Suppressors of the unc-73 gene of Caenorhabditis elegans

The unc-73 gene of Caenorhabditis elegans is necessary for proper axon guidance. Animals mutant in this gene are severely uncoordinated and also exhibit defects in cell migration and cell lineages. We have isolated coordinated revertants of unc-73 (e936). These fall into three classes: intragenic revertants, extragenic dominant suppressors (sup-39), and a single apparently intragenic mutation that is a dominant suppressor with a linked recessive lethal phenotype. sup-39 mutations cause early embryonic lethality, but escapers have a wild-type movement phenotype as larvae and adults. Gonads of sup-39 mutant animals show a novel defect: normal gonads have a single row of oocytes, but sup-39 gonads often have two rows of oocytes. This result suggests that the mutant gonad is defective in choosing on its surface only a single site form which nuclei will emerge to form oocytes. These results are interpreted in terms of an effect of unc-73 on determination of cell polarity.     

unc-8, a DEG/ENaC family member, encodes a subunit of a candidate mechanically gated channel that modulates C. elegans locomotion

Mechanically gated ion channels are important modulators of coordinated movement, yet little is known of their molecular properties. We report that C. elegans unc-8, originally identified by gain-of-function mutations that induce neuronal swelling and severe uncoordination, encodes a DEG/ENaC family member homologous to subunits of a candidate mechanically gated ion channel. unc-8 is expressed in several sensory neurons, interneurons, and motor neurons. unc-8 null mutants exhibit previously unrecognized but striking defects in the amplitude and wavelength of sinusoidal tracks inscribed as they move through an E. coli lawn. We hypothesize that UNC-8 channels could modulate coordinated movement in response to body stretch. del-1, a second DEG/ENaC family member coexpressed with unc-8 in a subset of motor neurons, might also participate in a channel that contributes to nematode proprioception.     

Morphogenesis of the C. elegans hermaphrodite uterus

We have undertaken electron micrographic reconstruction of the Caenorhabditis elegans hermaphrodite uterus and determined the correspondence between cells defined by their lineage history and differentiated cell types. In this organ, many cells do not move during morphogenesis and the cell lineage may function to put cells where they are needed. Differentiated uterine cell types include the toroidal ut cells that make structural epithelium, and specialized utse and uv cells that make the connection between the uterus and the vulva. A cell fate decision in which the anchor cell (AC) induces adjacent ventral uterine intermediate precursor cells to adopt the pi fate, rather than the ground state rho, has profound consequences for terminal differentiation: all pi progeny are directly involved in making the uterine-vulval connection whereas all rho progeny contribute to ut toroids or the uterine-spermathecal valve. In addition to specifying certain uterine cell fates, the AC also induces the vulva. Its multiple inductions thereby function to coordinate the connection of an internal to an external epithelium. The AC induces the pi cells and ultimately fuses with a subset of their progeny. This is an example of reciprocal cell-cell interaction that can be studied at single cell resolution. The AC is thus a transitory cell type that plays a pivotal role in organizing the morphogenesis of the uterine-vulval connection.     

Identification of chicken and C. elegans fibulin-1 homologs and characterization of the C. elegans fibulin-1 gene

Fibulin-1, a member of the emerging family of fibulin proteins, is a component of elastic extracellular matrix fibers, basement membranes and blood. Homologs of fibulin-1 have been described in man, mouse and zebrafish. In this study, we describe the isolation and sequencing of chicken fibulin-1C and D cDNA variants. We also describe identification of a C. elegans cDNA encoding fibulin-1D and cosmids containing the C. elegans fibulin-1 gene. Using the cDNA, RT-PCR and computer-based analysis of genomic sequences, the exon/intron organization of the C. elegans fibulin-1 gene was determined. The C. elegans fibulin-1 gene is located on chromosome IV, is approximately 6 kb in length, contains 16 exons and encodes fibulin-1C and D variants. Comparative analysis of the deduced amino acid sequences of nematode and chicken fibulin-1 variants with other known vertebrate fibulin-1 polypeptides showed that the number and organization of structural modules are identical. The results of this study indicate that the structure of the fibulin-1 protein has remained highly conserved over a large period of evolution, suggestive of functional conservation.     

