest that CDR expression is required for optimal viability but does not function in osmoregulation.     

Characterization of Caenorhabditis elegans homologs of the Down syndrome candidate gene DYRK1A

The pathology of trisomy 21/Down syndrome includes cognitive and memory deficits. Increased expression of the dual-specificity protein kinase DYRK1A kinase (DYRK1A) appears to play a significant role in the neuropathology of Down syndrome. To shed light on the cellular role of DYRK1A and related genes we identified three DYRK/minibrain-like genes in the genome sequence of Caenorhabditis elegans, termed mbk-1, mbk-2, and hpk-1. We found these genes to be widely expressed and to localize to distinct subcellular compartments. We isolated deletion alleles in all three genes and show that loss of mbk-1, the gene most closely related to DYRK1A, causes no obvious defects, while another gene, mbk-2, is essential for viability. The overexpression of DYRK1A in Down syndrome led us to examine the effects of overexpression of its C. elegans ortholog mbk-1. We found that animals containing additional copies of the mbk-1 gene display behavioral defects in chemotaxis toward volatile chemoattractants and that the extent of these defects correlates with mbk-1 gene dosage. Using tissue-specific and inducible promoters, we show that additional copies of mbk-1 can impair olfaction cell-autonomously in mature, fully differentiated neurons and that this impairment is reversible. Our results suggest that increased gene dosage of human DYRK1A in trisomy 21 may disrupt the function of fully differentiated neurons and that this disruption is reversible.     

Distinct developmental function of two Caenorhabditis elegans homologs of the cohesin subunit Scc1/Rad21

Cohesin, which mediates sister chromatid cohesion, is composed of four subunits, named Scc1/Rad21, Scc3, Smc1, and Smc3 in yeast. Caenorhabditis elegans has a single homolog for each of Scc3, Smc1, and Smc3, but as many as four for Scc1/Rad21 (COH-1, SCC-1/COH-2, COH-3, and REC-8). Except for REC-8 required for meiosis, function of these C. elegans proteins remains largely unknown. Herein, we examined their possible involvement in mitosis and development. Embryos depleted of the homolog of either Scc3, or Smc1, or Smc3 by RNA interference revealed a defect in mitotic chromosome segregation but not in chromosome condensation and cytokinesis. Depletion of SCC-1/COH-2 caused similar phenotypes. SCC-1/COH-2 was present in cells destined to divide. It localized to chromosomes in a cell cycle-dependent manner. Worms depleted of COH-1 arrested at either the late embryonic or the larval stage, with no indication of mitotic dysfunction. COH-1 associated chromosomes throughout the cell cycle in all somatic cells undergoing late embryogenesis or larval development. Thus, SCC-1/COH-2 and the homologs of Scc3, Smc1, and Smc3 facilitate mitotic chromosome segregation during the development, presumably by forming a cohesin complex, whereas COH-1 seems to play a role important for development but unrelated to mitosis.     

Physiological roles of STIM1 and Orai1 homologs and CRAC channels in the genetic model organism Caenorhabditis elegans

The nematode Caenorhabditis elegans provides numerous experimental advantages for developing an integrative molecular understanding of physiological processes and has proven to be a valuable model for characterizing Ca(2+) signaling mechanisms. This review will focus on the role of Ca(2+) release activated Ca(2+) (CRAC) channel activity in function of the worm gonad and intestine. Inositol 1,4,5-trisphosphate (IP(3))-dependent oscillatory Ca(2+) signaling regulates contractile activity of the gonad and rhythmic posterior body wall muscle contraction (pBoc) required for ovulation and defecation, respectively. The C. elegans genome contains a single homolog of both STIM1 and Orai1, proteins required for CRAC channel function in mammalian and Drosophila cells. C. elegans STIM-1 and ORAI-1 are coexpressed in the worm gonad and intestine and give rise to robust CRAC channel activity when coexpressed in HEK293 cells. STIM-1 or ORAI-1 knockdown causes complete sterility demonstrating that the genes are essential components of gonad Ca(2+) signaling. Knockdown of either protein dramatically inhibits intestinal cell CRAC channel activity, but surprisingly has no effect on pBoc, intestinal Ca(2+) oscillations or intestinal ER Ca(2+) store homeostasis. CRAC channels thus do not play obligate roles in all IP(3)-dependent signaling processes in C. elegans. Instead, we suggest that CRAC channels carry out highly specialized and cell specific signaling roles and that they may function as a failsafe mechanism to prevent Ca(2+) store depletion under pathophysiological and stress conditions.     

