The Caenorhabditis elegans rsd-2 and rsd-6 genes are required for chromosome functions during exposure to unfavorable environments

In Caenorhabditis elegans, exogenous dsRNA can elicit systemic RNAi, a process that requires the function of many genes. Considering that the activities of many of these genes are also required for normal development, it is surprising that exposure to high concentrations of dsRNA does not elicit adverse consequences to animals. Here, we report inducible phenotypes in attenuated C. elegans strains reared in environments that include nonspecific dsRNA and elevated temperature. Under these conditions, chromosome integrity is compromised in RNAi-defective strains harboring mutations in rsd-2 or rsd-6. Specifically, rsd-2 mutants display defects in transposon silencing, while meiotic chromosome disjunction is affected in rsd-6 mutants. RSD-2 proteins localize to multiple cellular compartments, including the nucleolus and cytoplasmic compartments that, in part, are congruent with calreticulin and HAF-6. We considered that the RNAi defects in rsd-2 mutants might have relevance to membrane-associated functions; however, endomembrane compartmentalization and endocytosis/exocytosis markers in rsd-2 and rsd-6 mutants appear normal. The mutants also possess environmentally sensitive defects in cell-autonomous RNAi elicited from transgene-delivered dsRNAs. Thus, the ultimate functions of rsd-2 and rsd-6 in systemic RNAi are remarkably complex and environmentally responsive.     

The SynMuv genes of Caenorhabditis elegans in vulval development and beyond

For a nonessential diminutive organ comprised of only 22 nuclei, the Caenorhabditis elegans vulva has done very well for itself. The status of the vulva as an overachiever is in part due to its inherent structural simplicity as well as to the intricate regulation of its induction and development. Studies over the past twenty years have shown the vulva to be a microcosm for organogenesis and a model for the integration of complex signaling pathways. Furthermore, many of these signaling molecules are themselves associated with cancer in mammals. This review focuses on what is perhaps the most intriguing and complex story to emerge from these studies thus far, the role of the Synthetic Multivulval (SynMuv) genes in controlling vulval cell-fate adoption. Recent advances have led to a greater mechanistic understanding of how these genes function during vulval development and have also identified roles for these genes in diverse developmental processes.     

The gon-1 gene is required for gonadal morphogenesis in Caenorhabditis elegans

In wild-type Caenorhabditis elegans, the gonad is a complex epithelial tube that consists of long arms composed predominantly of germline tissue as well as somatic structures specialized for particular reproductive functions. In gon-1 mutants, the adult gonad is severely disorganized with essentially no arm extension and no recognizable somatic structure. The developmental defects in gon-1 mutants are limited to the gonad; other cells, tissues, and organs appear to develop normally. Previous work defined the regulatory "leader" cells as crucial for extension of the gonadal arms (J. E. Kimble and J. G. White, 1981, Dev. Biol. 81, 208-219). In gon-1 mutants, the leader cells are specified correctly, but they fail to migrate and gonadal arms are not generated. In addition, gon-1 is required for morphogenesis of the gonadal somatic structures. This second role appears to be independent of that required for leader migration. Parallel studies have shown that gon-1 encodes a secreted metalloprotease (R. Blelloch and J. Kimble, 1999, Nature 399, 586-590). We discuss how a metalloprotease may control two aspects of gonadal morphogenesis.     

The Caenorhabditis elegans NR4A nuclear receptor is required for spermatheca morphogenesis

The gene nhr-6 encodes the Caenorhabditis elegans ortholog of the NR4A nuclear receptor. We determined the biological functions of NHR-6 through the isolation and characterization of a deletion allele of nhr-6, lg6001. We demonstrate that nhr-6 has an essential role in the development of the C. elegans somatic gonad. Specifically, nhr-6 is required for the development of the hermaphrodite spermatheca, a somatic gonad organ that serves as the site of sperm storage and oocyte fertilization. Using a variety of spermatheca cell markers, we have determined that loss of nhr-6 function causes severe morphological defects in the spermatheca and associated spermathecal valves. This appears to be due to specific requirements for nhr-6 in regulating cell proliferation and cell differentiation during development of these structures. The improper development of these structures in nhr-6(lg6001) mutants leads to defects in ovulation and significantly reduced fecundity of C. elegans hermaphrodites. The phenotypes of nhr-6(lg6001) mutants are consistent with a role for nhr-6 in organogenesis, similar to the functions of its mammalian homologs.     

