Professor Emeritus, Developmental Biology
Please see my lab website for description of research and publications. I do not update this site often.
The normal aging process is associated with stereotyped changes in gene expression, but the regulators responsible for these age-dependent changes are poorly understood. Using a novel genomics approach, we identified HOX co-factor unc-62 (Homothorax) as a developmental regulator that binds proximal to age-regulated genes and modulates lifespan. Although unc-62 is expressed in diverse tissues, its functions in the intestine play a particularly important role in modulating lifespan, as intestine-specific knockdown of unc-62 by RNAi increases lifespan. An alternatively-spliced, tissue-specific isoform of unc-62 is expressed exclusively in the intestine and declines with age. Through analysis of the downstream consequences of unc-62 knockdown, we identify multiple effects linked to aging. First, unc-62 RNAi decreases the expression of yolk proteins (vitellogenins) that aggregate in the body cavity in old age. Second, unc-62 RNAi results in a broad increase in expression of intestinal genes that typically decrease expression with age, suggesting that unc-62 activity balances intestinal resource allocation between yolk protein expression and fertility on the one hand and somatic functions on the other. Finally, in old age, the intestine shows increased expression of several aberrant genes; these UNC-62 targets are expressed predominantly in neuronal cells in developing animals, but surprisingly show increased expression in the intestine of old animals. Intestinal expression of some of these genes during aging is detrimental for longevity; notably, increased expression of insulin ins-7 limits lifespan by repressing activity of insulin pathway response factor DAF-16/FOXO in aged animals. These results illustrate how unc-62 regulation of intestinal gene expression is responsible for limiting lifespan during the normal aging process.
View details for DOI 10.1371/journal.pgen.1003325
View details for PubMedID 23468654
We have taken an engineering approach to extending the lifespan of Caenorhabditis elegans. Aging stands out as a complex trait, because events that occur in old animals are not under strong natural selection. As a result, lifespan can be lengthened rationally using bioengineering to modulate gene expression or to add exogenous components. Here, we engineered longer lifespan by expressing genes from zebrafish encoding molecular functions not normally present in worms. Additionally, we extended lifespan by increasing the activity of four endogenous worm aging pathways. Next, we used a modular approach to extend lifespan by combining components. Finally, we used cell- and worm-based assays to analyze changes in cell physiology and as a rapid means to evaluate whether multi-component transgenic lines were likely to have extended longevity. Using engineering to add novel functions and to tune endogenous functions provides a new framework for lifespan extension that goes beyond the constraints of the worm genome.
View details for DOI 10.1371/journal.pgen.1002780
View details for PubMedID 22737090
A common property of aging in all animals is that chronologically and genetically identical individuals age at different rates. To unveil mechanisms that influence aging variability, we identified markers of remaining lifespan for Caenorhabditis elegans. In transgenic lines, we expressed fluorescent reporter constructs from promoters of C. elegans genes whose expression change with age. The expression levels of aging markers in individual worms from a young synchronous population correlated with their remaining lifespan. We identified eight aging markers, with the superoxide dismutase gene sod-3 expression being the best single predictor of remaining lifespan. Correlation with remaining lifespan became stronger if expression from two aging markers was monitored simultaneously, accounting for up to 49% of the variation in individual lifespan. Visualizing the physiological age of chronologically-identical individuals allowed us to show that a major source of lifespan variability is different pathogenicity from individual to individual and that the mechanism involves variable activation of the insulin-signaling pathway.
View details for DOI 10.1371/journal.pgen.1002047
View details for PubMedID 21533182
To define the C. elegans aging process at the molecular level, we used DNA microarray experiments to identify a set of 1294 age-regulated genes and found that the GATA transcription factors ELT-3, ELT-5, and ELT-6 are responsible for age regulation of a large fraction of these genes. Expression of elt-5 and elt-6 increases during normal aging, and both of these GATA factors repress expression of elt-3, which shows a corresponding decrease in expression in old worms. elt-3 regulates a large number of downstream genes that change expression in old age, including ugt-9, col-144, and sod-3. elt-5(RNAi) and elt-6(RNAi) worms have extended longevity, indicating that elt-3, elt-5, and elt-6 play an important functional role in the aging process. These results identify a transcriptional circuit that guides the rapid aging process in C. elegans and indicate that this circuit is driven by drift of developmental pathways rather than accumulation of damage.
View details for DOI 10.1016/j.cell.2008.05.044
View details for PubMedID 18662544
We present the AGEMAP (Atlas of Gene Expression in Mouse Aging Project) gene expression database, which is a resource that catalogs changes in gene expression as a function of age in mice. The AGEMAP database includes expression changes for 8,932 genes in 16 tissues as a function of age. We found great heterogeneity in the amount of transcriptional changes with age in different tissues. Some tissues displayed large transcriptional differences in old mice, suggesting that these tissues may contribute strongly to organismal decline. Other tissues showed few or no changes in expression with age, indicating strong levels of homeostasis throughout life. Based on the pattern of age-related transcriptional changes, we found that tissues could be classified into one of three aging processes: (1) a pattern common to neural tissues, (2) a pattern for vascular tissues, and (3) a pattern for steroid-responsive tissues. We observed that different tissues age in a coordinated fashion in individual mice, such that certain mice exhibit rapid aging, whereas others exhibit slow aging for multiple tissues. Finally, we compared the transcriptional profiles for aging in mice to those from humans, flies, and worms. We found that genes involved in the electron transport chain show common age regulation in all four species, indicating that these genes may be exceptionally good markers of aging. However, we saw no overall correlation of age regulation between mice and humans, suggesting that aging processes in mice and humans may be fundamentally different.
