Parental DNA strands segregate randomly during embryonic development of Caenorhabditis elegans

The fate of gamete DNA was followed in the next generation embryos of the nematode C. elegans. Either male worms or spermless hermaphrodites were grown on bromodeoxyuridine-containing E. coli in order to label germ-line DNA. Matings then produced embryos in which only the DNA strands provided by the gametes contained label. This original gamete DNA could be detected during embryonic development by using a fluorescently labeled monoclonal antibody specific to bromodeoxyuridine. Both the number and position of fluorescent spots in the embryo indicate that gamete DNA strands segregate randomly during development. Random segregation of parental DNA strands rules out models of development that invoke chromosome imprinting or immortal DNA strands.     

Differential expression pattern of coq-8 gene during development in Caenorhabditis elegans

Coenzyme Q (Q) and the genes involved in its biosynthesis are involved in aging and development of Caenorhabditis elegans. Q is synthesized by at least eight highly conserved nuclear coq genes, but this biosynthesis pathway and its regulation is not known. The coq-8 gene sequence has homology to the ABC-1 family kinases and is the only known candidate for a possible regulation of this pathway. To study coq-8 expression pattern, we have developed a C. elegans transgenic strain expressing ubiquinone biosynthesis coq-8 gene promoter and GFP construct. We show here an age-dependent specific pattern from embryo to senescence for COQ-8 protein expression. Expression in embryo was triggered by a defined group of blastomers before morphogenesis. In elderly nematodes expression was only observed in nervous system, whilst expression in larvae was also detected in hypodermis, muscles and coelomocytes. Global expression provide a regulated pattern during life cycle of the nematode.     

Clathrin interaction and subcellular localization of Ce-DAB-1, an adaptor for protein secretion in Caenorhabditis elegans

Growth factors must be secreted appropriately to co-ordinate cell proliferation, specification and movement during development and to control cell numbers and migrations in adult animals. Previous results showed that the secretion of the Caenorhabditis elegans fibroblast growth factor homologue, EGL-17, from vulval precursor cells in vivo involves the cytoplasmic adaptor protein Ce-DAB-1 and two lipoprotein receptors that bind Ce-DAB-1 and EGL-17. Here, we confirm the Ce-DAB-1 requirement for EGL-17 secretion using mutant animals. In vitro, Ce-DAB-1 binds to clathrin and APT-4, the C. elegans homologue of the alpha-adaptin subunit of adaptor protein 2 (AP2), and weakly to the gamma-appendage domains of APT-1 (AP1gamma-adaptin) and APT-9 (GGA protein). In tissue-culture cells, Ce-DAB-1 localizes to various compartments, including AP2-containing vesicles near the cell surface and perinuclear vesicles that contain AP1. The latter also contain Rab8, but not Rab5 or Rab11, as well as proteins en route from the trans Golgi network (TGN) to the surface. In vivo, EGL-17 secretion was inhibited by depletion of apt-1, apt-9 or ce-rab-8 and partially inhibited by RNAi of ce-rab-5, consistent with an important role for these proteins in the secretion of EGL-17 in vivo. These results suggest that Ce-DAB-1 might co-ordinate the assembly of endocytic or secretory vesicles in vivo and may mediate EGL-17 secretion directly, by recruiting clathrin to lipoprotein receptors at the TGN, or indirectly, by affecting lipoprotein receptor endocytosis and recycling.     

Spatiotemporal localization of D-amino acid oxidase and D-aspartate oxidases during development in Caenorhabditis elegans

Recent investigations have shown that a variety of D-amino acids are present in living organisms and that they possibly play important roles in physiological functions in the body. D-Amino acid oxidase (DAO) and D-aspartate oxidase (DDO) are degradative enzymes stereospecific for D-amino acids. They have been identified in various organisms, including mammals and the nematode Caenorhabditis elegans, although the significance of these enzymes and the relevant functions of D-amino acids remain to be elucidated. In this study, we investigated the spatiotemporal localization of C. elegans DAO and DDOs (DDO-1, DDO-2, and DDO-3) and measured the levels of several D- and L-amino acids in wild-type C. elegans and four mutants in which each gene for DAO and the DDOs was partially deleted and thereby inactivated. Furthermore, several phenotypes of these mutant strains were characterized. The results reported in this study indicate that C. elegans DAO and DDOs are involved in egg-laying events and the early development of C. elegans. In particular, DDOs appear to play important roles in the development and maturation of germ cells. This work provides novel and useful insights into the physiological functions of these enzymes and D-amino acids in multicellular organisms.     