Promoter trapping identifies real genes in C. elegans

Rats were fed on a low fat diet or on high fat diets which included coconut oil, olive oil, safflower oil, evening primrose oil or fish oil as the principal fat source. The level of phosphatidylinositol-4, 5-bisphosphate in spleen lymphocytes was unaffected by diet. However, the fish oil diet significantly decreased the concentration of inositol-1,4,5-trisphosphate in stimulated lymphocytes; this concentration was also reduced following olive oil feeding. Diet did not significantly affect the level of phospholipase C-gamma1 in spleen lymphocytes but the tyrosine phosphorylation state of this enzyme in stimulated lymphocytes, as well as that of a range of other proteins, was decreased following feeding the fish oil and, to a lesser extent, the olive oil diets. It is concluded that fish oil feeding appears to result in inhibition of one or more tyrosine kinases.     

Genetic analysis of sterile mutants in the dpy-5 unc-13 (I) genomic region of Caenorhabditis elegans

Essential genes were identified in the 1.5-map unit dpy-5 unc-13 region of chromosome I in the Caenorhabditis elegans genome by rescuing lethal mutations using the duplication sDp2. In this paper, we report the mapping and complementation testing of lethal mutations, 45 of which identify 18 new, essential genes. This analysis brings the number of essential genes defined by the sDp2 rescue of lethal mutants to 97; 64 of these map between dpy-5 and unc-13. 61% of these essential genes are identified by more than one allele. Positioning of the mutations was done using the breakpoints of six duplications. The mutant phenotypes of 14 loci essential for fertility were characterized by Nomarski microscopy and DAPI staining. None of the mutants were rescued by wild-type male sperm. The cytological data showed that four genes produced mutants with defects in gonadogenesis. let-395. let-603, let-605 and let-610. Mutations in seven genes, let-355, let-367, let-384, let-513, let-544, let-545 and let-606, affected germ cell proliferation or gametogenesis. Mutants for the remaining three genes, let-370, let-599 and let-604, produced eggs that failed to develop or hatch. thereby acting as maternal effect lethals. We observed a nonrandom distribution of arrest phenotypes with regard to map position.     

Mammalian homologue of the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth is a protein kinase C zeta-interacting protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C zeta (PKCzeta) as a bait, we have cloned a gene coding for a novel PKCzeta-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH2-terminal variable region (V1) of PKCzeta and weakly with that of PKCepsilon. In the COS-7 cells coexpressing FEZ1 and PKCzeta, FEZ1 was present mainly in the plasma membrane, associating with PKCzeta and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCzeta. When the constitutively active mutant of PKCzeta was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCzeta activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCzeta stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCzeta.     

C. elegans neuroscience: genetics to genome

From their earliest experiments, researchers using Caenorhabditis elegans have been interested in the role of genes in the development and function of the nervous system. As the C. elegans Genome Project completes the genomic sequence, we review the accomplishments of these researchers and the impact that the Genome Project has bad on their research. We also speculate on future directions in this research that are enabled by the efforts of the Genome Project.     

A sperm-supplied factor required for embryogenesis in C. elegans

The paternal-effect embryonic-lethal gene, spe-11, is required for normal development of early C. elegans embryos. Spe-11 embryos fail to complete meiosis, form a weak eggshell, fail to orient properly the first mitotic spindle, and fail to undergo cytokinesis. Here we report cloning and sequencing of the spe-11 gene, which encodes a novel protein. As predicted by the paternal-effect mutant phenotype, the gene is expressed during spermatogenesis but is not detectable in females undergoing oogenesis, and the protein is present in mature sperm. To investigate whether SPE-11's essential function is during spermatogenesis or whether sperm-delivered SPE-11 functions in the newly fertilized embryo, we engineered animals to supply SPE-11 to the embryo through the oocyte rather than through the sperm. We found that maternal expression is sufficient for embryonic viability. This result demonstrates that SPE-11 is not required during spermatogenesis, and suggests that SPE-11 is a sperm-supplied factor that participates directly in development of the early embryo. In contrast to the many known maternal factors required for embryogenesis, SPE-11 is the first paternally contributed factor to be genetically identified and molecularly characterized.     

Sex and reproduction in the nematode C. elegans]