View details for DOI 10.1371/journal.pgen.0030201
View details for PubMedID 18081424
We analyzed expression of 81 normal muscle samples from humans of varying ages, and have identified a molecular profile for aging consisting of 250 age-regulated genes. This molecular profile correlates not only with chronological age but also with a measure of physiological age. We compared the transcriptional profile of muscle aging to previous transcriptional profiles of aging in the kidney and the brain, and found a common signature for aging in these diverse human tissues. The common aging signature consists of six genetic pathways; four pathways increase expression with age (genes in the extracellular matrix, genes involved in cell growth, genes encoding factors involved in complement activation, and genes encoding components of the cytosolic ribosome), while two pathways decrease expression with age (genes involved in chloride transport and genes encoding subunits of the mitochondrial electron transport chain). We also compared transcriptional profiles of aging in humans to those of the mouse and fly, and found that the electron transport chain pathway decreases expression with age in all three organisms, suggesting that this may be a public marker for aging across species.
View details for DOI 10.1371/journal.pgen.0020115
View details for PubMedID 16789832
In this study, we found 985 genes that change expression in the cortex and the medulla of the kidney with age. Some of the genes whose transcripts increase in abundance with age are known to be specifically expressed in immune cells, suggesting that immune surveillance or inflammation increases with age. The age-regulated genes show a similar aging profile in the cortex and the medulla, suggesting a common underlying mechanism for aging. Expression profiles of these age-regulated genes mark not only age, but also the relative health and physiology of the kidney in older individuals. Finally, the set of aging-regulated kidney genes suggests specific mechanisms and pathways that may play a role in kidney degeneration with age.
View details for DOI 10.1371/journal.pbio.0020427
View details for Web of Science ID 000226099600020
View details for PubMedID 15562319
View details for PubMedCentralID PMC532391
To elucidate gene function on a global scale, we identified pairs of genes that are coexpressed over 3182 DNA microarrays from humans, flies, worms, and yeast. We found 22,163 such coexpression relationships, each of which has been conserved across evolution. This conservation implies that the coexpression of these gene pairs confers a selective advantage and therefore that these genes are functionally related. Many of these relationships provide strong evidence for the involvement of new genes in core biological functions such as the cell cycle, secretion, and protein expression. We experimentally confirmed the predictions implied by some of these links and identified cell proliferation functions for several genes. By assembling these links into a gene-coexpression network, we found several components that were animal-specific as well as interrelationships between newly evolved and ancient modules.
View details for DOI 10.1126/science.1087447
View details for Web of Science ID 000185825900032
View details for PubMedID 12934013
Chromosomes are divided into domains of open chromatin, where genes have the potential to be expressed, and domains of closed chromatin, where genes are not expressed. Classic examples of open chromatin domains include 'puffs' on polytene chromosomes in Drosophila and extended loops from lampbrush chromosomes. If multiple genes were typically expressed together from a single open chromatin domain, the position of co-expressed genes along the chromosomes would appear clustered. To investigate whether co-expressed genes are clustered, we examined the chromosomal positions of the genes expressed in the muscle of Caenorhabditis elegans at the first larval stage. Here we show that co-expressed genes in C. elegans are clustered in groups of 2-5 along the chromosomes, suggesting that expression from a chromatin domain can extend over several genes. These observations reveal a higher-order organization of the structure of the genome, in which the order of the genes along the chromosome id correlated with their expression in specific tissues.
View details for DOI 10.1038/nature01012
View details for PubMedID 12214599
We have assembled data from Caenorhabditis elegans DNA microarray experiments involving many growth conditions, developmental stages, and varieties of mutants. Co-regulated genes were grouped together and visualized in a three-dimensional expression map that displays correlations of gene expression profiles as distances in two dimensions and gene density in the third dimension. The gene expression map can be used as a gene discovery tool to identify genes that are co-regulated with known sets of genes (such as heat shock, growth control genes, germ line genes, and so forth) or to uncover previously unknown genetic functions (such as genomic instability in males and sperm caused by specific transposons).
View details for PubMedID 11557892
We develop a new method for large-scale frequentist multiple testing with Bayesian prior information. We find optimal [Formula: see text]-value weights that maximize the average power of the weighted Bonferroni method. Due to the nonconvexity of the optimization problem, previous methods that account for uncertain prior information are suitable for only a small number of tests. For a Gaussian prior on the effect sizes, we give an efficient algorithm that is guaranteed to find the optimal weights nearly exactly. Our method can discover new loci in genome-wide association studies and compares favourably to competitors. An open-source implementation is available.
View details for DOI 10.1093/biomet/asv050
View details for Web of Science ID 000366379000001
View details for PubMedCentralID PMC4813057
Aging in Caenorhabditis elegans is characterized by widespread physiological and molecular changes, but the mechanisms that determine the rate at which these changes occur are not well understood. In this study, we identify a novel link between reproductive aging and somatic aging in C. elegans. By measuring global age-related changes in the proteome, we identify a previously uncharacterized group of secreted proteins in the adult uterus that dramatically increase in abundance with age. This accumulation is blunted in animals with an extended reproductive period and accelerated in sterile animals lacking a germline. Uterine proteins are not removed in old post-reproductive animals or in young vulvaless worms, indicating that egg-laying is necessary for their rapid removal in wild-type young animals. Together, these results suggest that age-induced infertility contributes to extracellular protein accumulation in the uterus with age. Finally, we show that knocking down multiple age-increased proteins simultaneously extends lifespan. These results provide a mechanistic example of how the cessation of reproduction contributes to detrimental changes in the soma, and demonstrate how the timing of reproductive decline can influence the rate of aging.