PAR-1 is required for morphogenesis of the Caenorhabditis elegans vulva

The Caenorhabditis elegans vulva provides a simple model for the genetic analysis of pattern formation and organ morphogenesis during metazoan development. We have discovered an essential role for the polarity protein PAR-1 in the development of the vulva. Postembryonic RNA interference of PAR-1 causes a protruding vulva phenotype. We found that depleting PAR-1 during the development of the vulva has no detectable effect on fate specification or precursor proliferation, but instead seems to specifically alter morphogenesis. Using an apical junction-associated GFP marker, we discovered that PAR-1 depletion causes a failure of the two mirror-symmetric halves of the vulva to join into a single, coherent organ. The cells that normally form the ventral vulval rings fail to make contact or adhere and consequently form incomplete toroids, and dorsal rings adopt variably abnormal morphologies. We also found that PAR-1 undergoes a redistribution from apical junctions to basolateral domains during morphogenesis. Despite a known role for PAR-1 in cell polarity, we have observed no detectable differences in the distribution of various markers of epithelial cell polarity. We propose that PAR-1 activity at the cell cortex is critical for mediating cell shape changes, cell surface composition, or cell signaling during vulval morphogenesis.     

Unfolded protein response genes regulated by CED-1 are required for Caenorhabditis elegans innate immunity

The endoplasmic reticulum stress response, also known as the unfolded protein response (UPR), has been implicated in the normal physiology of immune defense and in several disorders, including diabetes, cancer, and neurodegenerative disease. Here, we show that the apoptotic receptor CED-1 and a network of PQN/ABU proteins involved in a noncanonical UPR response are required for proper defense to pathogen infection in Caenorhabditis elegans. A full-genome microarray analysis indicates that CED-1 functions to activate the expression of pqn/abu genes. We also show that ced-1 and pqn/abu genes are required for the survival of C. elegans exposed to live Salmonella enterica, and that overexpression of pqn/abu genes confers protection against pathogen-mediated killing. The results indicate that unfolded protein response genes, regulated in a CED-1-dependent manner, are involved in the C. elegans immune response to live bacteria.     

Autophagy genes unc-51 and bec-1 are required for normal cell size in Caenorhabditis elegans

Here we show that in the nematode Caenorhabditis elegans mutational inactivation of two autophagy genes unc-51/atg1 and bec-1/atg6/beclin1 results in small body size without affecting cell number. Furthermore, loss-of-function mutations in unc-51 and bec-1 suppress the giant phenotype of mutant animals with aberrant insulin-like growth factor-1 (insulin/IGF-1) or transforming growth factor-beta (TGF-beta) signaling. This function for unc-51 and bec-1 in cell size control and their interaction with these two growth modulatory pathways may represent a link between the hormonal and nutritional regulation of cell growth.     

Identification of Caenorhabditis elegans genes required for neuronal differentiation and migration.

To understand the mechanisms that guide migrating cells, we have been studying the embryonic migrations of the C. elegans canal-associated neurons (CANs). Here, we describe two screens used to identify genes involved in CAN migration. First, we screened for mutants that died as clear larvae (Clr) or had withered tails (Wit), phenotypes displayed by animals lacking normal CAN function. Second, we screened directly for mutants with missing or misplaced CANs. We isolated and characterized 30 mutants that defined 14 genes necessary for CAN migration. We found that one of the genes, ceh-10, specifies CAN fate. ceh-10 had been defined molecularly as encoding a homeodomain protein expressed in the CANs. Mutations that reduce ceh-10 function result in Wit animals with CANs that are partially defective in their migrations. Mutations that eliminate ceh-10 function result in Clr animals with CANs that fail to migrate or express CEH-23, a CAN differentiation marker. Null mutants also fail to express CEH-10, suggesting that CEH-10 regulates its own expression. Finally, we found that ceh-10 is necessary for the differentiation of AIY and RMED, two additional cells that express CEH-10.     