SNPs in the neural cell adhesion molecule 1 gene (NCAM1) may be associated with human neural tube defects

Neural tube defects (NTDs) are common birth defects, occurring in approximately 1/1,000 births; both genetic and environmental factors are implicated. To date, no major genetic risk factors have been identified. Throughout development, cell adhesion molecules are strongly implicated in cell–cell interactions, and may play a role in the formation and closure of the neural tube. To evaluate the role of neural cell adhesion molecule 1 (NCAM1) in risk of human NTDs, we screened for novel single-nucleotide polymorphisms (SNPs) within the gene. Eleven SNPs across NCAM1 were genotyped using TaqMan. We utilized a family-based approach to evaluate evidence for association and/or linkage disequilibrium. We evaluated American Caucasian simplex lumbosacral myelomeningocele families (n=132 families) using the family based association test (FBAT) and the pedigree disequilibrium test (PDT). Association analysis revealed a significant association between risk for NTDs and intronic SNP rs2298526 using both the FBAT test (P=0.0018) and the PDT (P=0.0025). Using the HBAT version of the FBAT to look for haplotype association, all pairwise comparisons with SNP rs2298526 were also significant. A replication study set, consisting of 72 additional families showed no significant association; however, the overall trend for overtransmission of the less common allele of SNP rs2298526 remained significant in the combined sample set. In addition, we analyzed the expression pattern of the NCAM1 protein in human embryos, and while NCAM1 is not expressed within the neural tube at the time of closure, it is expressed in the surrounding and later in differentiated neurons of the CNS. These results suggest variations in NCAM1 may influence risk for human NTDs.Other members of NTD Collaborative Group involved in this study are listed in the appendix     

Molecular analysis of mutations in the Caenorhabditis elegans collagen gene dpy-7.

Collagens are a family of proteins contributing to the body structure of eukaryotes. They are encoded by a large and diverse gene family in the nematode Caenorhabditis elegans but by only a few genes in vertebrates. We have studied mutant alleles of the C. elegans dpy-7 gene, one of a large group of genes whose mutant phenotype is altered body form and several of which have previously been shown to encode cuticular collagens. We made use of the C. elegans physical map to screen specifically for collagen genes in the region of the X chromosome to which dpy-7 maps. This yielded a wild-type collagen gene clone which we showed, by micro-injection, could repair the dpy-7 mutant phenotype in transgenic animals. We cloned the homologous sequence from four dpy-7 mutant strains and by sequence analysis identified a single mutation in each case. All four mutations result in the substitution of a glycine with a larger residue in the conserved Gly-X-Y collagen domains. Similar substitutions in vertebrate collagens cause the heritable brittle bone disorder osteogenesis imperfecta. Whereas the human mutations are dominant, the dpy-7 mutations are recessive, and this may reflect different levels of complexity of collagenous macromolecular structures in the two organisms.     

Molecular basis of loss-of-function mutations in the glp-1 gene of Caenorhabditis elegans.