View details for DOI 10.1371/journal.pgen.1005725
View details for Web of Science ID 000368518400054
View details for PubMedCentralID PMC4676719
We developed a new statistical framework to find genetic variants associated with extreme longevity. The method, informed GWAS (iGWAS), takes advantage of knowledge from large studies of age-related disease in order to narrow the search for SNPs associated with longevity. To gain support for our approach, we first show there is an overlap between loci involved in disease and loci associated with extreme longevity. These results indicate that several disease variants may be depleted in centenarians versus the general population. Next, we used iGWAS to harness information from 14 meta-analyses of disease and trait GWAS to identify longevity loci in two studies of long-lived humans. In a standard GWAS analysis, only one locus in these studies is significant (APOE/TOMM40) when controlling the false discovery rate (FDR) at 10%. With iGWAS, we identify eight genetic loci to associate significantly with exceptional human longevity at FDR < 10%. We followed up the eight lead SNPs in independent cohorts, and found replication evidence of four loci and suggestive evidence for one more with exceptional longevity. The loci that replicated (FDR < 5%) included APOE/TOMM40 (associated with Alzheimer's disease), CDKN2B/ANRIL (implicated in the regulation of cellular senescence), ABO (tags the O blood group), and SH2B3/ATXN2 (a signaling gene that extends lifespan in Drosophila and a gene involved in neurological disease). Our results implicate new loci in longevity and reveal a genetic overlap between longevity and age-related diseases and traits, including coronary artery disease and Alzheimer's disease. iGWAS provides a new analytical strategy for uncovering SNPs that influence extreme longevity, and can be applied more broadly to boost power in other studies of complex phenotypes.
View details for DOI 10.1371/journal.pgen.1005728
View details for Web of Science ID 000368518400057
View details for PubMedCentralID PMC4683064
Stress is a fundamental aspect of aging, as accumulated damage from a lifetime of stress can limit lifespan and protective responses to stress can extend lifespan. In this study, we identify a conserved Caenorhabditis elegans GATA transcription factor, egl-27, that is involved in several stress responses and aging. We found that overexpression of egl-27 extends the lifespan of wild-type animals. Furthermore, egl-27 is required for the pro-longevity effects from impaired insulin/IGF-1 like signaling (IIS), as reduced egl-27 activity fully suppresses the longevity of worms that are mutant for the IIS receptor, daf-2. egl-27 expression is inhibited by daf-2 and activated by pro-longevity factors daf-16/FOXO and elt-3/GATA, suggesting that egl-27 acts at the intersection of IIS and GATA pathways to extend lifespan. Consistent with its role in IIS signaling, we found that egl-27 is involved in stress response pathways. egl-27 expression is induced in the presence of multiple stresses, its targets are significantly enriched for many types of stress genes, and altering levels of egl-27 itself affects survival to heat and oxidative stress. Finally, we found that egl-27 expression increases between young and old animals, suggesting that increased levels of egl-27 in aged animals may act to promote stress resistance. These results identify egl-27 as a novel factor that links stress and aging pathways.
View details for DOI 10.1371/journal.pgen.1003108
View details for PubMedID 23271974
The decreasing cost of genotyping and genome sequencing has ushered in an era of genomic personalized medicine. More than 100,000 individuals have been genotyped by direct-to-consumer genetic testing services, which offer a glimpse into the interpretation and exploration of a personal genome. However, these interpretations, which require extensive manual curation, are subject to the preferences of the company and are not customizable by the individual. Academic institutions teaching personalized medicine, as well as genetic hobbyists, may prefer to customize their analysis and have full control over the content and method of interpretation. We present the Interpretome, a system for private genome interpretation, which contains all genotype information in client-side interpretation scripts, supported by server-side databases. We provide state-of-the-art analyses for teaching clinical implications of personal genomics, including disease risk assessment and pharmacogenomics. Additionally, we have implemented client-side algorithms for ancestry inference, demonstrating the power of these methods without excessive computation. Finally, the modular nature of the system allows for plugin capabilities for custom analyses. This system will allow for personal genome exploration without compromising privacy, facilitating hands-on courses in genomics and personalized medicine.
View details for PubMedID 22174289
There has been a recent explosion in the wealth of genomic data available to C. elegans researchers, as efforts to characterize gene expression and its regulators at a molecular level have borne significant fruit. Detailed measurement of gene expression at a variety of developmental stages, and in numerous individual tissues, has dramatically increased our understanding of cell-type-specific gene expression networks. Characterization of the targets of transcription factors, chromatin-binding proteins, and miRNAs has provided genome-wide insights into the mechanisms governing gene expression. Development of new techniques have allowed this characterization to begin to shift from whole-organism studies to tissue-level, and even single-cell-level profiling, creating a first glimpse into gene regulatory circuits at the single-cell level in a living organism. Integration of these datasets has yielded novel insights into evolution, gene expression regulation, and the link between sequence and phenotype.
View details for DOI 10.1016/j.gde.2011.08.007
View details for PubMedID 21963133
The inherent simplicity of Caenorhabditis elegans and its extensive genetic toolkit make it ideal for studying complex biological processes. Recent developments further increase the usefulness of the worm, including new methods for: altering gene expression, altering physiology using optogenetics, manipulating large numbers of worms, automating laborious processes and processing high-resolution images. These developments both enhance the worm as a model for studying processes such as development and ageing and make it an attractive model in areas such as neurobiology and behaviour.
