Positioning and maintenance of embryonic body wall muscle attachments in C. elegans requires the mup-1 gene

As part of a general study of genes specifying a pattern of muscle attachments, we identified and genetically characterised mutants in the mup-1 gene. The body wall muscles of early stage mup-1 embryos have a wild-type myofilament pattern but may extend ectopic processes. Later in embryogenesis, some body wall muscles detach from the hypodermis. Genetic analysis suggests that mup-1 has both a maternal and a zygotic component and is not required for postembryonic muscle growth and attachment. mup-1 mutants are suppressed by mutations in several genes that encode extracellular matrix components. We propose that mup-1 may encode a cell surface/extracellular matrix molecule required both for the positioning of body wall muscle attachments in early embryogenesis and the subsequent maintenance of these attachments to the hypodermis until after cuticle synthesis.     

UNC-108/Rab2 regulates postendocytic trafficking in Caenorhabditis elegans

After endocytosis, membrane proteins are often sorted between two alternative pathways: a recycling pathway and a degradation pathway. Relatively little is known about how trafficking through these alternative pathways is differentially regulated. Here, we identify UNC-108/Rab2 as a regulator of postendocytic trafficking in both neurons and coelomocytes. Mutations in the Caenorhabditis elegans Rab2 gene unc-108, caused the green fluorescent protein (GFP)-tagged glutamate receptor GLR-1 (GLR-1::GFP) to accumulate in the ventral cord and in neuronal cell bodies. In neuronal cell bodies of unc-108/Rab2 mutants, GLR-1::GFP was found in tubulovesicular structures that colocalized with markers for early and recycling endosomes, including Syntaxin-13 and Rab8. GFP-tagged Syntaxin-13 also accumulated in the ventral cord of unc-108/Rab2 mutants. UNC-108/Rab2 was not required for ubiquitin-mediated sorting of GLR-1::GFP into the multivesicular body (MVB) degradation pathway. Mutations disrupting the MVB pathway and unc-108/Rab2 mutations had additive effects on GLR-1::GFP levels in the ventral cord. In coelomocytes, postendocytic trafficking of the marker Texas Red-bovine serum albumin was delayed. These results demonstrate that UNC-108/Rab2 regulates postendocytic trafficking, most likely at the level of early or recycling endosomes, and that UNC-108/Rab2 and the MVB pathway define alternative postendocytic trafficking mechanisms that operate in parallel. These results define a new function for Rab2 in protein trafficking.     

Novel genes controlling ventral cord asymmetry and navigation of pioneer axons in C. elegans

The ventral cord in C. elegans is the major longitudinal axon tract containing essential components of the motor circuit. In genetic screens using transgenic animals expressing neuron specific GFP reporters, we identified twelve genes required for the correct outgrowth of interneuron axons of the motor circuit. In mutant animals, axons fail to navigate correctly towards the ventral cord or fail to fasciculate correctly within the ventral cord. Several of those mutants define previously uncharacterized genes. Two of the genes, ast-4 and ast-7, are involved in the generation of left-right asymmetry of the two ventral cord axon tracts. Three other genes specifically affect pioneer-follower relationships between early and late outgrowing axons, controlling either differentiation of a pioneer neuron (lin-11) or the ability of axons to follow a pioneer (ast-2, unc-130). Navigation of the ventral cord pioneer neuron AVG itself is defective in ast-4, ast-6 and unc-130 mutants. Correlation of these defects with navigation defects in different classes of follower axons revealed a true pioneer role for AVG in the guidance of interneurons in the ventral cord. Taken together, these genes provide a basis to address different aspects of axon navigation within the ventral cord of C. elegans.     

