C. elegans PlexinA PLX-1 mediates a cell contact-dependent stop signal in vulval precursor cells

PLX-1 is a PlexinA transmembrane protein in Caenorhabditis elegans, and the transmembrane-type semaphorin, SMP-1, is a ligand for PLX-1. The SMP-1/PLX-1 system has been shown to be necessary for proper epidermal morphogenesis in the male tail and seam cells. Here, we show that the SMP-1/PLX-1 system also regulates vulval morphogenesis. In plx-1 and smp-1 mutants, hermaphrodites sometimes exhibit a protruding vulva or multiple vulva-like protrusions. Throughout the vulval development of plx-1 and smp-1 mutants, the arrangement of vulval cells is often disrupted. In the initial step of vulval morphogenesis, vulval precursor cells (VPCs) are generated normally but are subsequently arranged abnormally in mutants. Continuous observation revealed that plx-1 VPC fails to terminate longitudinal extension after making contact with neighbor VPCs. The arrangement defects of VPCs in plx-1 and smp-1 mutants are rescued by expressing the respective cDNA in VPCs. plx-1::egfp and smp-1::egfp transgenes are both expressed in all vulval cells, including VPCs, throughout vulval development. We propose that the SMP-1/PLX-1 system is responsible for a cell contact-mediated stop signal for VPC extension. Analyses using cell fate-specific markers showed that the arrangement defects of VPCs also affect cell fate specification and cell lineages, but in a relatively small fraction of plx-1 mutants.     

Characterization of C. elegans RING finger protein 1, a binding partner of ubiquitin-conjugating enzyme 1

In a yeast two-hybrid screen, RING finger protein 1 (RFP-1) and UBR1 were identified as potential binding partners of C. elegans UBC-1, a ubiquitin-conjugating enzyme with a high degree of identity to S. cerevisiae UBC2/RAD6. The interaction of RFP-1 and UBC-1 was confirmed by co-immunoprecipitation experiments. Yeast interaction trap experiments mapped the region of interaction to the basic N-terminal 313 residues of RFP-1. The acidic carboxy-terminal extension of UBC-1 was not required for the interaction with RFP-1. Western blot analysis and indirect immunohistochemical staining show that RFP-1 is present in embryos, larvae, and adults, where it is found in intestinal, nerve ring, pharyngeal, gonadal, and oocyte cell nuclei. Double-stranded RNA interference experiments against rfp-1 indicate that this gene is required for L1 development, vulval development, and for egg laying. By contrast, RNA interference against ubc-1 gave no obvious phenotype, suggesting that ubc-1 is nonessential or is functionally redundant.     

Identification of cis-regulatory elements from the C. elegans Hox gene lin-39 required for embryonic expression and for regulation by the transcription factors LIN-1, LIN-31 and LIN-39

Expression of the Caenorhabditis elegans Hox gene lin-39 begins in the embryo and continues in multiple larval cells, including the P cell lineages that generate ventral cord neurons (VCNs) and vulval precursor cells (VPCs). lin-39 is regulated by several factors and by Wnt and Ras signaling pathways; however, no cis-acting sites mediating lin-39 regulation have been identified. Here, we describe three elements controlling lin-39 expression: a 338-bp upstream fragment that directs embryonic expression in P5-P8 and their descendants in the larva, a 247-bp intronic region sufficient for VCN expression, and a 1.3-kb upstream cis-regulatory module that drives expression in the VPC P6.p in a Ras-dependent manner. Three trans-acting factors regulate expression via the 1.3-kb element. A single binding site for the ETS factor LIN-1 mediates repression in VPCs other than P6.p; however, loss of LIN-1 decreases expression in P6.p. Therefore, LIN-1 acts both negatively and positively on lin-39 in different VPCs. The Forkhead domain protein LIN-31 also acts positively on lin-39 in P6.p via this module. Finally, LIN-39 itself binds to this element, suggesting that LIN-39 autoregulates its expression in P6.p. Therefore, we have begun to unravel the cis-acting sites regulating lin-39 Hox gene expression and have shown that lin-39 is a direct target of the Ras pathway acting via LIN-1 and LIN-31.     

lin-17/Frizzled and lin-18 regulate POP-1/TCF-1 localization and cell type specification during C. elegans vulval development

The Caenorhabditis elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. We show that most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. We demonstrate that lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva.     