View details for DOI 10.1038/nrg3050
View details for PubMedID 21969037
This paper takes a close look at balanced permutations, a recently developed sample reuse method with applications in bioinformatics. It turns out that balanced permutation reference distributions do not have the correct null behavior, which can be traced to their lack of a group structure. We find that they can give p-values that are too permissive to varying degrees. In particular the observed test statistic can be larger than that of all B balanced permutations of a data set with a probability much higher than 1/(B + 1), even under the null hypothesis.
View details for DOI 10.1089/cmb.2008.0144
View details for PubMedID 19361331
Using DNA microarrays to generate transcriptional profiles of the aging process is a powerful tool for identifying biomarkers of aging. In Caenorhabditis elegans, a number of whole-genome profiling studies identified genes that change expression levels with age. High-throughput RNAi screens in worms determined a number of genes that modulate lifespan when silenced. Transcriptional profiling of the fly head identified a molecular pathway, the 'response to light' gene set, that increases expression with age and could be directly related to the tendency for a reduction in light levels to extend fly's lifespan. In mouse, comparing the gene expression profiles of several drugs to the gene expression profile of caloric restriction identified metformin as a drug whose action could potentially mimic caloric restriction in vivo. Finally, genes in the mitochondrial electron transport chain group decrease expression with age in the human, mouse, fly, and worm.
View details for DOI 10.1016/j.copbio.2007.07.004
View details for PubMedID 17681777
Development of functional genomics tools has made it possible to define the aging process by performing genome-wide scans for transcriptional differences between the young and the old. Global screens for age regulation have been performed for worms and flies, as well as many tissues in mice and humans. Recent work has begun to analyze the similarities and differences in transcriptional changes in aging among different species. Most age-related expression changes are specific for a given species, but genes in one pathway (the electron transport chain pathway) show common age regulation in species from worms to humans. Evolutionary theories of aging provide a basis to understand how age regulation of a genetic pathway might be preserved between distantly related species.
View details for DOI 10.1242/jeb.004887
View details for PubMedID 17449826
We used mRNA tagging to identify genes expressed in the intestine of C. elegans. Animals expressing an epitope-tagged protein that binds the poly-A tail of mRNAs (FLAG::PAB-1) from an intestine-specific promoter (ges-1) were used to immunoprecipitate FLAG::PAB-1/mRNA complexes from the intestine. A total of 1938 intestine-expressed genes (P<0.001) were identified using DNA microarrays. First, we compared the intestine-expressed genes with those expressed in the muscle and germline, and identified 510 genes enriched in all three tissues and 624 intestine-, 230 muscle- and 1135 germ line-enriched genes. Second, we showed that the 1938 intestine-expressed genes were physically clustered on the chromosomes, suggesting that the order of genes in the genome is influenced by the effect of chromatin domains on gene expression. Furthermore, the commonly expressed genes showed more chromosomal clustering than the tissue-enriched genes, suggesting that chromatin domains may influence housekeeping genes more than tissue-specific genes. Third, in order to gain further insight into the regulation of intestinal gene expression, we searched for regulatory motifs. This analysis found that the promoters of the intestine genes were enriched for the GATA transcription factor consensus binding sequence. We experimentally verified these results by showing that the GATA motif is required in cis and that GATA transcription factors are required in trans for expression of these intestinal genes.
View details for DOI 10.1242/dev.02185
View details for PubMedID 16354718
One of the most important uses of whole-genome expression data is for the discovery of new genes with similar function to a given list of genes (the query) already known to have closely related function. We have developed an algorithm, called the gene recommender, that ranks genes according to how strongly they correlate with a set of query genes in those experiments for which the query genes are most strongly coregulated. We used the gene recommender to find other genes coexpressed with several sets of query genes, including genes known to function in the retinoblastoma complex. Genetic experiments confirmed that one gene (JC8.6) identified by the gene recommender acts with lin-35 Rb to regulate vulval cell fates, and that another gene (wrm-1) acts antagonistically. We find that the gene recommender returns lists of genes with better precision, for fixed levels of recall, than lists generated using the C. elegans expression topomap.
View details for DOI 10.1101/gr.1125403
View details for PubMedID 12902378
The dauer is a developmental stage in C. elegans that exhibits increased longevity, stress resistance, nictation and altered metabolism compared with normal worms. We have used DNA microarrays to profile gene expression differences during the transition from the dauer state to the non-dauer state and after feeding of starved L1 animals, and have identified 1984 genes that show significant expression changes. This analysis includes genes that encode transcription factors and components of signaling pathways that could regulate the entry to and exit from the dauer state, and genes that encode components of metabolic pathways important for dauer survival and longevity. Homologs of C. elegans dauer-enriched genes may be involved in the disease process in parasitic nematodes.
View details for DOI 10.1242/dev.00363
View details for PubMedID 12620986
A report on the Keystone Symposium 'Functional Genomics: Global Analysis of Complex Biological Systems', Santa Fe, USA, 20-24 February 2003.