The Caenorhabditis elegans odr-2 gene encodes a novel Ly-6-related protein required for olfaction

Caenorhabditis elegans odr-2 mutants are defective in the ability to chemotax to odorants that are recognized by the two AWC olfactory neurons. Like many other olfactory mutants, they retain responses to high concentrations of AWC-sensed odors; we show here that these residual responses are caused by the ability of other olfactory neurons (the AWA neurons) to be recruited at high odor concentrations. odr-2 encodes a membrane-associated protein related to the Ly-6 superfamily of GPI-linked signaling proteins and is the founding member of a C. elegans gene family with at least seven other members. Alternative splicing of odr-2 yields three predicted proteins that differ only at the extreme amino terminus. The three isoforms have different promoters, and one isoform may have a unique role in olfaction. An epitope-tagged ODR-2 protein is expressed at high levels in sensory neurons, motor neurons, and interneurons and is enriched in axons. The AWC neurons are superficially normal in their development and structure in odr-2 mutants, but their function is impaired. Our results suggest that ODR-2 may regulate AWC signaling within the neuronal network required for chemotaxis.     

eat-2 and eat-18 are required for nicotinic neurotransmission in the Caenorhabditis elegans pharynx

Mutations in eat-2 and eat-18 cause the same defect in C. elegans feeding behavior: the pharynx is unable to pump rapidly in the presence of food. EAT-2 is a nicotinic acetylcholine receptor subunit that functions in the pharyngeal muscle. It is localized to the synapse between pharyngeal muscle and the main pharyngeal excitatory motor neuron MC, and it is required for MC stimulation of pharyngeal muscle. eat-18 encodes a small protein that has no homology to previously characterized proteins. It has a single transmembrane domain and a short extracellular region. Allele-specific genetic interactions between eat-2 and eat-18 suggest that EAT-18 interacts physically with the EAT-2 receptor. While eat-2 appears to be required specifically for MC neurotransmission, eat-18 also appears to be required for the function of other nicotinic receptors in the pharynx. In eat-18 mutants, the gross localization of EAT-2 at the MC synapse is normal, suggesting that it is not required for trafficking. These data indicate that eat-18 could be a novel component of the pharyngeal nicotinic receptor.     

Overcoming redundancy: an RNAi enhancer screen for morphogenesis genes in Caenorhabditis elegans

Morphogenesis is an important component of animal development. Genetic redundancy has been proposed to be common among morphogenesis genes, posing a challenge to the genetic dissection of morphogenesis mechanisms. Genetic redundancy is more generally a challenge in biology, as large proportions of the genes in diverse organisms have no apparent loss of function phenotypes. Here, we present a screen designed to uncover redundant and partially redundant genes that function in an example of morphogenesis, gastrulation in Caenorhabditis elegans. We performed an RNA interference (RNAi) enhancer screen in a gastrulation-sensitized double-mutant background, targeting genes likely to be expressed in gastrulating cells or their neighbors. Secondary screening identified 16 new genes whose functions contribute to normal gastrulation in a nonsensitized background. We observed that for most new genes found, the closest known homologs were multiple other C. elegans genes, suggesting that some may have derived from rounds of recent gene duplication events. We predict that such genes are more likely than single copy genes to comprise redundant or partially redundant gene families. We explored this prediction for one gene that we identified and confirmed that this gene and five close relatives, which encode predicted substrate recognition subunits (SRSs) for a CUL-2 ubiquitin ligase, do indeed function partially redundantly with each other in gastrulation. Our results implicate new genes in C. elegans gastrulation, and they show that an RNAi-based enhancer screen in C. elegans can be used as an efficient means to identify important but redundant or partially redundant developmental genes.     