Differential subcellular localization of RIC-3 isoforms and their role in determining 5-HT3 receptor composition

RIC-3 has been identified as a chaperone molecule involved in promoting the functional expression of nicotinic acetylcholine and 5-HT(3) receptors in mammalian cells. In this study, we examined the effects of RIC-3a (isoform a) and a truncated isoform (isoform d) on RIC-3 localization, mobility, and aggregation and its effect on 5-HT3 receptor composition in mammalian cells. Human RIC-3a possesses an amino-terminal signal sequence that targets it to the endoplasmic reticulum where it is distributed within the reticular network, often forming large diffuse "slicks" and bright "halo" structures. RIC-3a is highly mobile within and between these compartments. Despite the propensity for RIC-3a to aggregate, its expression enhances the level of surface 5-HT3A (homomeric) receptors. In contrast, RIC-3a exerts an inhibitory action on the surface expression of heteromeric 5-HT3A/B receptors. RIC-3d exhibits an altered subcellular distribution, being localized to the endoplasmic reticulum, large diffuse slicks, tubulo-vesicular structures, and the Golgi. Bidirectional trafficking between the endoplasmic reticulum and Golgi suggests that RIC-3d constitutively cycles between these two compartments. In support of the large coiled-coil domain of RIC-3a being responsible for protein aggregation, RIC-3d, lacking this cytoplasmic domain, does not aggregate or induce the formation of bright aggregates. Regardless of these differences, isoform d is still capable of enhancing homomeric, and inhibiting heteromeric, 5-HT3 receptor expression. Thus, both isoforms of RIC-3 play a role in determining 5-HT3 receptor composition.     

Differential localization of conventional protein kinase C isoforms during mouse oocyte development

Protein kinase C (PKC), the major cell target for tumor-promoting phorbol esters, plays a central role in signal transduction pathways. In many biological systems where Ca(2+) serves as a second messenger, regulatory control is mediated by PKC. The activation of PKC depends on its binding to RACK1 receptor, which is an intracellular protein anchor for activated PKC. We demonstrate that the conventional PKC (cPKC) isoforms, PKC-alpha, PKC-betaI, and PKC-betaII, as well as RACK1, are expressed in mouse oocytes (germinal vesicle [GV]) and mature eggs (metaphase II [MII]). In GV oocytes, PKC-alpha, PKC-betaII, and RACK1 were uniformly distributed in the cytoplasm, while PKC-betaI was localized in the cytoplasm and in the plasma membrane as well. Treatment of GV oocytes with the biologically active phorbol ester, 12-o-tetradecanoyl phorbol-13-acetate (TPA), resulted in a rapid translocation of the cytosolic PKC-alpha, but not PKC-betaI, PKC-betaII, or RACK1, to the plasma membrane. This was associated with inhibition of GV breakdown. In MII eggs (17 h post-hCG), PKC-alpha was uniformly distributed in the cytoplasm while PKC-betaI and -betaII were distributed in the cytoplasm and in the plasma membrane as well. Treatment with TPA resulted in a rapid translocation of PKC-alpha from the cytoplasm to the plasma membrane and a significant decrease of PKC-betaI throughout the cytoplasm, while it also remained in the cell periphery. No change in the distribution of PKC-betaII or RACK1 was observed. TPA also induced pronucleus formation. Physiological activation of MII eggs by sperm induced cortical granule exocytosis associated with significant translocation of PKC-alpha and -betaI, but not -betaII, to the plasma membrane. Overall, these results suggest a possible involvement of cPKC isoforms in the mechanism of mouse oocyte maturation and egg activation.     

Distinct roles for RDE-1 and RDE-4 during RNA interference in Caenorhabditis elegans.

RNA interference (RNAi) is a cellular defense mechanism that uses double-stranded RNA (dsRNA) as a sequence-specific trigger to guide the degradation of homologous single-stranded RNAs. RNAi is a multistep process involving several proteins and at least one type of RNA intermediate, a population of small 21-25 nt RNAs (called siRNAs) that are initially derived from cleavage of the dsRNA trigger. Genetic screens in Caenorhabditis elegans have identified numerous mutations that cause partial or complete loss of RNAi. In this work, we analyzed cleavage of injected dsRNA to produce the initial siRNA population in animals mutant for rde-1 and rde-4, two genes that are essential for RNAi but that are not required for organismal viability or fertility. Our results suggest distinct roles for RDE-1 and RDE-4 in the interference process. Although null mutants lacking rde-1 show no phenotypic response to dsRNA, the amount of siRNAs generated from an injected dsRNA trigger was comparable to that of wild-type. By contrast, mutations in rde-4 substantially reduced the population of siRNAs derived from an injected dsRNA trigger. Injection of chemically synthesized 24- or 25-nt siRNAs could circumvent RNAi resistance in rde-4 mutants, whereas no bypass was observed in rde-1 mutants. These results support a model in which RDE-4 is involved before or during production of siRNAs, whereas RDE-1 acts after the siRNAs have been formed.     