View details for PubMedID 12734004
Numerous gerontogene mutants leading to dramatic life extensions have been identified in the nematode Caenorhabditis elegans over the last 20 years. Analysis of these mutants has provided a basis for understanding the mechanisms driving the aging process(es). Several distinct mechanisms including an altered rate of aging, increased resistance to stress, decreased metabolic rate, or alterations in a program causing organismic aging and death have been proposed to underlie these mutants.Whole-genome analysis of gene expression during chronological aging of the worm provides a rich database of age-specific changes in gene expression and represents one way to distinguish among these models. Using a rigorous statistical model with multiple replicates, we find that a relatively small number of genes (only 164) show statistically significant changes in transcript levels as aging occurs (<1% of the genome). Expression of heat shock proteins decreases, while expression of certain transposases increases in older worms, and these findings are consistent with a higher mortality risk due to a failure in homeostenosis and destabilization of the genome in older animals. Finally, a specific subset of genes is coordinately altered both during chronological aging and in the transition from the reproductive form to the dauer, demonstrating a mechanistic overlap in aging between these two processes.We have performed a whole-genome analysis of changes in gene expression during aging in C. elegans that provides a molecular description of C. elegans senescence.
View details for PubMedID 12372248
We have implemented a functional genomics strategy to identify genes involved in chromosome morphogenesis and nuclear organization during meiotic prophase in the Caenorhabditis elegans germline. This approach took advantage of a gene-expression survey that used DNA microarray technology to identify genes preferentially expressed in the germline. We defined a subset of 192 germline-enriched genes whose expression profiles were similar to those of previously identified meiosis genes and designed a screen to identify genes for which inhibition by RNA interference (RNAi) elicited defects in function or development of the germline. We obtained strong germline phenotypes for 27% of the genes tested, indicating that this targeted approach greatly enriched for genes that function in the germline. In addition to genes involved in key meiotic prophase events, we identified genes involved in meiotic progression, germline proliferation, and chromosome organization and/or segregation during mitotic growth.
View details for PubMedID 12242227
In Caenorhabditis elegans, let-60 Ras controls many cellular processes, such as differentiation of vulval epithelial cells, function of chemosensory neurons, and meiotic progression in the germ line. Although much is known about the let-60 Ras signaling pathway, relatively little is understood about the target genes induced by let-60 Ras signaling that carry out terminal effector functions leading to morphological change. We have used DNA microarrays to identify 708 genes that change expression in response to activated let-60 Ras.
View details for DOI 10.1006/dbio.2002.0692
View details for PubMedID 12074557
Caenorhabditis elegans is a powerful animal model for the study of functional genomics. The completed and well-annotated DNA sequence is available and a systematic study of gene function by RNA-interference-mediated knockdown of every gene is in progress. Full-genome DNA microarrays and DNA chips can be used to determine expression changes at different stages of development and in different mutant backgrounds, and a protein-interaction map based on the yeast two-hybrid approach is in progress. These high-capacity approaches to studying gene function will provide new insights into invertebrate and vertebrate biology.
View details for PubMedID 11533717
Following the completion of the genome sequence of Caenorhabditis elegans, four independent studies have now assessed the functions of more than a third of the worm's genes by analysing the phenotypes caused when each of a large set of genes is inactivated by RNA interference.
View details for PubMedID 11231164
We have constructed DNA microarrays containing 17,871 genes, representing about 94% of the 18,967 genes currently annotated in the Caenorhabditis elegans genome. These DNA microarrays can be used as a tool to define a nearly complete molecular profile of gene expression levels associated with different developmental stages, growth conditions, or worm strains. Here, we used these full-genome DNA microarrays to show the relative levels of gene expression for nearly every gene during development, from eggs through adulthood. These expression data can help reveal when a gene may act during development. We also compared gene expression in males to that of hermaphrodites and found a total of 2,171 sex-regulated genes (P < 0.05). The sex-regulated genes provide a global view of the differences between the sexes at a molecular level and identify many genes likely to be involved in sex-specific differentiation and behavior.
View details for PubMedID 11134517
We generated a mutant of the red fluorescent protein drFP583. The mutant (E5) changes its fluorescence from green to red over time. The rate of color conversion is independent of protein concentration and therefore can be used to trace time-dependent expression. We used in vivo labeling with E5 to measure expression from the heat shock-dependent promoter in Caenorhabditis elegans and from the Otx-2 promoter in developing Xenopus embryos. Thus, E5 is a "fluorescent timer" that can be used to monitor both activation and down-regulation of target promoters on the whole-organism scale.
View details for PubMedID 11090358
The Caenorhabditis elegans vulva develops from the progeny of three vulval precursor cells (VPCs) induced to divide and differentiate by a signal from the somatic gonad. Evolutionarily conserved Ras and Notch extracellular signaling pathways are known to function during this process. To identify novel loci acting in vulval development, we carried out a genetic screen for mutants having a protruding-vulva (Pvl) mutant phenotype. Here we report the initial genetic characterization of several novel loci: bar-1, pvl-4, pvl-5, and pvl-6. In addition, on the basis of their Pvl phenotypes, we show that the previously identified genes lin-26, mom-3/mig-14, egl-18, and sem-4 also function during vulval development. Our characterization indicates that (1) pvl-4 and pvl-5 are required for generation/survival of the VPCs; (2) bar-1, mom-3/mig-14, egl-18, and sem-4 play a role in VPC fate specification; (3) lin-26 is required for proper VPC fate execution; and (4) pvl-6 acts during vulval morphogenesis. In addition, two of these genes, bar-1 and mom-3/mig-14, are known to function in processes regulated by Wnt signaling, suggesting that a Wnt signaling pathway is acting during vulval development.