The Mup-4 Locus in Caenorhabditis Elegans Is Essential for Hypodermal Integrity, Organismal Morphogenesis and Embryonic Body Wall Muscle Position

mup-4 is a member of a set of genes essential for correct embryonic body wall muscle cell positions in Caenorhabditis elegans. The mup-4 phenotype is variably expressed and three discrete arrest phenotypes arise during the phase of embryonic development when the worm elongates from a ball of cells to its worm shape (organismal morphogenesis). Mutants representing two of the phenotypic classes arrest without successful completion of elongation. Mutants of the third phenotypic class arrest after completion of elongation. Mutants that arrest after elongation display profound dorsal and ventral body wall muscle cell position abnormalities and a characteristic kinked body shape (the Mup phenotype) due to the muscle cell position abnormalities. Significantly, genetic mosaic analysis of mup-4 mutants demonstrates that mup-4 gene function is essential in the AB lineage, which generates most of the hypodermis (epidermis), a tissue with which muscle interacts. Consistent with the genetic mosaic data, phenotypic characterizations reveal that mutants have defects in hypodermal integrity and morphology. Our analyses support the conclusion that mup-4 is essential for hypodermal function and that this function is necessary for organismal morphogenesis and for the maintenance of body wall muscle position.     

Developmental genetic analysis of troponin T mutations in striated and nonstriated muscle cells of Caenorhabditis elegans

We have been investigating a set of genes, collectively called mups, that are essential to striated body wall muscle cell positioning in Caenorhabditis elegans. Here we report our detailed characterization of the mup-2 locus, which encodes troponin T (TnT). Mutants for a heat- sensitive allele, called mup-2(e2346ts), and for a putative null, called mup-2(up1), are defective for embryonic body wall muscle cell contraction, sarcomere organization, and cell positioning. Characterizations of the heat-sensitive allele demonstrate that mutants are also defective for regulated muscle contraction in larval and adult body wall muscle, defective for function of the nonstriated oviduct myoepithelial sheath, and defective for epidermal morphogenesis. We cloned the mup-2 locus and its corresponding cDNA. The cDNA encodes a predicted 405-amino acid protein homologous to vertebrate and invertebrate TnT and includes an invertebrate-specific COOH-terminal tail. The mup-2 mutations lie within these cDNA sequences: mup-2(up1) is a termination codon near NH2 terminus (Glu94) and mup-2(e2346ts) is a termination codon in the COOH-terminal invertebrate-specific tail (Trp342). TnT is a muscle contractile protein that, in association with the thin filament proteins tropomyosin, troponin I and troponin C, regulates myosin-actin interaction in response to a rise in intracellular Ca2+. Our findings demonstrate multiple essential functions for TnT and provide a basis to investigate the in vivo functions and protein interactions of TnT in striated and nonstriated muscles.     

Regulation of body length and male tail ray pattern formation of Caenorhabditis elegans by a member of TGF-beta family

We have identified a new member of the TGF-beta superfamily, CET-1, from Caenorhabditis elegans, which is expressed in the ventral nerve cord and other neurons. cet-1 null mutants have shortened bodies and male tail abnormal phenotype resembling sma mutants, suggesting cet-1, sma-2, sma-3 and sma-4 share a common pathway. Overexpression experiments demonstrated that cet-1 function requires wild-type sma genes. Interestingly, CET-1 appears to affect body length in a dose-dependent manner. Heterozygotes for cet-1 displayed body lengths ranging between null mutant and wild type, and overexpression of CET-1 in wild-type worms elongated body length close to lon mutants. In male sensory ray patterning, lack of cet-1 function results in ray fusions. Epistasis analysis revealed that mab-21 lies downstream and is negatively regulated by the cet-1/sma pathway in the male tail. Our results show that cet-1 controls diverse biological processes during C. elegans development probably through different target genes.     

A genetic screen for temperature-sensitive cell-division mutants of Caenorhabditis elegans.