Regulatory machinery of UNC-33 Ce-CRMP localization in neurites during neuronal development in Caenorhabditis elegans

In Caenorhabditis elegans, unc-33 encodes an orthologue of the vertebrate collapsin response mediator protein (CRMP) family. We previously reported that CRMP-2 accumulated in the distal part of the growing axon of vertebrate neurons and played critical roles in axon elongation. unc-33 mutants show axonal outgrowth defects in several neurons. It has been reported that UNC-33 accumulates in neurites, whereas a missense mutation causes the mislocalization of UNC-33 from neurites to cell body, which suggests that the localization of UNC-33 in neurites is important for axonal outgrowth. However, it is unclear how UNC-33 accumulates in neurites and regulates neuronal development. In this study, to understand the regulatory mechanisms of localization of UNC-33 in neurites, we screened for the mutants that were involved in the localization of UNC-33, and identified three mutants: unc-14 (RUN domain protein), unc-51 (ULK kinase) and unc-116 (kinesin heavy chain). UNC-14 is known to associate with UNC-51. UNC-116 forms a complex with KLC-2 as Kinesin-1, a microtubule-dependent motor complex. We found that UNC-33 interacted with UNC-14 and KLC-2 in vivo. These results suggest that the UNC-14/UNC-51 complex and Kinesin-1 are involved in the localization of UNC-33 in neurites.     

Cyclin E expression during development in Caenorhabditis elegans

Our interest in the coordination of cell cycle control and differentiation has led us to investigate the Caenorhabditis elegans cye-1 gene encoding the G(1) cell cycle regulator cyclin E. We have studied the expression and function of cye-1 by using monoclonal antibodies directed against CYE-1 protein, cye-1::GFP reporter genes, and a cye-1 chromosomal deletion mutation. We show that a ubiquitous embryonic pattern of expression becomes restricted and dynamic during postembryonic development. Promoter analysis reveals a relatively small region of cis-acting sequences that are necessary for the complex pattern of expression of this gene. Our studies demonstrate that two other G(1) cell cycle genes, encoding cyclin D and CDK4/6, have similarly compact promoter requirements. This suggests that a relatively simple mechanism of regulation may underlie the dynamic developmental patterns of expression exhibited by these three G(1) cell cycle genes. Our analysis of a new cye-1 deletion allele confirms and extends previous studies of two point mutations in the gene.     

Four subunit a isoforms of Caenorhabditis elegans vacuolar H+-ATPase. Cell-specific expression during development.

We have identified four genes (vha-5, vha-6, vha-7, and unc-32) coding for vacuolar-type proton-translocating ATPase (V-ATPase) subunit a in Caenorhabditis elegans, the first example of four distinct isoforms in eukaryotes. Their products had nine putative transmembrane regions, exhibited 43-60% identity and 62-84% similarity with the bovine subunit a1 isoform, and retained 11 amino acid residues essential for yeast V-ATPase activity (Leng, X. H., Manolson, M. F., and Forgac, M. (1998) J. Biol. Chem. 273, 6717-6723). The similarities, together with the results of immunoprecipitation, suggest that these isoforms are components of V-ATPase. Transgenic and immunofluorescence analyses revealed that these genes were strongly expressed in distinct cells; vha-5 was strongly expressed in an H-shaped excretory cell, vha-6 was strongly expressed in intestine, vha-7 was strongly expressed in hypodermis, and unc-32 was strongly expressed in nerve cells. Furthermore, the vha-7 and unc-32 genes were also expressed in the uteri of hermaphrodites. RNA interference analysis showed that the double-stranded RNA for unc-32 caused embryonic lethality similar to that seen with other subunit genes (vha-1, vha-4, and vha-11) (Oka, T., and Futai, M. (2000) J. Biol. Chem. 275, 29556-29561). The progenies of worms injected with the vha-5 or vha-6 double-stranded RNA became died at a specific larval stage, whereas the vha-7 double-stranded RNA showed no effect on development. These results suggest that V-ATPases with these isoforms generate acidic compartments essential for worm development in a cell-specific manner.     

