Analysis of smu-1, a gene that regulates the alternative splicing of unc-52 pre-mRNA in Caenorhabditis elegans.

Mutations in the smu-1 gene of Caenorhabditis elegans were previously shown to suppress mutations in the genes mec-8 and unc-52. mec-8 encodes a putative RNA binding protein that affects the accumulation of specific alternatively spliced mRNA isoforms produced by unc-52 and other genes. unc-52 encodes a set of basement membrane proteins, homologs of mammalian perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis, and cuticle. We show that a presumptive null mutation in smu-1 suppresses nonsense mutations in exon 17 but not exon 18 of unc-52 and enhances the phenotype conferred by an unc-52 splice site mutation in intron 16. We have used reverse transcription-PCR and RNase protection to show that loss-of-function smu-1 mutations enhance accumulation in larvae of an alternatively spliced isoform that skips exon 17 but not exon 18 of unc-52. We have identified smu-1 molecularly; it encodes a nuclearly localized protein that contains five WD motifs and is ubiquitously expressed. The SMU-1 amino acid sequence is more than 60% identical to a predicted human protein of unknown function. We propose that smu-1 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and other genes.

Functional overlap between the mec-8 gene and five sym genes in Caenorhabditis elegans.

Earlier work showed that the Caenorhabditis elegans gene mec-8 encodes a regulator of alternative RNA splicing and that mec-8 null mutants have defects in sensory neurons and body muscle attachment but are generally viable and fertile. We have used a genetic screen to identify five mutations in four genes, sym-1-sym-4, that are synthetically lethal with mec-8 loss-of-function mutations. The phenotypes of sym single mutants are essentially wild type. mec-8; sym-1 embryos arrest during embryonic elongation and exhibit defects in the attachment of body muscle to extracellular cuticle. sym-1 can encode a protein containing a signal sequence and 15 contiguous leucine-rich repeats. A fusion of sym-1 and the gene for green fluorescent protein rescued the synthetic lethality of mec-8; sym-1 mutants; the fusion protein was secreted from the apical hypodermal surface of the embryo. We propose that SYM-1 helps to attach body muscle to the extracellular cuticle and that another gene that is dependent upon mec-8 for pre-mRNA processing overlaps functionally with sym-1. RNA-mediated interference experiments indicated that a close relative of sym-1 functionally overlaps both sym-1 and mec-8 in affecting muscle attachment. sym-2, sym-3, and sym-4 appear to provide additional functions that are essential in the absence of mec-8(+).

EGL-27 is similar to a metastasis-associated factor and controls cell polarity and cell migration in C. elegans.

Mutations in the C. elegans gene egl-27 cause defects in cell polarity and cell migration: the polarity of the asymmetric T cell division is disrupted and the descendants of the migratory QL neuroblast migrate incorrectly because they fail to express the Hox gene mab-5. Both of these processes are known to be controlled by Wnt pathways. Mosaic analysis indicates that egl-27 function is required in the T cell for proper cell polarity. We cloned egl-27 and discovered that a domain of the predicted EGL-27 protein has similarity to Mta1, a mammalian factor overexpressed in metastatic cells. Overlaps in the phenotypes of egl-27 and Wnt pathway mutants suggest that the EGL-27 protein interacts with Wnt signaling pathways in C. elegans.

Changing styles in C. elegans genetics.

The past 30 years have taken the nematode Caenorhabditis elegans from obscurity, as a nondescript member of a large but unglamorous invertebrate phylum, to a position as one of the major model organisms. This year, it will acquire a particular celeberity as the owner of the first animal genome to be sequenced in its entirety. In this review we consider the ways in which genetical investigations of this species have begun to change and what some of the consequences of the completion of the sequence are likely to be.

UNC-115, a conserved protein with predicted LIM and actin-binding domains, mediates axon guidance in C. elegans.

Axon guidance receptors modulate the growth cone cytoskeleton through signaling pathways that are not well understood. Here, we describe the C. elegans unc-115 gene, which encodes a candidate cytoskeletal linker protein that acts in axon guidance. unc-115 mutants have defects in a subset of axons, particularly as the affected axons change environments during outgrowth. The unc-115 gene encodes a putative actin-binding protein that is similar to the human actin-binding protein abLIM/limatin; it has a villin headpiece domain and three LIM domains that could mediate protein interactions. unc-115 is expressed in neurons during their development and is required cell-autonomously in certain neurons for normal axon guidance. We propose that UNC-115 modulates the growth cone actin cytoskeleton in response to signals received by growth cone receptors.