The glp-1 gene encodes a membrane protein required for inductive cell interactions during development of the nematode Caenorhabditis elegans. Here we report the molecular characterization of 15 loss-of-function (lf) mutations of glp-1. Two nonsense mutations appear to eliminate glp-1 activity; both truncate the glp-1 protein in its extracellular domain and have a strong loss-of-function phenotype. Twelve missense mutations and one in-frame deletion map to sites within the repeated motifs of the glp-1 protein (10 epidermal growth factor [EGF]-like and 3 LNG repeats extracellularly and 6 cdc10/SWI6, or ankyrin, repeats intracellularly). We find that all three types of repeated motifs are critical to glp-1 function, and two individual EGF-like repeats may have distinct functions. Intriguingly, all four missense mutations in one phenotypic class map to the N-terminal EGF-like repeats and all six missense mutations in a second phenotypic class reside in the intracellular cdc10/SWI6 repeats. These two clusters of mutations may identify functional domains within the glp-1 protein.     

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.     

Illuminating neural circuits and behaviour in Caenorhabditis elegans with optogenetics

The development of optogenetics, a family of methods for using light to control neural activity via light-sensitive proteins, has provided a powerful new set of tools for neurobiology. These techniques have been particularly fruitful for dissecting neural circuits and behaviour in the compact and transparent roundworm Caenorhabditis elegans. Researchers have used optogenetic reagents to manipulate numerous excitable cell types in the worm, from sensory neurons, to interneurons, to motor neurons and muscles. Here, we show how optogenetics applied to this transparent roundworm has contributed to our understanding of neural circuits.     

Quantitative genetic analysis of life-history traits of Caenorhabditis elegans in stressful environments

In Figure 1 of [Harvey et al (Evolutionary Biology 2008, 8:15)] the plotted data were inverted. The correct Figure is shown below. The text and statistical analyses in [Harvey et al (Evolutionary Biology 2008, 8:15)] are correct.     

Molecular and genetic characterization of osmosensing and signal transduction in the nematode Caenorhabditis elegans

Osmotic homeostasis is a fundamental requirement for life. In general, the effector mechanisms that mediate cellular and extracellular osmoregulation in animals are reasonably well defined. However, at the molecular level, little is known about how animals detect osmotic and ionic perturbations and transduce them into regulatory responses. The nematode Caenorhabditis elegans provides numerous powerful experimental advantages for defining the genes and integrated gene networks that underlie basic biological processes. These advantages include a fully sequenced and well-annotated genome, forward and reverse genetic and molecular tractability, and a relatively simple anatomy. C. elegans normally inhabits soil environments where it is exposed to repeated osmotic stress. In the laboratory, nematodes readily acclimate to and recover from extremes of hypertonicity. We review recent progress in defining the molecular mechanisms that underlie osmosensing and associated signal transduction in C. elegans. Some of these mechanisms are now known to be highly conserved. Therefore, studies of osmosensing in nematodes have provided, and will undoubtedly continue to provide, new insights into similar processes in more complex organisms including mammals.     

Large-scale analysis of gene function in Caenorhabditis elegans by high-throughput RNAi

Genome-wide analysis of gene function is essential for the post-genome era, and development of efficient and economical technology suitable for it has been in demand. Here we report a large-scale inactivation of the expressed genes in the nematode Caenorhabditis elegans. For this purpose, we have established a high-throughput "RNAi-by-soaking" methodology by modifying the conventional RNAi method [1, 2]. A set of tag-sequenced, nonredundant cDNAs corresponding to approximately 10,000 genes [3] (representing half of the predicted genes [4]) was used for the systematic RNAi analysis. We have processed approximately 2500 genes to date. In development, 27% of them showed detectable phenotypes, such as embryonic lethality, post-embryonic lethality, sterility, and morphological abnormality. Of these, we analyzed the phenotypes of F1 sterility in detail, and we have identified 24 genes that might play important roles in germline development. Combined with the ongoing analysis of expression patterns of these cDNAs [3, 5], the functional information obtained in this work will provide a starting point for the further analysis of each gene. Another finding from this screening is that the incidence of essential genes is significantly lower in the X chromosome than in the autosomes.     