Regulation and cell autonomy during postembryonic development of Caenorhabditis elegans

The role of cell-cell interaction in the postembryonic development of nongonadal tissues in the nematode Caenorhabditis elegans has been explored by selective cell ablation with a laser microbeam. Examples have been found of induction and of regulation in cell lineage and fate. Regulation in which one cell precisely or partially replaces another is seen, but only in certain groups of hypodermal cells which resemble one another closely; cells which are unique are not replaced in this way. The regulation of cell form is more widespread and less restrictive.     

Control of cell migration during Caenorhabditis elegans development.

In Caenorhabditis elegans, cell migration is guided by localized cues, including molecules such as EGL-17/FGF and UNC-6/netrin. These external cues are linked to an intracellular response to migrate, at least in part, by CED-5, a homolog of DOCK180/MBC, and MIG-2, a Rac-like GTPase. In addition, metalloproteases are required for a cell migration that controls organ shape.     

Ribosomal DNA of Caenorhabditis elegans

We have characterized the organization of the genes coding for 18 S, 5·8 S and 26 S ribosomal RNAs in the nematode Caenorhabditis elegans. These ribosomal genes, present in about 55 copies per haploid genome, alternate in a repeating tandem array. The repeating unit is only 7000 base-pairs, containing a non-transcribed spacer of no more than 1000 base-pairs. Most of the repeating units have identical restriction maps, but one repeat contains a deletion of 2900 base-pairs, which eliminates all or part of the 18 S coding region. We have found no difference in the major ribosomal DNA restriction endonuclease cleavage patterns between two interbreeding strains of C. elegans, but found differences between C. elegans and the closely related Caenorhabditis briggsae.     

LET-23-mediated signal transduction during Caenorhabditis elegans development

We are using Caenorhabditis elegans vulval induction to study intercellular signaling and its regulation. Genes required for vulval induction include the LIN-3 transforming α-like growth factor, the LET-23 epidermal growth factor (EGF)-receptor-like transmembrane tyrosine kinase, the SEM-5 adaptor protein, LET-60 Ras, and the LIN-45 Raf serine/threonine kinase. Inactivation of this pathway results in a failure of vulval differentiation, the “vulvaless” phenotype. Activation of this pathway either by overexpression of LIN-3, a point mutation in the LET-23 extracellular domain, or hyperactivity of LET-60 Ras results in excessive vulval differentiation, the “multivulva” phenotype. In addition to searching for new genes that act positively in this signaling pathway, we have also characterized genes that negatively regulate this inductive signaling pathway. We find that such negative regulators are functionally redundant: mutation of only one of these negative regulators has no effect on vulval differentiation; however, if particular combinations of these genes are inactivated, excessive vulval differentiation occurs. The LIN-15 locus encodes two functionally redundant products, LIN-15A and LIN-15B, that formally act upstream of the LET-23 receptor to prevent its activity in the absence of inductive signal. The LIN-15A and B proteins are novel and unrelated to each other. The unc-101, sli-1, and rok-1 genes encode a distinct set of negative regulators of vulval differentiation. The unc-101 gene encodes an adaptin, proposed to be involved in intracellular protein trafficking. The sli-1 gene encodes a protein with similarity to c-cbl, a mammalian proto-oncogene not previously linked with a tyrosine kinase-Ras-mediated signaling pathway. LIN-3 and LET-23 are required for several aspects of C. elegans development—larval viability, P12 neuroectoblast specification, hermaphrodite vulval induction and fertility, and three inductions during male copulatory spicule development. Fertility and vulval differentiation appear to be mediated by distinct parts of the cytoplasmic tail of LET-23, and by distinct signal transduction pathways. © 1995 wiley-Liss, Inc.     

Cellular and subcellular localization, N-terminal acylation, and calcium binding of Caenorhabditis elegans protein phosphatase with EF-hands

The RdgC/PPEF family of serine/threonine protein phosphatases is distinguished by the presence of C-terminal EF-hands and neuron-specific expression, including frequent expression in primary sensory neurons. Here we report that the sole Caenorhabditis elegans PPEF (CePPEF) homolog is also highly expressed in primary sensory neurons and is not found outside the nervous system. Neurons expressing CePPEF include the ciliary chemosensory neurons AWB and AWC; and within these neurons, CePPEF is highly enriched in the sensory cilia. In transgenic C. elegans and in transfected 293 cells, CePPEF is membrane-associated, and the N terminus of CePPEF is necessary and sufficient for this membrane association. [(3)H]Myristate and [(3)H]palmitate labeling studies in 293 cells demonstrated that this association was mediated by myristoylation at Gly(2) and palmitoylation at Cys(3). Introducing the G2A or C3S mutation into CePPEF greatly reduced membrane association in 293 cells and in transgenic nematodes. A recombinant C-terminal fragment of CePPEF containing two putative EF-hands bound between one and two Ca(2+) ions/protein, and mutation of residues presumed to ligand calcium in the two putative EF-hands led to diminished calcium binding. These results establish the first direct evidence for fatty acylation and calcium binding of a PPEF family member and demonstrate a remarkable conservation of sensory neuron expression among the members of this distinctive family of protein phosphatases.     