View details for PubMedID 11063687
We used DNA microarrays to profile gene expression patterns in the C. elegans germline and identified 1416 germline-enriched transcripts that define three groups. The sperm-enriched group contains an unusually large number of protein kinases and phosphatases. The oocyte-enriched group includes potentially new components of embryonic signaling pathways. The germline-intrinsic group, defined as genes expressed similarly in germlines making only sperm or only oocytes, contains a family of piwi-related genes that may be important for stem cell proliferation. Finally, examination of the chromosomal location of germline transcripts revealed that sperm-enriched and germline-intrinsic genes are nearly absent from the X chromosome, but oocyte-enriched genes are not.
View details for PubMedID 11030340
View details for PubMedID 10644223
In Caenorhabditis elegans, the EGF receptor (encoded by let-23) is localized to the basolateral membrane domain of the epithelial vulval precursor cells, where it acts through a conserved Ras/MAP kinase signaling pathway to induce vulval differentiation. lin-10 acts in LET-23 receptor tyrosine kinase basolateral localization, because lin-10 mutations result in mislocalization of LET-23 to the apical membrane domain and cause a signaling defective (vulvaless) phenotype. We demonstrate that the previous molecular identification of lin-10 was incorrect, and we identify a new gene corresponding to the lin-10 genetic locus. lin-10 encodes a protein with regions of similarity to mammalian X11/mint proteins, containing a phosphotyrosine-binding and two PDZ domains. A nonsense lin-10 allele that truncates both PDZ domains only partially reduces lin-10 gene activity, suggesting that these protein interaction domains are not essential for LIN-10 function in vulval induction. Immunocytochemical experiments show that LIN-10 is expressed in vulval epithelial cells and in neurons. LIN-10 is present at low levels in the cytoplasm and at the plasma membrane and at high levels at or near the Golgi. LIN-10 may function in secretion of LET-23 to the basolateral membrane domain, or it may be involved in tethering LET-23 at the basolateral plasma membrane once it is secreted.
View details for PubMedID 10359617
The molecular basis by which commonly used signaling pathways are able to elicit tissue-specific responses in multicellular organisms is an important yet poorly understood problem. In this review, we use the receptor tyrosine kinase (RTK)/RAS/MAP kinase signaling cascade as a model to discuss various hypotheses that have been proposed to explain signaling specificity. Specificity can arise at the level of the receptor, through the modulation of signaling kinetics, through the interaction of different signaling pathways, and at the level of downstream signaling components. Mechanisms of specificity used by the RTK/RAS/MAP kinase signaling pathway might apply to other signaling pathways as well, and might help explain how multicellular organisms are able to generate tissues of diverse forms and functions from a small set of common signaling pathways.
View details for PubMedID 10203824
In C. elegans, the LET-23 receptor tyrosine kinase is localized to the basolateral membranes of polarized vulval epithelial cells. lin-2, lin-7, and lin-10 are required for basolateral localization of LET-23, since LET-23 is mislocalized to the apical membrane in lin-2, lin-7, and lin-10 mutants. Yeast two-hybrid, in vitro binding, and in vivo coimmunoprecipitation experiments show that LIN-2, LIN-7, and LIN-10 form a protein complex. Furthermore, compensatory mutations in lin-7 and let-23 exhibit allele-specific suppression of apical mislocalization and signaling-defective phenotypes. These results present a mechanism for basolateral localization of LET-23 receptor tyrosine kinase by direct binding to the LIN-2/LIN-7/LIN-10 complex. Each of the binding interactions within this complex is conserved, suggesting that this complex may also mediate basolateral localization in mammals.
View details for PubMedID 9753323
The Caenorhabditis elegans mpk-1 gene encodes a MAP kinase protein that plays an important role in Ras-mediated induction of vulval cell fates. We show that mutations that eliminate mpk-1 activity result in a highly penetrant, vulvaless phenotype. A double mutant containing a gain-of-function mpk-1 mutation and a gain-of-function mek mutation (MEK phosphorylates and activates MPK-1) exhibits a multivulva phenotype. These results suggest that mpk-1 may transduce most or all of the anchor cell signal. Epistasis analysis suggests that mpk-1 acts downstream of mek-2 (encodes a MEK homolog) and upstream of lin-1 (encodes an Ets transcription factor) in the anchor cell signaling pathway. Finally, mpk-1 may act together with let-60 ras in multiple developmental processes, as mpk-1 mutants exhibit nearly the same range of developmental phenotypes as let-60 ras mutants.
View details for PubMedID 9725833
In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Our results suggest that a Wnt signal transduced through a pathway involving the beta-catenin homolog BAR-1 controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. Our genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target LIN-39 Hox.
View details for PubMedID 9716532
The let-23 receptor/mpk-1 MAP kinase signaling pathway induces the vulva in C. elegans. We show that MPK-1 directly regulates both the LIN-31 winged-helix and the LIN-1 Ets transcription factors to specify the vulval cell fate. lin-31 and lin-1 act genetically downstream of mpk-1, and both proteins can be directly phosphorylated by MAP kinase. LIN-31 binds to LIN-1, and the LIN-1/LIN-31 complex inhibits vulval induction. Phosphorylation of LIN-31 by MPK-1 disrupts the LIN-1/LIN-31 complex, relieving vulval inhibition. Phosphorylated LIN-31 may also act as a transcriptional activator, promoting vulval cell fates. LIN-31 is a vulval-specific effector of MPK-1, while LIN-1 acts as a general effector. The partnership of tissue-specific and general effectors may confer specificity onto commonly used signaling pathways, creating distinct tissue-specific outcomes.
