Systematic interactome mapping and genetic perturbation analysis of a C. elegans TGF-beta signaling network

To initiate a system-level analysis of C. elegans DAF-7/TGF-beta signaling, we combined interactome mapping with single and double genetic perturbations. Yeast two-hybrid (Y2H) screens starting with known DAF-7/TGF-beta pathway components defined a network of 71 interactions among 59 proteins. Coaffinity purification (co-AP) assays in mammalian cells confirmed the overall quality of this network. Systematic perturbations of the network using RNAi, both in wild-type and daf-7/TGF-beta pathway mutant animals, identified nine DAF-7/TGF-beta signaling modifiers, seven of which are conserved in humans. We show that one of these has functional homology to human SNO/SKI oncoproteins and that mutations at the corresponding genetic locus daf-5 confer defects in DAF-7/TGF-beta signaling. Our results reveal substantial molecular complexity in DAF-7/TGF-beta signal transduction. Integrating interactome maps with systematic genetic perturbations may be useful for developing a systems biology approach to this and other signaling modules.     

A genetic analysis of axon guidance in the C elegans pharynx

We wish to understand how the trajectories of the twenty pharyngeal neurons of C. elegans are established. In this study we focused on the two bilateral M2 pharyngeal motorneurons, which each have their cell body located in the posterior bulb and send one axon through the isthmus and into the metacorpus. We used a GFP reporter to visualize these neurons in cell-autonomous and cell-non-autonomous axon guidance mutant backgrounds, as well as other mutant classes. Our main findings are: 1). Mutants with impaired growth cone functions, such as unc-6, unc-51, unc-73 and sax-3, often exhibit abnormal terminations and inappropriate trajectories at the distal ends of the M2 axons, i.e. within the metacorpus; and 2). Growth cone function mutants never exhibit abnormalities in the proximal part of the M2 neuron trajectories, i.e. between the cell body and the metacorpus. Our results suggest that the proximal and distal trajectories are established using distinct mechanisms, including a growth cone-independent process to establish the proximal trajectory. We isolated five novel mutants in a screen for worms exhibiting abnormal morphology of the M2 neurons. These mutants define a new gene class designated mnm (M neuron morphology abnormal).     

High-throughput behavioral analysis in C. elegans

We designed a real-time computer vision system, the Multi-Worm Tracker (MWT), which can simultaneously quantify the behavior of dozens of Caenorhabditis elegans on a Petri plate at video rates. We examined three traditional behavioral paradigms using this system: spontaneous movement on food, where the behavior changes over tens of minutes; chemotaxis, where turning events must be detected accurately to determine strategy; and habituation of response to tap, where the response is stochastic and changes over time. In each case, manual analysis or automated single-worm tracking would be tedious and time-consuming, but the MWT system allowed rapid quantification of behavior with minimal human effort. Thus, this system will enable large-scale forward and reverse genetic screens for complex behaviors.     

The initiation of spermiogenesis in the nematode Caenorhabditis elegans

Spermiogenesis in nematodes involves the activation of sessile spherical spermatids to motile bipolar amoeboid spermatozoa. In Caenorhabditis elegans males spermiogenesis is normally induced by copulation. Spermatids transferred to hermaphrodites as well as some of those left behind in the male become spermatozoa a few minutes after mating. Spermiogenesis can also be induced in vitro by the ionophore monensin (G.A. Nelson and S. Ward, 1980, Cell 19, 457-464) and by weak bases such as triethanolamine. Both triethanolamine and monensin cause a rapid increase in intracellular pH from 7.1 to 7.5 or 8.0. This pH increase precedes the subsequent morphological events of spermiogenesis. Triethanolamine or monensin must be present throughout spermiogenesis for all cells to form pseudopods, but once pseudopods are formed the inducers are unnecessary for subsequent motility. The pH induced spermiogenesis is inhibited by drugs that block mitochondria or glycolysis. Protease treatment can also induce spermiogenesis without increasing intracellular pH, apparently bypassing the pH-dependent steps in activation and the requirement for glycolysis. These results show that the initiation of spermiogenesis in C. elegans, like some steps in egg activation and the initiation of sea urchin sperm motility, can be induced by an increase in intracellular pH, but this pH change can be bypassed by proteolysis.     