RAD-51-dependent and -independent roles of a Caenorhabditis elegans BRCA2-related protein during DNA double-strand break repair

The BRCA2 tumor suppressor is implicated in DNA double-strand break (DSB) repair by homologous recombination (HR), where it regulates the RAD51 recombinase. We describe a BRCA2-related protein of Caenorhabditis elegans (CeBRC-2) that interacts directly with RAD-51 via a single BRC motif and that binds preferentially to single-stranded DNA through an oligonucleotide-oligosaccharide binding fold. Cebrc-2 mutants fail to repair meiotic or radiation-induced DSBs by HR due to inefficient RAD-51 nuclear localization and a failure to target RAD-51 to sites of DSBs. Genetic and cytological comparisons of Cebrc-2 and rad-51 mutants revealed fundamental phenotypic differences that suggest a role for Cebrc-2 in promoting the use of an alternative repair pathway in the absence of rad-51 and independent of nonhomologous end joining (NHEJ). Unlike rad-51 mutants, Cebrc-2 mutants also accumulate RPA-1 at DSBs, and abnormal chromosome aggregates that arise during the meiotic prophase can be rescued by blocking the NHEJ pathway. CeBRC-2 also forms foci in response to DNA damage and can do so independently of rad-51. Thus, CeBRC-2 not only regulates RAD-51 during HR but can also function independently of rad-51 in DSB repair processes.     

Genetic analysis of the Caenorhabditis elegans pax-6 locus: roles of paired domain-containing and nonpaired domain-containing isoforms

PAX-6 proteins are involved in eye and brain development in many animals. In the nematode Caenorhabditis elegans the pax-6 locus encodes multiple PAX-6 isoforms both with and without a paired domain. Mutations in the C. elegans pax-6 locus can be grouped into three classes. Mutations that affect paired domain-containing isoforms cause defects in epidermal morphogenesis, epidermal cell fates, and gonad cell migration and define the class I (vab-3) complementation group. The class II mutation mab-18(bx23) affects nonpaired domain-containing isoforms and transforms the fate of a sensory organ in the male tail. Class III mutations affect both paired domain and nonpaired domain isoforms; the most severe class III mutations are candidate null mutations in pax-6. Class III mutant phenotypes do not resemble a simple sum of class I and class II phenotypes. A comparison of class I and class III phenotypes indicates that PAX-6 isoforms can interact additively, synergistically, or antagonistically, depending on the cellular context.     

Patterns of proteins synthesized during development of Caenorhabditis elegans.

Two-dimensional gel electrophoresis has been used to analyze proteins synthesized during postembryonic development of the nematode Caenorhabditis elegans. This organism is favorable for these studies because it has a limited number of cells, it is genetically well-defined, and its development is currently under investigation in several laboratories. ³⁵S-Labeled E. coli was used for continuous and pulse labeling of C. elegans during its four juvenile larval stages and as a gravid adult. After continuous labeling or pulse labeling for 1 hr, 600–800 individual spots can be resolved on a 2D gel using fluorography and 2 weeks of exposure. Proteins that represent 0.0017% of the total sample can be detected. Exposure for 12 weeks reveals only 100 additional spots even though the films are not saturated. It therefore appears that the frequency distribution of proteins decreases significantly beyond these 800 most abundant proteins that can be fractionated on an O'Farrell gel. When the patterns of pulse-labeled proteins of the five developmental stages were compared, 113 proteins could be seen to undergo modulation at one or more of the developmental stages. A maximum number of changes was seen in the transition from the L4 to the adult stages when 11% of the total spots either appeared, disappeared, or changed in intensity. As controls, different preparations of the same developmental stage were compared and revealed considerable fluctuation, 2.6–4.8%. These fluctuations are presumed to be due to variations in growth conditions during culture of the organism. Continuous label experiments reveal a distinct set of proteins that undergo turnover and/or modification during development. Some of these proteins are absent in only one stage, indicating that stable proteins are also modulated. But nearly all of the proteins seen in a continuous label are also seen in a pulse label indicating that most of the major proteins are always present and always synthesized.