MEG-1 and MEG-2 are embryo-specific P-granule components required for germline development in Caenorhabditis elegans

In Caenorhabditis elegans, germ granules called P granules are directly inherited from mother to daughter and segregate with the germ lineage as it separates from the soma during initial embryonic cell divisions. Here we define meg-1 and meg-2 (maternal-effect germ-cell defective), which are expressed in the maternal germline and encode proteins that localize exclusively to P granules during embryonic germline segregation. Localization of MEG-1 to P granules depends upon the membrane-bound protein MES-1. meg-1 mutants exhibit multiple germline defects: P-granule mis-segregation in embryos, underproliferation and aberrant P-granule morphology in larval germ cells, and ultimately, sterility as adults. The penetrance of meg-1 phenotypes increases when meg-2 is also absent. Loss of the P-granule component pgl-1 in meg-1 mutants increases germ-cell proliferation, while loss of glh-1 decreases proliferation. Because meg-1 is provided maternally but its action is required in the embryonic germ lineage during segregation from somatic lineages, it provides a critical link for ensuring the continuity of germline development from one generation to the next.     

The Caenorhabditis elegans P21-activated kinases are differentially required for UNC-6/netrin-mediated commissural motor axon guidance

P21 activated kinases (PAKs) are major downstream effectors of rac-related small GTPases that regulate various cellular processes. We have identified the new PAK gene max-2 in a screen for mutants disrupted in UNC-6/netrin-mediated commissural axon guidance. There are three Caenorhabditis elegans PAKs. We find that each C. elegans PAK represents a distinct group previously identified in other species. Here we examine their roles in the postembryonic migration of the P cell neuroblasts and the axon guidance of the ventral cord commissural motoneurons (VCCMNs). We find that the two PAKs, max-2 and pak-1, are redundantly required for P cell migration and function with UNC-73/Trio and the rac GTPases (CED-10 and MIG-2). During axon guidance of the VCCMNs, PAK-1 also acts with the rac GTPases, CED-10 and MIG-2, and is completely redundant with MAX-2. Interestingly, we find that unlike MAX-2 activity during P cell migration, for motoneuron axon guidance max-2 is also required in parallel to this PAK-1 pathway, independent of rac GTPase signaling. Finally, we provide evidence that MAX-2 functions downstream of the UNC-6/netrin receptor UNC-5 during axon repulsion and is an integral part of its signaling.     

The Caenorhabditis elegans Six/sine oculis class homeobox gene ceh-32 is required for head morphogenesis

Repulsion plays a fundamental role in the establishment of a topographic map of the chick retinotectal projections. This has been highlighted by studies demonstrating the role of opposing gradients of the EphA3 receptor tyrosine kinase on retinal axons and two of its ligands, ephrin-A2 and ephrin-A5, in the tectum. We have analyzed the distribution of these two ephrins in other retinorecipient structures in the chick diencephalon and mesencephalon during the period when visual connections are being established. We have found that both ephrin-A2 and ephrin-A5 and their receptors EphA4 and EphA7 are expressed in gradients whose orientation is consistent with the topography of the nasotemporal axis of the respective retinofugal projections. In addition, their distribution suggests that receptor-ligand interactions may be involved in the organization of connections between the different primary visual centers and, thus, in the topographic organization of secondary visual projections. Interestingly, where projections lack a clear topographic representation, a uniform expression of the Eph-ephrin molecules was observed. Finally, we also show that a similar patterning mechanism may be implicated in the transfer of visual information to the telencephalon. These results suggest a conserved function for EphA receptors and their ligands in the elaboration of topographic maps at multiple levels of the visual pathway.     

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.     

The egg surface LDL receptor repeat-containing proteins EGG-1 and EGG-2 are required for fertilization in Caenorhabditis elegans

The molecular machinery that mediates sperm-egg interactions at fertilization is largely unknown. We identify two partially redundant egg surface LDL receptor repeat-containing proteins (EGG-1 and EGG-2) that are required for Caenorhabditis elegans fertility in hermaphrodites, but not males. Wild-type sperm cannot enter the morphologically normal oocytes produced by hermaphrodites that lack egg-1 and egg-2 function despite direct gamete contact. Furthermore, we find that levels of meiotic maturation/ovulation and sperm migratory behavior are altered in egg-1 mutants. These observations suggest an unexpected regulatory link between fertilization and other events necessary for reproductive success. egg-1 and egg-2 are the result of a gene duplication in the nematode lineage leading to C. elegans. The two closely related species C. briggsae and C. remanei encode only a single egg-1/egg-2 homolog that is required for hermaphrodite/female fertility. In addition to being the first identified egg components of the nematode fertilization machinery, the egg-1 and egg-2 gene duplication could be vital with regards to maximizing C. elegans fecundity and understanding the evolutionary differentiation of molecular function and speciation.     