Hakuna Nematoda: genetic and phenotypic diversity in African isolates of Caenorhabditis elegans and C. briggsae

Caenorhabditis elegans and C. briggsae have many parallels in terms of morphology, life history and breeding system. Both species also share similar low levels of molecular diversity, although the global sampling of natural populations has been limited and geographically biased. In this study, we describe the first cultured isolates of C. elegans and C. briggsae from sub-Saharan Africa. We characterize these samples for patterns of nucleotide polymorphism and vulva precursor cell lineage, and conduct a series of hybrid crosses in C. briggsae to test for genetic incompatibilities. The distribution of genetic diversity confirms a lack of geographic structure to C. elegans sequences but shows genetic differentiation of C. briggsae into three distinct clades that may correspond to three latitudinal ranges. Despite low levels of molecular diversity, we find considerable variation in cell division frequency in African C. elegans for the P3.p vulva precursor cell, and in African C. briggsae for P4.p, a variation that was not previously observed in this species. Hybrid crosses did not reveal major incompatibilities between C. briggsae strains from Africa and elsewhere, and there was some evidence of inbreeding depression. These new African isolates suggest that important ecological factors may be shaping the patterns of diversity in C. briggsae, and that despite many similarities between C. elegans and C. briggsae, there may be more subtle differences in their natural histories than previously appreciated.     

Axon regeneration pathways identified by systematic genetic screening in C. elegans

The mechanisms underlying the ability of axons to regrow after injury remain poorly explored at the molecular genetic level. We used a laser injury model in Caenorhabditis elegans mechanosensory neurons to screen 654 conserved genes for regulators of axonal regrowth. We uncover several functional clusters of genes that promote or repress regrowth, including genes classically known to affect axon guidance, membrane excitability, neurotransmission, and synaptic vesicle endocytosis. The conserved Arf Guanine nucleotide Exchange Factor (GEF), EFA-6, acts as an intrinsic inhibitor of regrowth. By combining genetics and in?vivo imaging, we show that EFA-6 inhibits regrowth via microtubule dynamics, independent of its Arf GEF activity. Among newly identified regrowth inhibitors, only loss of function in EFA-6 partially bypasses the requirement for DLK-1 kinase. Identification of these pathways significantly expands our understanding of the genetic basis of axonal injury responses and repair.     

Developmental apoptosis in C. elegans: a complex CEDnario

Apoptosis, an evolutionarily conserved programme of cellular self-destruction, is essential for the development and survival of most multicellular animals. It is required to ensure functional organ architecture and to maintain tissue homeostasis. During development of the simple nematode Caenorhabditis elegans, apoptosis claims over 10% of the somatic cells that are generated - these cells were healthy but unnecessary. Exciting insights into the regulation and execution of apoptosis in C. elegans have recently been made. These new findings will undoubtedly influence our perception of developmental apoptosis in more complex species, including humans.     

Investigating C. elegans development through mosaic analysis

The analysis of genetically mosaic worms, in which some cells carry a wild-type gene and others are homozygous mutant, can reveal where in the animal a gene acts to prevent the appearance of a mutant phenotype. In this primer article, we describe how Caenorhabditis elegans genetic mosaics are generated, identified and analyzed, and we discuss examples in which the analysis of mosaic worms has provided important information about the development of this organism.     