BACKGROUND: An exploratory, case-control study was used to investigate a new hypothesis about suicide among farm operators. This hypothesis suggested a biologically plausible link between exposures to certain pesticides and the occurrence of suicide among farm operators. These analyses were based on data from the Canadian Farm Operator Cohort. METHODS: Canadian male farm operators who committed suicide between 1971-1987 (n = 1,457) were compared with a frequency matched (by age and province) sample of control farm operators (n = 11,656) who were alive at the time of death of individual cases. Comparisons focused on past exposures to pesticides reported to the 1971 Canada Census of Agriculture. RESULTS: Multivariate logistic regression analyses indicated no associations between suicide and (1) acres sprayed with herbicides, (2) acres sprayed with insecticides, and (3) the costs of agricultural chemicals purchased; after controlling for important covariates. There was, however, a suggestive increase in risk for suicide associated with herbicide and insecticide spraying among a subgroup of farm operators who were most likely to be directly exposed to pesticides: OR = 1.71 (95% CI = 1.08-2.71) for 1-48 vs. 0 acres sprayed. Additional risk factors that were identified included seasonal vs. year-round farm work (OR = 1.68; 95% CI = 1.15-2.46); and high levels of paid labor on the farm (e.g., OR = 1.61; 95% CI = 1.24-2.10, for > 13 vs. 0 weeks per year). Factors that were protective included marriage (odds ratio (OR) = 0.69; 95% confidence interval (CI) = 0.58-0.81), having more than one person resident in the farm house (e.g., two vs. one person; OR = 0.62; 95% CI = 0.42-0.92); and higher levels of education (e.g., postsecondary vs. primary; OR = 0.40; 95% CI = 0.17-0.96). CONCLUSIONS: This study does not provide strong support for the main hypothesis under study, that exposure to pesticides is an important risk factor for suicide among farmers. Although secondary to the main hypothesis, a number of other risk factors for suicide were suggested. These have implications for the future study and targeting of suicide prevention programs in rural Canada.     

Active currents regulate sensitivity and dynamic range in C. elegans neurons

Little is known about the physiology of neurons in Caenorhabditis elegans. Using new techniques for in situ patch-clamp recording in C. elegans, we analyzed the electrical properties of an identified sensory neuron (ASER) across four developmental stages and 42 unidentified neurons at one stage. We find that ASER is nearly isopotential and fails to generate classical Na+ action potentials. Rather, ASER displays a high sensitivity to input currents coupled to a depolarization-dependent reduction in sensitivity that may endow ASER with a wide dynamic range. Voltage clamp revealed depolarization-activated K+ and Ca2+ currents that contribute to high sensitivity near the zero-current potential. The depolarization-dependent reduction in sensitivity can be attributed to activation of K+ current at voltages where it dominates the net membrane current. The voltage dependence of membrane current was similar in all neurons examined, suggesting that C. elegans neurons share a common mechanism of sensitivity and dynamic range.     

Genes necessary for C. elegans cell and growth cone migrations

The migrations of cells and growth cones contribute to form and pattern during metazoan development. To study the mechanisms that regulate cell motility, we have screened for C. elegans mutants defective in the posteriorly directed migrations of the canal-associated neurons (CANs). Here we describe 14 genes necessary for CAN cell migration. Our characterization of the mutants has led to three conclusions. First, the mutations define three gene classes: genes necessary for cell fate specification, genes necessary for multiple cell migrations and a single gene necessary for final positioning of migrating cells. Second, cell interactions between the CAN and HSN, a neuron that migrates anteriorly to a position adjacent to the CAN, control the final destination of the HSN cell body. Third, C. elegans larval development requires the CANs. In the absence of CAN function, larvae arrest development, with excess fluid accumulating in their pseudocoeloms. This phenotype may reflect a role of the CANs in osmoregulation.     

Conservation of the C.elegans tra-2 3'UTR translational control

The Caenorhabditis elegans sex-determination gene, tra-2, is translationally regulated by two 28 nt elements (DREs) located in the 3'UTR that bind a factor called DRF. This regulation requires the laf-1 gene activity. We demonstrate that the nematode Caenorhabditis briggsae tra-2 gene and the human oncogene GLI are translationally regulated by elements that are functionally equivalent to DREs. Here, we rename the DREs to TGEs (tra-2 and GLI elements). Similarly to the C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs repress translation of a reporter transgene in a laf-1 dependent manner. Furthermore, they regulate poly(A) tail length and bind DRF. We also find that the C.elegans TGEs control translation and poly(A) tail length in C.briggsae and rodent cells. Moreover, these same organisms contain a factor that specifically associates with the C.elegans TGEs. These findings are consistent with the TGE control being present in C.briggsae and rodent cells. Three lines of evidence indicate that C.briggsae tra-2 and GLI are translationally controlled in vivo by TGEs. First, like C.elegans tra-2 TGEs, the C.briggsae tra-2 and GLI TGEs control translation and poly(A) tail lengths in C.briggsae and rodent cells, respectively. Second, the same factor in C.briggsae and mammalian cells that binds to the C.elegans tra-2 TGEs binds the C.briggsae tra-2 and GLI TGEs. Third, deletion of the GLI TGE increases GLI's ability to transform cells. These findings suggest that TGE control is conserved and regulates the expression of other mRNAs.