A novel screen to isolate conditional cell-division mutants in Caenorhabditis elegans has been developed. The screen is based on the phenotypes associated with existing cell-division mutations: some disrupt postembryonic divisions and affect formation of the gonad and ventral nerve cord-resulting in sterile, uncoordinated animals-while others affect embryonic divisions and result in lethality. We obtained 19 conditional mutants that displayed these phenotypes when shifted to the restrictive temperature at the appropriate developmental stage. Eighteen of these mutations have been mapped; 17 proved to be single alleles of newly identified genes, while 1 proved to be an allele of a previously identified gene. Genetic tests on the embryonic lethal phenotypes indicated that for 13 genes, embryogenesis required maternal expression, while for 6, zygotic expression could suffice. In all cases, maternal expression of wild-type activity was found to be largely sufficient for embryogenesis. Cytological analysis revealed that 10 mutants possessed embryonic cell-division defects, including failure to properly segregate DNA, failure to assemble a mitotic spindle, late cytokinesis defects, prolonged cell cycles, and improperly oriented mitotic spindles. We conclude that this approach can be used to identify mutations that affect various aspects of the cell-division cycle.     

Evolutionarily conserved nuclear migration genes required for early embryonic development in Caenorhabditis elegans

The nudF and nudC genes of the fungus Aspergillus nidulans encode proteins that are members of two evolutionarily conserved families. In A. nidulans these proteins mediate nuclear migration along the hyphae. The human ortholog of nudF is Lis1, a gene essential for neuronal migration in the developing cerebral cortex. The mammalian ortholog of nudC encodes a protein that interacts with Lis1. We have identified orthologs of nudC and Lis1 from the nematode Caenorhabditis elegans. Heterologous expression of the C. elegans nudC ortholog, nud-1, complements the A. nidulans nudC3 mutant, demonstrating evolutionary conservation of function. A C. elegans nud-1::GFP fusion produces sustained fluorescence in sensory neurons and embryos, and transient fluorescence in the gonad, gut, vulva, ventral cord, and hypodermal seam cells. Fusion of GFP to C. elegans lis-1 revealed expression in all major neuronal processes of the animal as well as the multinucleate spermathecal valves and adult seam cells. Phenotypic analysis of either nud-1 and lis-1 by RNA interference yielded similar phenotypes, including embryonic lethality, sterility, altered vulval morphology, and uncoordinated movement. Digital time-lapse video microscopy was used to determine that RNAi-treated embryos exhibited nuclear positioning defects in early embryonic cell division similar to those reported for dynein/dynactin depletion. These results demonstrate that the LIS-1/NUDC-like proteins of C. elegans represent a link between nuclear positioning, cell division, and neuronal function.     

mua-6, a gene required for tissue integrity in Caenorhabditis elegans, encodes a cytoplasmic intermediate filament

Locomotion in Caenorhabditis elegans requires force transmission through a network of proteins linking the skeletal muscle, via an intervening basal lamina and epidermis (hypodermis), to the cuticle. Mutations in mua-6 result in hypodermal rupture, muscle detachment from the bodywall, and progressive paralysis. It is shown that mua-6 encodes the cytoplasmic intermediate filament (cIF) A2 protein and that a MUA-6/IFA-2::GFP fusion protein that rescues the presumptive mua-6 null allele localizes to hypodermal hemidesmosomes. This result is consistent with what is known about the function of cIFs in vertebrates. Although MUA-6/IFA-2 is expressed embryonically, and plays an essential postembryonic role in tissue integrity, it is not required for embryonic development of muscle-cuticle linkages nor for the localization of other cIFs or hemidesmosome-associated proteins in the embryo. Finally, the molecular lesion in the mua-6(rh85) allele suggests that the head domain of the MUA-6/IFA-2 is dispensable for its function.     

