[Show abstract][Hide abstract] ABSTRACT: Discovering the structure and dynamics of transcriptional regulatory events in the genome with cellular and temporal resolution is crucial to understanding the regulatory underpinnings of development and disease. We determined the genomic distribution of binding sites for 92 transcription factors and regulatory proteins across multiple stages of Caenorhabditis elegans development by performing 241 ChIP-seq (chromatin immunoprecipitation followed by sequencing) experiments. Integration of regulatory binding and cellular-resolution expression data produced a spatiotemporally resolved metazoan transcription factor binding map. Using this map, we explore developmental regulatory circuits that encode combinatorial logic at the levels of co-binding and co-expression of transcription factors, characterizing the genomic coverage and clustering of regulatory binding, the binding preferences of, and biological processes regulated by, transcription factors, the global transcription factor co-associations and genomic subdomains that suggest shared patterns of regulation, and identifying key transcription factors and transcription factor co-associations for fate specification of individual lineages and cell types.
[Show abstract][Hide abstract] ABSTRACT: The coupling of transgenes to heat shock promoters is a widely applied method for regulating gene expression. In Caenorhabditis elegans gene induction can be controlled temporally through timing of heat shock, and spatially via targeted rescue in heat shock mutants. Here we present a method for evoking gene expression in arbitrary cells, with single-cell resolution. We use a focused pulsed infrared laser to locally induce a heat shock response in specific cells. Our method builds on and extends a previously reported method using a continuous-wave laser. In our technique the pulsed laser illumination enables a much higher degree of spatial selectivity due to diffusion of heat between pulses. We apply our method to induce transient and long-term transgene expression in embryonic, larval, and adult cells. Our method allows highly selective spatiotemporal control of transgene expression and is a powerful tool for model organism biology.
[Show abstract][Hide abstract] ABSTRACT: Understanding the in vivo dynamics of protein localization and their physical interactions is important for many problems in biology. To enable systematic protein function interrogation in a multicellular context, we built a genome-scale transgenic platform for in vivo expression of fluorescent- and affinity-tagged proteins in Caenorhabditis elegans under endogenous cis regulatory control. The platform combines computer-assisted transgene design, massively parallel DNA engineering, and next-generation sequencing to generate a resource of 14,637 genomic DNA transgenes, which covers 73% of the proteome. The multipurpose tag used allows any protein of interest to be localized in vivo or affinity purified using standard tag-based assays. We illustrate the utility of the resource by systematic chromatin immunopurification and automated 4D imaging, which produced detailed DNA binding and cell/tissue distribution maps for key transcription factor proteins.
[Show abstract][Hide abstract] ABSTRACT: Cells perform wide varieties of functions that are facilitated, in part, by adopting unique shapes. Many of the genes and pathways that promote cell fate specification have been elucidated. However, relatively few transcription factors have been identified that promote shape acquisition after fate specification. Here we show that the Nkx5/HMX homeodomain protein MLS-2 is required for cellular elongation and shape maintenance of two tubular epithelial cells in the C. elegans excretory system, the duct and pore cells. The Nkx5/HMX family is highly conserved from sea urchins to humans, with known roles in neuronal and glial development. MLS-2 is expressed in the duct and pore, and defects in mls-2 mutants first arise when the duct and pore normally adopt unique shapes. MLS-2 cooperates with the EGF-Ras-ERK pathway to turn on the LIN-48/Ovo transcription factor in the duct cell during morphogenesis. These results reveal a novel interaction between the Nkx5/HMX family and the EGF-Ras pathway and implicate a transcription factor, MLS-2, as a regulator of cell shape.
[Show abstract][Hide abstract] ABSTRACT: The secondary structure of RNA is necessary for its maturation, regulation, processing, and function. However, the global influence of RNA folding in eukaryotes is still unclear. Here, we use a high-throughput, sequencing-based, structure-mapping approach to identify the paired (double-stranded RNA [dsRNA]) and unpaired (single-stranded RNA [ssRNA]) components of the Drosophila melanogaster and Caenorhabditis elegans transcriptomes, which allows us to identify conserved features of RNA secondary structure in metazoans. From this analysis, we find that ssRNAs and dsRNAs are significantly correlated with specific epigenetic modifications. Additionally, we find key structural patterns across protein-coding transcripts that indicate that RNA folding demarcates regions of protein translation and likely affects microRNA-mediated regulation of mRNAs in animals. Finally, we identify and characterize 546 mRNAs whose folding pattern is significantly correlated between these metazoans, suggesting that their structure has some function. Overall, our findings provide a global assessment of RNA folding in animals.
[Show abstract][Hide abstract] ABSTRACT: Caenorhabditis elegans has been a key model organism for biomedical research. Light microscopy has played a central role in C. elegans biology. C. elegans is transparent throughout its life cycle, and its physical size, from 50 µm (embryos) to 1 mm (adults), is well suited for light microscopy. Furthermore, it has an invariant body plan that arises from an invariant cell lineage. A wide range of biological processes, from patterns of gene expression to cell migration to neuronal activity, can be readily observed in single cells with a well-defined developmental context. This protocol describes how to collect and mount young C. elegans embryos for live imaging throughout embryogenesis.