The Caenorhabditis elegans FancD2 ortholog is required for survival following DNA damage

Fanconi anemia (FA) is an autosomal recessive disease characterized by bone-marrow failure, congenital abnormalities, and cancer susceptibility. There are 11 FA complementation groups in human where 8 genes have been identified. We found that FancD2 is conserved in evolution and present in the genome of the nematode Caenorhabditis elegans. The gene Y41E3.9 (CeFancD2) encodes a structural ortholog of human FANCD2 and is composed of 10 predicted exons. Our analysis showed that exons 6 and 7 were absent from a CeFancD2 EST suggesting the presence of a splice variant. In an attempt to characterize its role in DNA damage, we depleted worms of CeFANCD2 using RNAi. When the CeFANCD2(RNAi) worms were treated with a crosslinking agent, a significant drop in the progeny survival was noted. These worms were also sensitive, although to a lesser extent, to ionizing radiation (IR). Therefore, these data support an important role for CeFANCD2 in DNA damage response as for its human counterpart. The data also support the usefulness of C. elegans to study the Fanconi anemia pathway, and emphasize the biological importance of FANCD2 in DNA damage response throughout evolution.     

Tropomyosin and troponin are required for ovarian contraction in the Caenorhabditis elegans reproductive system

Ovulation in the nematode Caenorhabditis elegans is coordinated by interactions between the somatic gonad and germ cells. Myoepithelial sheath cells of the proximal ovary are smooth muscle-like cells, but the regulatory mechanism of their contraction is unknown. We show that contraction of the ovarian muscle requires tropomyosin and troponin, which are generally major actin-linked regulators of contraction of striated muscle. RNA interference of tropomyosin or troponin C caused sterility by inhibiting ovarian contraction that is required for expelling mature oocytes into the spermatheca where fertilization takes place, thus causing accumulation of endomitotic oocytes in the ovary. Tropomyosin and troponin C were associated with actin filaments in the myoepithelial sheath, and the association of troponin C with actin was dependent on tropomyosin. A mutation in the actin depolymerizing factor/cofilin gene suppressed the ovulation defects by RNA interference of tropomyosin or troponin C. These results strongly suggest that tropomyosin and troponin are the actin-linked regulators for contraction of ovarian muscle in the C. elegans reproductive system.     

Cholesterol and the biosynthesis of glycosphingolipids are required for sperm activation in Caenorhabditis elegans

Ejaculated mammalian sperm must acquire fertilization capacity after residing into the female reproductive tract, a process collectively known as capacitation. Cholesterol efflux was required for sperm maturation. Different from flagellated sperm, C. elegans sperm are crawling cells. C. elegans sperm are highly enriched with cholesterol though this animal species lacks biosynthetic pathway for cholesterol and its survival requires an exogenous cholesterol supply. The low abundance of cholesterol in C. elegans lipid extract is thought insufficient to form lipid microdomains ubiquitously in this organism. We present evidence that cholesterol is enriched in the plasma membrane of C. elegans spermatids and that cholesterol- and glycosphingolipids (GSLs)-enriched membrane microdomains (lipid microdomains) mediate sperm activation. Disruption of sperm lipid microdomains by acute manipulation of cholesterol in vitro blocks the sperm activation. Restriction of cholesterol uptake also results in the abnormal sperm activation in both males and hermaphrodites. Manipulation of the integrity of lipid microdomains by targeting the biosynthesis of GSLs inhibits sperm activation and the inhibition can be rescued by the addition of exogenous GSLs. The cleavage of glycosylphosphatidylinositol (GPI)-anchored proteins, which are exclusively found in lipid microdomains, also affects sperm activation. We conclude that localized signaling mediated by lipid microdomains is critical for worm sperm activation. Lipid microdomains composed of cholesterol and GSLs have been observed in flagellated sperm of several animal species, thus cholesterol, before its efflux from the plasma membrane, might be needed to assemble into a platform for some more important upstream signal sorting during spermatogenesis than was previously thought.