C. elegans ankyrin repeat protein VAB-19 is a component of epidermal attachment structures and is essential for epidermal morphogenesis

Elongation of the epidermis of the nematode Caenorhabditis elegans involves both actomyosin-mediated changes in lateral epidermal cell shape and body muscle attachment to dorsal and ventral epidermal cells via intermediate-filament/hemidesmosome structures. vab-19 mutants are defective in epidermal elongation and muscle attachment to the epidermis. VAB-19 is a member of a conserved family of ankyrin repeat-containing proteins that includes the human tumor suppressor Kank. In epidermal cells, VAB-19::GFP localizes with components of epidermal attachment structures. In vab-19 mutants, epidermal attachment structures form normally but do not remain localized to muscle-adjacent regions of the epidermis. VAB-19 localization requires function of the transmembrane attachment structure component Myotactin. vab-19 mutants also display aberrant actin organization in the epidermis. Loss of function in the spectrin SMA-1 partly bypasses the requirement for VAB-19 in elongation, suggesting that VAB-19 and SMA-1/spectrin might play antagonistic roles in regulation of the actin cytoskeleton.     

The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets key cell cycle regulatory proteins for degradation. Blockade of APC activity causes mitotic arrest. Recent evidence suggests that the APC may have roles outside the cell cycle. Several studies indicate that ubiquitin plays an important role in regulating synaptic strength. We previously showed that ubiquitin is directly conjugated to GLR-1, a C. elegans non-NMDA (N-methyl-D-aspartate) class glutamate receptor (GluR), resulting in its removal from synapses. By contrast, endocytosis of rodent AMPA GluRs is apparently regulated by ubiquitination of associated scaffolding proteins. Relatively little is known about the E3 ligases that mediate these effects. We examined the effects of perturbing APC function on postmitotic neurons in the nematode C. elegans. Temperature-sensitive mutations in APC subunits increased the abundance of GLR-1 in the ventral nerve cord. Mutations that block clathrin-mediated endocytosis blocked the effects of the APC mutations, suggesting that the APC regulates some aspect of GLR-1 recycling. Overexpression of ubiquitin decreased the density of GLR-1-containing synapses, and APC mutations blunted this effect. APC mutants had locomotion defects consistent with increased synaptic strength. This study defines a novel function for the APC in postmitotic neurons.     

Identification of new members of Fertilisation Independent Seed Polycomb Group pathway involved in the control of seed development in Arabidopsis thaliana

In higher plants, double fertilisation initiates seed development. One sperm cell fuses with the egg cell and gives rise to the embryo, the second sperm cell fuses with the central cell and gives rise to the endosperm. The endosperm develops as a syncytium with the gradual organisation of domains along an anteroposterior axis defined by the position of the embryo at the anterior pole and by the attachment to the placenta at the posterior pole. We report that ontogenesis of the posterior pole in Arabidopsis thaliana involves oriented migration of nuclei in the syncytium. We show that this migration is impaired in mutants of the three founding members of the FERTILIZATION INDEPENDENT SEED (FIS) class, MEDEA (MEA), FIS2 and FERTILIZATION INDEPENDENT ENDOSPERM (FIE). A screen based on a green fluorescent protein (GFP) reporter line allowed us to identify two new loci in the FIS pathway, medicis and borgia. We have cloned the MEDICIS gene and show that it encodes the Arabidopsis homologue of the yeast WD40 domain protein MULTICOPY SUPRESSOR OF IRA (MSI1). The mutations at the new fis loci cause the same cellular defects in endosperm development as other fis mutations, including parthenogenetic development, absence of cellularisation, ectopic development of posterior structures and overexpression of the GFP marker.     

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.     