Quantitative analysis of germline mitosis in adult C. elegans

Certain aspects of the distal gonad of C. elegans are comparable to niche/stem cell systems in other organisms. The distal tip cell (DTC) caps a blind-ended tube; only the distal germ cells maintain proliferation in response to signaling from the DTC via the GLP-1/Notch signaling pathway in the germ line. Fruitful comparison between this system and other stem cell systems is limited by a lack of basic information regarding germ cell division behavior in C. elegans. Here, we explore the spatial pattern of cell division frequency in the adult C. elegans germ line relative to distance from the distal tip. We mapped the positions of actively dividing germline nuclei in over 600 fixed gonad preparations including the wild type and a gain-of-function ligand-responsive GLP-1 receptor mutant with an extended mitotic zone. One particularly surprising observation from these data is that the frequency of cell divisions is lower in distal-most cells-cells that directly contact the distal tip cell body-relative to cells further proximal, a difference that persists in the gain-of-function GLP-1 mutant. These results suggest that cell division frequency in the distal-most cells may be suppressed or otherwise controlled in a complex manner. Further, our data suggest that the presence of an active cell division influences the probability of observing simultaneous cell divisions in the same gonad arm, and that simultaneous divisions tend to cluster spatially. We speculate that this system behaves similarly to niche/stem cell/transit amplifying cell systems in other organisms.     

Systematic analysis of pleiotropy in C. elegans early embryogenesis

Pleiotropy refers to the phenomenon in which a single gene controls several distinct, and seemingly unrelated, phenotypic effects. We use C. elegans early embryogenesis as a model to conduct systematic studies of pleiotropy. We analyze high-throughput RNA interference (RNAi) data from C. elegans and identify "phenotypic signatures", which are sets of cellular defects indicative of certain biological functions. By matching phenotypic profiles to our identified signatures, we assign genes with complex phenotypic profiles to multiple functional classes. Overall, we observe that pleiotropy occurs extensively among genes involved in early embryogenesis, and a small proportion of these genes are highly pleiotropic. We hypothesize that genes involved in early embryogenesis are organized into partially overlapping functional modules, and that pleiotropic genes represent "connectors" between these modules. In support of this hypothesis, we find that highly pleiotropic genes tend to reside in central positions in protein-protein interaction networks, suggesting that pleiotropic genes act as connecting points between different protein complexes or pathways.     

Innexins in C. elegans

Innexins are functionally analogous to the vertebrate connexins, and the innexin family of gap junction proteins has been identified in many invertebrates, including Drosophila and C. elegans. The genome sequencing project has identified 25 innexins in C. elegans. We are particularly interested in the roles that gap junctions may play in embryonic development and in wiring of the nervous system. To identify the particular C. elegans innexins that are involved in these processes, we are examining their expression patterns using specific antibodies and translational GFP fusions. In addition we are investigating mutant, RNAi and overexpression phenotypes for many of these genes. To date, we have generated specific antibodies to the non-conserved carboxyl termini of 5 innexins. We have constructed GFP translational fusions for 17 innexins and observed expression patterns for 13 of these genes. In total we have characterized expression patterns representing 14 innexins. Mutations have been identified in 5 of these genes, and at least 3 others have RNAi mutant phenotypes. Generalities emerging from our studies include: 1) most tissues and many individual cells express more than one innexin, 2) some innexins are expressed widely, while others are expressed in only a few cells, and 3) there is a potential for functional pairing of innexins.     

Functional genomic analysis of apoptotic DNA degradation in C. elegans

Chromosomal DNA degradation is critical for cell death execution and is a hallmark of apoptosis, yet little is known about how this process is executed. Using an RNAi-based functional genomic approach, we have identified seven additional cell death-related nucleases (crn genes), which along with two known nucleases (CPS-6 and NUC-1) comprise at least two independent pathways that contribute to cell killing, and likely signaling for phagocytosis, by degrading chromosomal DNA. Several crn genes have human homologs that are important for RNA processing, protein folding, DNA replication, and DNA damage repair, suggesting dual roles for CRN nucleases in cell survival and cell death. It should now be possible to systematically decipher the mechanisms of apoptotic DNA degradation.     