Analysis of osm-6, a gene that affects sensory cilium structure and sensory neuron function in Caenorhabditis elegans.

Mutation in the Caenorhabditis elegans gene osm-6 was previously shown to result in defects in the ultrastructure of sensory cilia and defects in chemosensory and mechanosensory behaviors. We have cloned osm-6 by transposon tagging and transformation rescue and have identified molecular lesions associated with five osm-6 mutations. The osm-6 gene encodes a protein that is 40% identical in amino acid sequence to a predicted mammalian protein of unknown function. We fused osm-6 with the gene for green fluorescent protein (GFP); the fusion gene rescued the osm-6 mutant phenotype and showed accumulation of GFP in ciliated sensory neurons exclusively. The OSM-6::GFP protein was localized to cytoplasm, including processes and dendritic endings where sensory cilia are situated. Mutations in other genes known to cause ciliary defects led to changes in the appearance of OSM-6::GFP in dendritic endings or, in the case of daf-19, reduced OSM-6::GFP accumulation. We conclude from an analysis of genetic mosaics that osm-6 acts cell autonomously in affecting cilium structure.

The mec-8 gene of C. elegans encodes a protein with two RNA recognition motifs and regulates alternative splicing of unc-52 transcripts.

Mutations in the mec-8 gene of Caenorhabditis elegans were previously shown to affect the functions of body wall muscle and mechanosensory and chemosensory neurons. Mutations in mec-8 also strongly enhance the mutant phenotype of specific mutations in unc-52, a gene that encodes, via alternative splicing of pre-mRNA, a set of basement membrane proteins, homologs of perlecan, that are important for body wall muscle assembly and attachment to basement membrane, hypodermis and cuticle. We have cloned mec-8 and found that it encodes a protein with two RNA recognition motifs, characteristic of RNA binding proteins. We have used reverse transcription-PCR and RNase protection experiments to show that mec-8 regulates the accumulation of a specific subset of alternatively spliced unc-52 transcripts. We have also shown with antibodies to UNC-52 that mec-8 affects the abundance of a subset of UNC-52 isoforms. We propose that mec-8 encodes a trans-acting factor that regulates the alternative splicing of the pre-mRNA of unc-52 and one or more additional genes that affect mechanosensory and chemosensory neuron function.

Touch sensation in Caenorhabditis elegans.

The nematode C. elegans exhibits a variety of responses to touch. When specific sets of mechanosensory neurons are killed with a laser, specific touch responses are abolished. Many mutations that result in defective mechanosensation have been identified. Some of the mutations define genes that specify the fate of a set of mechanoreceptors called the touch cells, which mediate response to light touch to the body of the worm. Genes specifying touch cell fate appear to regulate genes that encode touch-cell differentiation proteins, including apparent subunits of a touch-cell-specific ion channel, rare mutant forms of which lead to swelling and lysis of the touch cells. Molecular attachments of the ion channel, both to extracellular matrix components and, intracellularly, to a special large-diameter microtubule, may be required for mechanical gating of the channel. A mechanoreceptor-interneuron-motorneuron reflex circuit for response to light touch has been proposed.

The ncl-1 gene and genetic mosaics of Caenorhabditis elegans.

A ncl-1 mutation results in enlarged nucleoli, which can be detected in nearly all cells of living animals by Nomarski microscopy. Spontaneous mitotic loss of a ncl-1(+)-containing free duplication in an otherwise homozygous ncl-1 mutant animal results in mosaicism for ncl-1 expression, and the patterns of mosaicism lead us to conclude that ncl-1 acts cell autonomously. The probability of mitotic loss of the duplication sDp3 is approximately constant over many cell divisions. About 60% of the losses of sDp3 at the first embryonic cell division involve nondisjunction. Frequencies of mitotic loss of different ncl-1(+)-bearing free duplications varied over a 200-fold range. The frequencies of mitotic loss were enhanced by a chromosomal him-10 mutation. We have used ncl-1 as a cell autonomous marker in the mosaic analysis of dpy-1 and lin-37. The focus of action of dpy-1 is in hypodermis. A mutation in lin-37 combined with a mutation in another gene results in a synthetic multivulva phenotype. We show that lin-37 acts cell nonautonomously and propose that it plays a role, along with the previously studied gene lin-15, in the generation of an intercellular signal by hyp7 that represses vulval development.