View details for PubMedID 9604932
Extracellular signals are normally presented to one surface of epithelial cells and to one end of neurons, and so neuronal and epithelial cell signaling is inherently polarized. Another aspect of signaling polarity is that receptors are often asymmetrically distributed on the surfaces of polarized cells. Recent evidence from studies of Caenorhabditis elegans shows that signaling polarity plays an important role in development. The underlying mesoderm induces the overlying ectoderm to form the vulva, and asymmetric distribution of the signal receptor on the basolateral surface of the epithelium is crucial for this signaling. In neurons, the localization of neurotransmitter receptors and ion channels at synapses allows neurons to be exquisitely sensitive to synaptic inputs. Exciting recent reports suggest that receptor localization to neuronal synapses and the basolateral membrane domains of epithelia may involve a common molecular mechanism involving localization by PDZ-containing proteins.
View details for PubMedID 9425351
During induction of the Caenorhabditis elegans hermaphrodite vulva, a signal from the anchor cell activates the LET-23 epidermal growth factor receptor (EGFR)/LET-60 Ras/MPK-1 MAP kinase signaling pathway in the vulval precursor cells. We have characterized two mechanisms that limit the extent of vulval induction. First, we found that gap-1 may directly inhibit the LET-60 Ras signaling pathway. We identified the gap-1 gene in a genetic screen for inhibitors of vulval induction. gap-1 is predicted to encode a protein similar to GTPase-activating proteins that likely functions to inhibit the signaling activity of LET-60 Ras. A loss-of-function mutation in gap-1 suppresses the vulvaless phenotype of mutations in the let-60 ras signaling pathway, but a gap-1 single mutant does not exhibit excess vulval induction. Second, we found that let-23 EGFR prevents vulval induction in a cell-nonautonomous manner, in addition to its cell-autonomous role in activating the let-60 ras/mpk-1 signaling pathway. Using genetic mosaic analysis, we show that let-23 activity in the vulval precursor cell closest to the anchor cell (P6.p) prevents induction of vulval precursor cells further away from the anchor cell (P3.p, P4.p, and P8.p). This result suggests that LET-23 in proximal vulval precursor cells might bind and sequester the inductive signal LIN-3 EGF, thereby preventing diffusion of the inductive signal to distal vulval precursor cells.
View details for PubMedID 9334333
The Caenorhabditis elegans let-60 gene encodes a Ras protein that mediates induction of the hermaphrodite vulva. To better understand how mutations constitutively activate Ras and cause unregulated cell division, we have characterized ga89, a temperature-sensitive, gain-of-function mutation in let-60 ras. At 25 degrees, ga89 increases let-60 activity resulting in a multivulva phenotype. At 15 degrees, ga89 decreases let-60 activity resulting in a vulvaless phenotype in let-60(ga89)/Df animals. The ga89 mutation causes a leucine (L) to phenylalanine (F) substitution at amino acid 19, a residue conserved in all Ras proteins. We introduced the L19F change into human H-Ras protein and found that the in vitro GTPase activity of H-Ras became temperature-dependent. Genetic experiments suggest that LET-60 (L19F) interacts with GAP and GNEF, since mutations that decrease GAP and GNEF activity affect the multivulva phenotype of let-60(ga89) animals. These results suggest that the L19F mutation primarily affects the intrinsic rate of GTP hydrolysis by Ras, and that this effect may be sufficient to account for the activated-Ras phenotype caused by let-60(ga89). Our results suggest that a mutation in a human ras gene analogous to ga89 might contribute to oncogenic transformation.
View details for PubMedID 9178006
We describe a genetic mosaic analysis procedure in which Caenorhabditis elegans mosaics are generated by spontaneous loss of an extrachromosomal array. This technique allows almost any C. elegans gene that can be used in germline transformation experiments to be used in mosaic analysis experiments. We identified a cosmid clone that rescues the mutant phenotype of ncl-1, so that this cell-autonomous marker could be used to analyze mosaic animals. To determine the sites of action for unc-29 and lin-31, an extrachromosomal array was constructed containing the ncl-1(+) cosmid linked to lin-31(+) and unc-29(+) cosmids. This array is mitotically unstable and can be lost to produce a clone of mutant cells. The specific cell division at which the extrachromosomal array had been lost was deduced by scoring the Ncl phenotypes of individual cells in genetic mosaics. The Unc-29 and Lin-31 phenotypes were then scored in these animals to determine in which cells these genes are required. This analysis showed that unc-29, which encodes a subunit of the acetylcholine receptor, acts in the body muscle cells. Furthermore, lin-31, which specifies cell fates during vulval induction and encodes a putative transcription factor similar to HNF-3/fork head, acts in the Pn.p cells.
View details for PubMedID 8807292
In C. elegans, the anchor cell signal induces Pn.p cells to form the vulva by activating a conserved receptor tyrosine kinase pathway. lin-2 and lin-7 mutants exhibit a vulvaless phenotype similar to the phenotype observed when this signaling pathway is defective. We have found that LIN-7 is a cell junction-associated protein that binds to the LET-23 receptor tyrosine kinase. LET-23 is also localized to the cell junctions, and both LIN-2 and LIN-7 are required for this localization. LET-23 overexpression rescues the lin-2 or lin-7 vulvaless phenotype, suggesting that increased receptor density can compensate for mislocalization. These results suggest that proper localization of LET-23 receptor to the Pn.p cell junctions is required for signaling activity.