Dissection of cell division processes in the one cell stage Caenorhabditis elegans embryo by mutational analysis

To identify novel components required for cell division processes in complex eukaryotes, we have undertaken an extensive mutational analysis in the one cell stage Caenorhabditis elegans embryo. The large size and optical properties of this cell permit observation of cell division processes with great detail in live specimens by simple differential interference contrast (DIC) microscopy. We have screened an extensive collection of maternal-effect embryonic lethal mutations on chromosome III with time-lapse DIC video microscopy. Using this assay, we have identified 48 mutations in 34 loci which are required for specific cell division processes in the one cell stage embryo. We show that mutations fall into distinct phenotypic classes which correspond, among others, to the processes of pronuclear migration, rotation of centrosomes and associated pronuclei, spindle assembly, chromosome segregation, anaphase spindle positioning, and cytokinesis. We have further analyzed pronuclear migration mutants by indirect immunofluorescence microscopy using antibodies against tubulin and ZYG-9, a centrosomal marker. This analysis revealed that two pronuclear migration loci are required for generating normal microtubule arrays and four for centrosome separation. All 34 loci have been mapped by deficiencies to distinct regions of chromosome III, thus paving the way for their rapid molecular characterization. Our work contributes to establishing the one cell stage C. elegans embryo as a powerful metazoan model system for dissecting cell division processes.     

Genes required for axon pathfinding and extension in the C. elegans nerve ring.

Over half of the neurons in Caenorhabditis elegans send axons to the nerve ring, a large neuropil in the head of the animal. Genetic screens in animals that express the green fluorescent protein in a subset of sensory neurons identified eight new sax genes that affect the morphology of nerve ring axons. sax-3/robo mutations disrupt axon guidance in the nerve ring, while sax-5, sax-9 and unc-44 disrupt both axon guidance and axon extension. Axon extension and guidance proceed normally in sax-1, sax-2, sax-6, sax-7 and sax-8 mutants, but these animals exhibit later defects in the maintenance of nerve ring structure. The functions of existing guidance genes in nerve ring development were also examined, revealing that SAX-3/Robo acts in parallel to the VAB-1/Eph receptor and the UNC-6/netrin, UNC-40/DCC guidance systems for ventral guidance of axons in the amphid commissure, a major route of axon entry into the nerve ring. In addition, SAX-3/Robo and the VAB-1/Eph receptor both function to prevent aberrant axon crossing at the ventral midline. Together, these genes define pathways required for axon growth, guidance and maintenance during nervous system development.     

Neuronal expression of a Caenorhabditis elegans elav-like gene and the effect of its ectopic expression.

We examined the expression of a Caenorhabditis elegans (C. elegans) elav-like gene, which we designated elr-1. The elr-1 gene encodes a predicted 456-amino-acid protein containing three putative RNA-binding domains and belongs to the ELAV family, which is functionally involved in neuronal differentiation. Northern blot analysis suggested that the levels of elr-1 mRNA are regulated developmentally. A elr-1::gfp reporter gene under the control of the elr-1 promoter was expressed specifically in the ring ganglia near the nerve ring, the ventral nerve cord (VNC), and the pre-anal and lumbar ganglia. In the VNC, GFP-positive cells were shown to be acetylcholine-producing motor neurons which increased in number as development proceeded, suggesting that elr-1 is expressed in mature neurons. Ectopic expression of ELR-1 protein at the L4 larval and adult stages, but not earlier stages, caused irreversible death, accompanied by uncoordinated movement (Unc), clear (Clr), and egg-laying defective (Egl) phenotypes, which are often observed in mutants with neuronal defects. These results suggest that ELR-1 may have important functions in specific mature neurons in C. elegans.     

Calcineurin may regulate multiple endocytic processes in C. elegans

Calcineurin is a serine/threonine protein phosphatase controlled by Ca(2+) and calmodulin that has been implicated in various signaling pathways. Previously, we reported that calcineurin regulates coelomocyte endocytosis in Caenorhabditis elegans. So far, simple and powerful in vivo approaches have been developed to study various endocytic processes in C. elegans. Using these in vivo assays, we further analyzed the endocytic phenotypes of calcineurin mutants. We observed that the calcineurin mutants were defective in apical endocytosis in the intestine as well as synaptic vesicle recycling in the nerve cord. However, we found that calcineurin mutants displayed normal receptor-mediated endocytosis in oocytes. Therefore, our results suggest that calcineurin may regulate specific sets of endocytic processes in nematode.