The C. elegans gene lin-44, which controls the polarity of certain asymmetric cell divisions, encodes a Wnt protein and acts cell nonautonomously.

Mutations in the C. elegans gene lin-44 lead to reversals in the polarity of certain asymmetric cell divisions. We have discovered that lin-44 is a member of the Wnt family of genes, which encode secretory glycoproteins implicated in intercellular signaling. Both in situ hybridization experiments using lin-44 transcripts and experiments using reporter constructs designed to mimic patterns of lin-44 expression indicate that lin-44 is expressed in hypodermal cells at the tip of the tail and posterior to the cells with polarities affected by lin-44 mutations. Our mosaic analysis indicates that lin-44 acts cell nonautonomously. We propose that LIN-44 protein is secreted by tail hypodermal cells and affects the polarity of asymmetric cell divisions that occur more anteriorly in the tail.

Mutations affecting the chemosensory neurons of Caenorhabditis elegans.

We have identified and characterized 95 mutations that reduce or abolish dye filling of amphid and phasmid neurons and that have little effect on viability, fertility or movement. Twenty-seven mutations occurred spontaneously in strains with a high frequency of transposon insertion. Sixty-eight were isolated after treatment with EMS. All of the mutations result in defects in one or more chemosensory responses, such as chemotaxis to ammonium chloride or formation of dauer larvae under conditions of starvation and overcrowding. Seventy-five of the mutations are alleles of 12 previously defined genes, mutations which were previously shown to lead to defects in amphid ultrastructure. We have assigned 20 mutations to 13 new genes, called dyf-1 through dyf-13. We expect that the genes represented by dye-filing defective mutants are important for the differentiation of amphid and phasmid chemosensilla.

The mec-8 gene of Caenorhabditis elegans affects muscle and sensory neuron function and interacts with three other genes: unc-52, smu-1 and smu-2.

Mutations in the Caenorhabditis elegans gene mec-8 were previously shown to cause defects in mechanosensation and in the structure and dye filling of certain chemosensory neurons. Using noncomplementation screens, we have identified eight new mec-8 alleles and a deficiency that uncovers the locus. Strong mec-8 mutants exhibit an incompletely penetrant cold-sensitive embryonic and larval arrest, which we have correlated with defects in the attachment of body muscle to the hypodermis and cuticle. Mutations in mec-8 strongly enhance the mutant phenotype of unc-52(viable) mutations; double mutants exhibit an unconditional arrest and paralysis at the twofold stage of embryonic elongation, a phenotype characteristic of lethal alleles of unc-52, a gene previously shown to encode a homolog of the core protein of heparan sulfate proteoglycan, found in basement membrane, and to be involved in the anchorage of myofilament lattice to the muscle cell membrane. We have identified and characterized four extragenic recessive suppressors of a mec-8; unc-52(viable) synthetic lethality. The suppressors, which define the genes smu-1 and smu-2, can weakly suppress all mec-8 mutant phenes. They also suppress the muscular dystrophy conferred by an unc-52(viable) mutation.

Molecular and genetic analysis of unc-7, a Caenorhabditis elegans gene required for coordinated locomotion.

Mutations in the Caenorhabditis elegans gene unc-7 confer an uncoordinated phenotype. Wild-type animals trace smooth, sinuous waves as they move; unc-7 mutants make irregular bends or kinks along their bodies, particularly when they move forward. The unc-7 locus has also been implicated in the nematode's response to volatile anesthetics. We have cloned unc-7 by transposon tagging: an unc-7 mutation was correlated with the insertion of the transposon Tc1, and reversion of the mutant phenotype was correlated with loss of the Tc1 element. We have physically mapped the region flanking the sites of Tc1 insertion and identified DNA rearrangements corresponding to eight additional unc-7 alleles. Northern analysis indicates that a 2.7-kb unc-7 message is present in all developmental stages but is most abundant in L1-L3 larvae. The 5' end of the message contains a trans-spliced leader SL1. An 18-kb intron is located upstream of the predicted translational start site of the gene, and DNA breakpoints of four gamma-ray-induced alleles were located within this intron. We determined the sequence of a cDNA corresponding to the unc-7 message. The message may encode a 60-kd protein whose amino acid sequence is unrelated to any other available protein sequence; a transmembrane location for the unc-7 protein is predicted. We predict from our analysis of unc-7 genetic mosaics that the unc-7 gene product is not required in muscle cells for wild-type coordination but is probably required in motor neurons (although a hypodermal role has not been excluded). We speculate that unc-7 may be involved in the function of neuronal ion channels.