Asymmetric localizations of LIN-17/Fz and MIG-5/Dsh are involved in the asymmetric B cell division in C. elegans

LIN-44/Wnt and LIN-17/Frizzled (Fz) function in a planar cell polarity (PCP)-like pathway to regulate the asymmetric B cell division in Caenorhabditis elegans. We observed asymmetric localization of LIN-17/Frizzled (Fz) and MIG-5/Dishevelled (Dsh) during the B cell division. LIN-17::GFP was asymmetrically localized within the B cell prior to and after the B cell division and correlated with B cell polarity. Asymmetric localization of LIN-17::GFP was dependent upon LIN-44/Wnt and MIG-5/Dsh function. The LIN-17 transmembrane domain and a portion of the cysteine-rich domain (CRD) were required for LIN-17 function and asymmetric distribution to the B cell daughters, while the conserved KTXXXW motif was only required for function. MIG-5::GFP was also asymmetrically localized within the B cell prior to and after the B cell division in a LIN-17- and LIN-44-dependent manner. Functions of the MIG-5 DEP, PDZ and DIX domains were also conserved. Thus, a novel PCP-like pathway, in which LIN-17 and MIG-5 are asymmetrically localized, is involved in the regulation of B cell polarity.     

The C. elegans homologue of the spastic paraplegia protein, spastin, disassembles microtubules

Mutations in human spastin (SPG4) cause an autosomal dominant form of hereditary spastic paraplegia. Sequence analysis revealed that spastin contains the AAA (ATPases associated with diverse cellular activities) domain in the C-terminal region. Recently, it was reported that spastin interacts dynamically with microtubules and displays microtubule-severing activity. A plausible Caenorhabditis elegans homologue of spastin (SPAS-1) has been identified by homology search and phylogenetic analyses. To understand the function of the spastin homologue, we characterized the spas-1 deletion mutant and analyzed spas-1 expression regulation in C. elegans. SPAS-1 was localized with cytoskeletons at the perinuclear region. We found that microtubules were intensely stained at the centrosomal region in the deletion mutant. Furthermore, overexpression of SPAS-1 caused disassembly of microtubule network in cultured cells, while ATPase-deficient SPAS-1 did not. These results indicate that C. elegans SPAS-1 plays an important role in microtubule dynamics. We also found that two kinds of products were generated from spas-1 by alternative splicing in a developmental stage-dependent manner.     

The Caenorhabditis elegans EGL-26 protein mediates vulval cell morphogenesis.

In screens for Caenorhabditis elegans mutants defective in vulval morphogenesis, we isolated multiple mutants in which the uterus and the vulva fail to make a functional connection, resulting in an egg-laying defective phenotype. Two of these connection of gonad defective (Cog) mutants carry alleles of the egl-26 gene. We demonstrate that vulval lineages in egl-26 mutant animals are normal, but one vulval cell, vulF, adopts an abnormal morphology. This results in formation of an abnormally thick layer of vulval tissue at the apex of the vulva and a physical blockage of the exit to the vulva from the uterus. egl-26 was cloned and is predicted to encode a novel protein. Mosaic analysis indicates that egl-26 activity is required in the primary vulval lineage for vulF morphogenesis. Expression of a functional translational fusion of EGL-26 to GFP was observed within the primary vulval lineage only in vulE, which neighbors vulF. EGL-26 is localized at the apical edge of the vulE cell. It is thus possible that vulE acts to instruct morphological changes in the neighboring cell, vulF, in an interaction mediated by EGL-26.     