Molecular cloning and characterization of the dpy-20 gene of Caenorhabditis elegans

We describe the molecular analysis of the dpy20 gene in Caenorhabditis elegans. Isolation of genomic sequences was facilitated by the availability of a mutation that resulted from insertion of a Tc1 transposable element into the dpy-20 gene. The Tc1 insertion site in the m474:: Tc1 allele was identified and was found to lie within the coding region of dpy-20. Three revertants (two wild-type and one partial revertant) resulted from the excision of this Tc1 element. Genomic dpy-20 clones were isolated from a library of wild-type DNA and were found to lie just to the left of the unc-22 locus on the physical map, compatible with the position of dpy-20 on the genetic map. Cosmid DNA containing the dpy-20 gene was successfully used to rescue the mutant phenotype of animals homozygous for another dpy-20 allele, e1282ts. Sequence analysis of the putative dpy-20 homologue in Caenorhabditis briggsae was performed to confirm identification of the coding regions of the C. elegans gene and to identify conserved regulatory regions. Sequence analysis of dpy-20 revealed that it was not similar to other genes encoding known cuticle components such as collagen or cuticulin. The dpy-20 gene product, therefore, identifies a previously unknown type of protein that may be directly or indirectly involved in cuticle function. Northern blot analysis showed that dpy-20 is expressed predominantly in the second larval stage and that the mRNA is not at all abundant. Data from temperature shift studies using the temperature-sensitive allele e1282ts showed that the sensitive period also occurs at approximately the second larval stage. Therefore, expression of dpy-20 mRNA and function of the DPY-20 protein are closely linked temporally.     

Molecular genetic analysis of the human sorbitol dehydrogenase gene

The polyol pathway comprises the enzymes aldose reductase and sorbitol dehydrogenase, which convert glucose to sorbitol and sorbitol to fructose, respectively, particularly in hyperglycemic states. The accumulation and toxicity of sorbitol in specific tissues has been implicated in the development of microvascular problems in some diabetic patients. Inappropriate sorbitol accumulation in some patients may be the result of polymorphic variation in the human sorbitol dehydrogenase gene, causing reduced expression levels or enzymatic activity. We now describe the structure and expression profile of the human sorbitol dehydrogenase gene and identify a range of polymorphic variants that may be useful for co-segregation studies in diabetic patients with and without severe clinical complications from their disease.     

The Caenorhabditis elegans autosomal dominant polycystic kidney disease gene homologs lov-1 and pkd-2 act in the same pathway.

Autosomal dominant polycystic kidney disease (ADPKD) strikes 1 in 1000 individuals and often results in end-stage renal failure. Mutations in either PKD1 or PKD2 account for 95% of all cases [1-3]. It has recently been demonstrated that polycystin-1 and polycystin-2 (encoded by PKD1 and PKD2, respectively) assemble to form a cation channel in vitro [4]. Here we determine that the Caenorhabditis elegans PKD1 and PKD2 homologs, lov-1 [5] and pkd-2, act in the same pathway in vivo. Mutations in either lov-1 or pkd-2 result in identical male sensory behavioral defects. Also, pkd-2;lov-1 double mutants are no more severe than either of the single mutants, indicating that lov-1 and pkd-2 act together. LOV-1::GFP and PKD-2::GFP are expressed in the same male-specific sensory neurons and are concentrated in cilia and cell bodies. Cytoplasmic, nonnuclear staining in cell bodies is punctate, suggesting that one pool of PKD-2 is localized to intracellular membranes while another is found in sensory cilia. In contrast to defects in the C. elegans autosomal recessive PKD gene osm-5 [6-8], the cilia of lov-1 and pkd-2 single mutants and of lov-1;pkd-2 double mutants are normal as judged by electron microscopy, demonstrating that lov-1 and pkd-2 are not required for ultrastructural development of male-specific sensory cilia.