Analysis of the Caenorhabditis elegans axonal guidance and outgrowth gene unc-33.

Mutations in the unc-33 gene of the nematode Caenorhabditis elegans lead to severely uncoordinated movement, abnormalities in the guidance and outgrowth of the axons of many neurons, and a superabundance of microtubules in neuronal processes. We have cloned unc-33 by tagging the gene with the transposable element Tc4. Three unc-33 messages, which are transcribed from a genomic region of at least 10 kb, were identified and characterized. The three messages have common 3' ends and identical reading frames. The largest (3.8-kb) message consists of the 22-nucleotide trans-spliced leader SL1 and 10 exons (I-X); the intermediate-size (3.3-kb) message begins with SL1 spliced to the 5' end of exon V and includes exons V-X; and the smallest (2.8-kb) message begins within exon VII and also includes exons VIII-X. A gamma-ray-induced deletion mutation situated within exon VIII reduces the sizes of all three messages by 0.5 kb. The three putative polypeptides encoded by the three messages overlap in C-terminal sequence but differ by the positions at which their N termini begin; none has significant similarity to any other known protein. A Tc4 insertion in exon VII leads to alterations in splicing that result in three approximately wild-type-size messages: the Tc4 sequence and 28 additional nucleotides are spliced out of the two larger messages; the Tc4 sequence is trans-spliced off the smallest message such that SL1 is added 13 nucleotides upstream of the normal 5' end of the smallest message.

Limitation of the size of the vulval primordium of Caenorhabditis elegans by lin-15 expression in surrounding hypodermis.

In the nematode Caenorhabditis elegans six hypodermal cells, the vulval precursor cells, are each competent to generate vulval cells. Normally only the three nearest precursor cells to the uterine anchor cell generate the vulva (22 nuclei), while the three others fuse with the non-specialized hypodermal syncytium (hyp7) surrounding each precursor cell and covering the body. Without an inductive signal from the anchor cell, all six vulval precursor cells fuse with hyp7 and no vulva is formed. But without activity of the vulval determination gene lin-15(+), all six cells undergo vulval divisions whether the anchor cell is present or not. Using mosaic analysis, we demonstrate here that lin-15(+) expression is necessary in cells other than the vulval precursor cells or the anchor cell, most probably in the hyp7 syncytium. We propose that lin-15(+) is active in hyp7 in order to repress an intrinsic vulval program in the precursor cells. The inductive signal from the anchor cell counteracts this repression for three precursor cells, allowing them to generate vulval cells. Such a two-signal (repressor/derepressor) mechanism may operate in other cases of tissue induction.

Recombination between small X chromosome duplications and the X chromosome in Caenorhabditis elegans.

Twelve new X chromosome duplications were identified and characterized. Eight are translocated to autosomal sites near four different telomeres, and four are free. Ten include unc-1(+), which in wild type is near the left end of the X chromosome, and two of these, mnDp72(X;IV) and mnDp73(X;f), extend rightward past dpy-3. Both mnDp72 and mnDp73 recombined with the one X chromosome in males in the unc-1-dpy-3 interval at a frequency 15- to 30-fold higher than was observed for X-X recombination in hermaphrodites in the same interval. Recombinant duplications and recombinant X chromosomes were both recovered. Recombination with the X chromosome in the unc-1-dpy-3 interval was also detected for five other unc-1(+) duplications, even though their right breakpoints lie within the interval. In hermaphrodites, mnDp72 and mnDp73 promoted meiotic X nondisjunction and recombined with an X chromosome in the unc-1-dpy-3 interval at frequencies comparable to that found for X-X recombination; mnDp72(X;IV) also promoted trisomy for chromosome IV. A mutation in him-8 IV was identified that severely reduced recombination between the two X chromosomes in hermaphrodites and between mnDp73 and the X chromosome in males. Recombination between the X chromosome and duplications of either the right end of the X or a region near but not including the left end was rare. We suggest that the X chromosome has one or more elements near its left end that promote meiotic chromosome pairing.