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.     

C. elegans HIM-8 functions outside of meiosis to antagonize EGL-13 Sox protein function

egl-13 encodes a Sox domain protein that is required for proper uterine seam cell development in Caenorhabditis elegans. We demonstrate that mutations of the C2H2 zinc fingers encoded by the him-8 (high incidence of males) gene partially suppress the egg-laying and connection-of-gonad morphology defects caused by incompletely penetrant alleles of egl-13. him-8 alleles have previously characterized recessive effects on recombination and segregation of the X chromosome during meiosis due to failure of X chromosome homolog pairing and subsequent synapsis. However, we show that him-8 alleles are semi-dominant suppressors of egl-13, and the semi-dominant effect is due to haplo-insufficiency of the him-8 locus. Thus, we conclude that the wild-type him-8 gene product acts antagonistically to EGL-13. Null alleles of egl-13 cannot be suppressed, suggesting that this antagonistic interaction most likely occurs either upstream of or in parallel with EGL-13. Moreover, we conclude that suppression of egl-13 is due to a meiosis-independent function of him-8 because suppression is observed in mutants that have severely reduced meiotic germ cell populations and suppression does not depend on the function of him-8 in the maternal germ line. We also show that the chromosomal context of egl-13 seems important in the him-8 suppression mechanism. Interactions between these genes can give insight into function of Sox family members, which are important in many aspects of metazoan development, and into functions of him-8 outside of meiosis.     

C. elegans CPB-3 interacts with DAZ-1 and functions in multiple steps of germline development

Cytoplasmic polyadenylation element-binding proteins (CPEBs) are well-conserved RNA-binding proteins, which regulate mRNA translation mainly through control of poly(A) elongation. Here, we show that CPB-3, one of the four CPEB homologs in C. elegans, positively regulates multiple aspects of oocyte production. CPB-3 protein was highly expressed in early meiotic regions of the hermaphrodite gonad. Worms deficient in cpb-3 were apparently impaired in germ cell proliferation and differentiation including sperm/oocyte switching and progression of female meiosis. We also show that cpb-3 is likely to promote the meiotic entry in parallel with gld-3, a component of one of the redundant but essential genetic pathways for the entry to and progression through meiosis. Taken together, CPEB appears to have a conserved role in the early phase of meiosis and in the sperm/oocyte specification, in addition to its reported function during meiotic progression.     

A possible role for FRM-1, a C. elegans FERM family protein, in embryonic development

FRM-1 is a member of the FERM protein superfamily containing a FERM domain, which is a highly conserved protein-protein interaction module found in most eukaryotes. Although FRM-1 is thought to be involved in linking intracellular proteins to membrane proteins, the specific role for FRM-1 remains to be elucidated. In an effort to explore the biological function of FRM-1, we examined the phenotype of frm-1(tm4168) mutant worms. We observed that frm-1(tm4168) worms have a delayed hatching phenotype. Twelve hours after being laid, when virtually all wild-type eggs had hatched, only 64% of frm-1(tm4168) eggs had hatched. About 3% of frm-1(tm4168) eggs failed to hatch, even 3 days after they had been laid. We also found that frm-1(tm4168) mutants displayed a temperature-sensitive sterility phenotype. About 13% of frm-1(tm4168) worms were unable to produce eggs or produced nonviable eggs at 25°C. In contrast, less than 1% of wild-type animals were sterile at this temperature. At 20°C, neither the mutant nor wild type appeared to be sterile. Western blot analysis indicates that FRM-1 is expressed throughout the developmental stages with the strongest expression at the egg stage. Immunostaining experiments revealed that FRM-1 is mainly localized to the plasma membrane of most if not all cells at an early embryonic stage and to the plasma membrane of P cells during the late embryonic stages. GFP fusion experiments showed that FRM-1 can be expressed in the pharynx and intestine at the larval and adult stages. Our data suggest that FRM-1 may participate in diverse biological processes, including embryonic development.