In vivo analysis of conserved C. elegans tomosyn domains

Neurosecretion is critically dependent on the assembly of a macromolecular complex between the SNARE proteins syntaxin, SNAP-25 and synaptobrevin. Evidence indicates that the binding of tomosyn to syntaxin and SNAP-25 interferes with this assembly, thereby negatively regulating both synaptic transmission and peptide release. Tomosyn has two conserved domains: an N-terminal encompassing multiple WD40 repeats predicted to form two β-propeller structures and a C-terminal SNARE-binding motif. To assess the function of each domain, we performed an in vivo analysis of the N- and C- terminal domains of C. elegans tomosyn (TOM-1) in a tom-1 mutant background. We verified that both truncated TOM-1 constructs were transcribed at levels comparable to rescuing full-length TOM-1, were of the predicted size, and localized to synapses. Unlike full-length TOM-1, expression of the N- or C-terminal domains alone was unable to restore inhibitory control of synaptic transmission in tom-1 mutants. Similarly, co-expression of both domains failed to restore TOM-1 function. In addition, neither the N- nor C-terminal domain inhibited release when expressed in a wild-type background. Based on these results, we conclude that the ability of tomosyn to regulate neurotransmitter release in vivo depends on the physical integrity of the protein, indicating that both N- and C-terminal domains are necessary but not sufficient for effective inhibition of release in vivo.     

C. elegans model identifies genetic modifiers of alpha-synuclein inclusion formation during aging

Inclusions in the brain containing alpha-synuclein are the pathological hallmark of Parkinson's disease, but how these inclusions are formed and how this links to disease is poorly understood. We have developed a C. elegans model that makes it possible to monitor, in living animals, the formation of alpha-synuclein inclusions. In worms of old age, inclusions contain aggregated alpha- synuclein, resembling a critical pathological feature. We used genome-wide RNA interference to identify processes involved in inclusion formation, and identified 80 genes that, when knocked down, resulted in a premature increase in the number of inclusions. Quality control and vesicle-trafficking genes expressed in the ER/Golgi complex and vesicular compartments were overrepresented, indicating a specific role for these processes in alpha-synuclein inclusion formation. Suppressors include aging-associated genes, such as sir-2.1/SIRT1 and lagr-1/LASS2. Altogether, our data suggest a link between alpha-synuclein inclusion formation and cellular aging, likely through an endomembrane-related mechanism. The processes and genes identified here present a framework for further study of the disease mechanism and provide candidate susceptibility genes and drug targets for Parkinson's disease and other alpha-synuclein related disorders.     

MyoD and myogenesis in C. elegans

One of the goals in developmental biology is the identification of key regulatory genes that govern the transition of embryonic cells from a pluripotent potential to a specific, committed cell fate. During vertebrate skeletal myogenesis, this transition is regulated by the MyoD family of genes. C. elegans has muscle analogous to vertebrate skeletal muscle and has a gene(hlh-1) related to the MyoD family. The molecular and genetic characterization of hlh-1 shows that it is very similar to the vertebrate MyoD family in many respects, including its expression pattern and DNA binding activity. The hlh-1 product is required for proper myogenesis, but it is not required for myogenic commitment during embryogenesis in the nematode. The role of this MyoD-related gene in nematode myogenesis is discussed and compared to those of the vertebrate MyoD family.     

spe-12 encodes a sperm cell surface protein that promotes spermiogenesis in Caenorhabditis elegans

During spermiogenesis, Caenorhabditis elegans spermatids activate and mature into crawling spermatozoa without synthesizing new proteins. Mutations in the spe-12 gene block spermatid activation, rendering normally self-fertile hermaphrodites sterile. Mutant males, however, are fertile. Surprisingly, when mutant hermaphrodites mate with a male, their self-spermatids activate and form functional spermatozoa, presumably due to contact with male seminal fluid. Here we show that, in addition to its essential role in normal activation of hermaphrodite-derived spermatids, SPE-12 also plays a supplementary but nonessential role in mating-induced activation. We have identified the spe-12 gene, which encodes a novel protein containing a single transmembrane domain. spe-12 mRNA is expressed in the sperm-producing germ line and the protein localizes to the spermatid cell surface. We propose that SPE-12 functions downstream of both hermaphrodite- and male-derived activation signals in a spermatid signaling pathway that initiates spermiogenesis.