View details for PubMedID 8612272
The lin-2 gene is required for the induction of the Caenorhabditis elegans vulva. Vulval development is initiated by a signal from the anchor cell that is transduced by a receptor tyrosine kinase/Ras pathway. We show that lin-2 acts in the vulval precursor cell P6.p, downstream of lin-3 EGF and upstream of let-60 ras, to allow expression of the 1 degrees cell fate. lin-2 encodes a protein of relative molecular mass 109,000 (LIN-2A) with regions of similarity to CaM kinase II and membrane-associated guanylate kinases. Mutant lin-2 transgenes designed to lack either protein kinase or guanylate kinase activity are functional, indicating that LIN-2A has a structural rather than an enzymatic role in vulval induction. Most or all identified membrane-associated guanylate kinases are components of cell junctions, including vertebrate tight junctions and arthropod septate junctions in epithelia. Thus, LIN-2A may be a component of the cell junctions of the epithelial vulval precursor cells that is required for signaling by the receptor tyrosine kinase LET-23. We propose that LIN-2A is required for the localization of one or more signal transduction proteins (such as LET-23) to either the basal membrane domain or the cell junctions, and that mislocalization of signal transduction proteins in lin-2 mutants interferes with vulval induction.
View details for PubMedID 8565857
Proteins that define a new family, termed membrane-associated guanylate kinases, have recently been identified as structural components of epithelial tight junctions and neuronal synapses, and as cell signaling proteins in Drosophila and Caenorhabditis elegans. In particular, the lin-2 gene has been shown to encode a membrane-associated guanylate kinase and to act in the let-23 receptor tyrosine kinase/let-60 ras signaling pathway that controls vulval induction in C. elegans. The combined data from recent biochemical and genetic analyses of membrane-associated guanylate kinases suggest that certain tight junction proteins can play an important role in cell signaling pathways. One possibility is that asymmetric segregation of signaling receptors to the basolateral membrane domain of polarized epithelial cells is crucial for proper cell signaling, and that membrane-associated guanylate kinases may be required for this localization.
View details for PubMedID 8573338
During the induction of the Caenorhabditis elegans vulva, cell signalling causes initially equipotent cells to express a reproducible pattern of cell fates. The position of the anchor cell determines the pattern of vulval precursor cell fates, such that the closest precursor cell (P6.p) expresses the primary cell fate, the next closest cells (P5.p and P7.p) both express the secondary cell fate, and each of the precursor cells located at a distance (P3.p, P4.p and P8.p) express the tertiary cell fate (Fig. 1a). We present data indicating that this stereotypical pattern of cell fates can be generated by sequential signals. We identified genetic mosaic animals in which P5.p and P7.p were defective in the anchor-cell signal-transduction pathway and observed that these cells adopted the secondary cell fate, indicating that anchor-cell signal transduction is not required for the expression of the secondary cell fate. These results suggest that the anchor cell induces P6.p to express the primary cell fate, and that P6.p subsequently induces P5.p and P7.p to express the secondary cell fate.
View details for PubMedID 7753169
A receptor tyrosine kinase/Ras signaling pathway controls the specification of vulval cell fates in Caenorhabditis elegans. Recently, C. elegans genes encoding proteins with similarity to mammalian Raf (lin-45), mitogen-activated protein kinase (mpk-1/sur-1), and an HNF-3 transcription factor (lin-31) have been identified and shown to act downstream of let-60 (ras) in this pathway. These genetically identified gene products bridge the gap between signal transduction at the plasma membrane and the control of cell fate specification in the nucleus.
View details for PubMedID 7950317
During development of the Caenorhabditis elegans hermaphrodite, the gonadal anchor cell induces nearby Pn.p cells to adopt vulval fates. The response to this signal is mediated by a receptor tyrosine kinase signal transduction pathway that has been remarkably well conserved during metazoan evolution. Because mitogen-activated protein (MAP) kinases are activated by receptor tyrosine kinase pathways in vertebrate cells, we hypothesized that C. elegans MAP kinase homologs may play a role in vulval induction. Two C. elegans MAP kinase genes, mpk-1 and mpk-2 (mpk, MAP kinase), were cloned using degenerate oligonucleotide primers and PCR amplification; in parallel, genes involved in vulval induction were identified by screening for mutations that suppress the vulval defects caused by an activated let-60 ras gene. One such suppressor mutation is an allele of mpk-1. We used a new type of mosaic analysis to show that mpk-1 acts cell autonomously in the Pn.p cells. Our results show that mpk-1 plays an important functional role as an activator in ras-mediated cell signaling in vivo.
View details for PubMedID 8299936
Cell-cell signaling controls the specification of vulval cell fates in Caenorhabditis elegans. Although previous studies have identified genes that function at early steps in the signaling pathway, the late steps are not well understood. Here, we begin to characterize those late events by showing that the lin-31 gene acts near the end of the vulval signaling pathway. We show that lin-31 acts downstream of the ras homolog let-60 and that lin-31 encodes a member of the HNF-3/fork head family of DNA-binding transcription factors. lin-31 regulates how vulval precursor cells choose their fate; in lin-31 mutants, these cells do not properly choose which fate to express and therefore adopt any one of the three possible vulval cell fates in a deregulated fashion. This interesting mutant phenotype suggests mechanisms for how vulval cell fates become determined.
View details for PubMedID 8504934
Programmed cell death is a physiological process that eliminates unwanted cells. The bcl-2 gene regulates programmed cell death in mammalian cells, but the way it functions is not known. Expression of the human bcl-2 gene in the nematode Caenorhabditis elegans reduced the number of programmed cell deaths, suggesting that the mechanism of programmed cell death controlled by bcl-2 in humans is the same as that in nematodes.
View details for PubMedID 1470921