[Show abstract][Hide abstract] ABSTRACT: Receptor tyrosine kinases and Notch are crucial for tube formation and branching morphogenesis in many systems, but the specific cellular processes that require signaling are poorly understood. Here we describe sequential roles for Notch and Epidermal growth factor (EGF)-Ras-ERK signaling in the development of epithelial tube cells in the C. elegans excretory (renal-like) organ. This simple organ consists of three tandemly connected unicellular tubes: the excretory canal cell, duct and G1 pore. lin-12 and glp-1/Notch are required to generate the canal cell, which is a source of LIN-3/EGF ligand and physically attaches to the duct during de novo epithelialization and tubulogenesis. Canal cell asymmetry and let-60/Ras signaling influence which of two equivalent precursors will attach to the canal cell. Ras then specifies duct identity, inducing auto-fusion and a permanent epithelial character; the remaining precursor becomes the G1 pore, which eventually loses epithelial character and withdraws from the organ to become a neuroblast. Ras continues to promote subsequent aspects of duct morphogenesis and differentiation, and acts primarily through Raf-ERK and the transcriptional effectors LIN-1/Ets and EOR-1. These results reveal multiple genetically separable roles for Ras signaling in tube development, as well as similarities to Ras-mediated control of branching morphogenesis in more complex organs, including the mammalian kidney. The relative simplicity of the excretory system makes it an attractive model for addressing basic questions about how cells gain or lose epithelial character and organize into tubular networks.
Development 08/2011; 138(16):3545-55. DOI:10.1242/dev.068148 · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulation of gene expression by sequence-specific transcription factors is central to developmental programs and depends on the binding of transcription factors with target sites in the genome. To date, most such analyses in Caenorhabditis elegans have focused on the interactions between a single transcription factor with one or a few select target genes. As part of the modENCODE Consortium, we have used chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) to determine the genome-wide binding sites of 22 transcription factors (ALR-1, BLMP-1, CEH-14, CEH-30, EGL-27, EGL-5, ELT-3, EOR-1, GEI-11, HLH-1, LIN-11, LIN-13, LIN-15B, LIN-39, MAB-5, MDL-1, MEP-1, PES-1, PHA-4, PQM-1, SKN-1, and UNC-130) at diverse developmental stages. For each factor we determined candidate gene targets, both coding and non-coding. The typical binding sites of almost all factors are within a few hundred nucleotides of the transcript start site. Most factors target a mixture of coding and non-coding target genes, although one factor preferentially binds to non-coding RNA genes. We built a regulatory network among the 22 factors to determine their functional relationships to each other and found that some factors appear to act preferentially as regulators and others as target genes. Examination of the binding targets of three related HOX factors--LIN-39, MAB-5, and EGL-5--indicates that these factors regulate genes involved in cellular migration, neuronal function, and vulval differentiation, consistent with their known roles in these developmental processes. Ultimately, the comprehensive mapping of transcription factor binding sites will identify features of transcriptional networks that regulate C. elegans developmental processes.
Genome Research 02/2011; 21(2):245-54. DOI:10.1101/gr.114587.110 · 13.85 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers, with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions to most of the conserved genome.
[Show abstract][Hide abstract] ABSTRACT: We systematically generated large-scale data sets to improve genome annotation for the nematode Caenorhabditis elegans, a key model organism. These data sets include transcriptome profiling across a developmental time course, genome-wide identification
of transcription factor–binding sites, and maps of chromatin organization. From this, we created more complete and accurate
gene models, including alternative splice forms and candidate noncoding RNAs. We constructed hierarchical networks of transcription
factor–binding and microRNA interactions and discovered chromosomal locations bound by an unusually large number of transcription
factors. Different patterns of chromatin composition and histone modification were revealed between chromosome arms and centers,
with similarly prominent differences between autosomes and the X chromosome. Integrating data types, we built statistical
models relating chromatin, transcription factor binding, and gene expression. Overall, our analyses ascribed putative functions
to most of the conserved genome.
[Show abstract][Hide abstract] ABSTRACT: MicroRNAs (miRNAs) have been found to regulate gene expression across eukaryotic species, but the function of most miRNA genes remains unknown. Here we describe how the analysis of the expression patterns of a well-conserved miRNA gene, mir-57, at cellular resolution for every minute during early development of Caenorhabditis elegans provided key insights in understanding its function. Remarkably, mir-57 expression shows strong positional bias but little tissue specificity, a pattern reminiscent of Hox gene function. Despite the minor defects produced by a loss of function mutation, overexpression of mir-57 causes dramatic posterior defects, which also mimic the phenotypes of mutant alleles of a posterior Hox gene, nob-1, an Abd homolog. More importantly, nob-1 expression is found in the same two posterior AB sublineages as those expressing mir-57 but with an earlier onset. Intriguingly, nob-1 functions as an activator for mir-57 expression; it is also a direct target of mir-57. In agreement with this, loss of mir-57 function partially rescues the nob-1 allele defects, indicating a negative feedback regulatory loop between the miRNA and Hox gene to provide positional cues. Given the conservation of the miRNA and Hox gene, the regulatory mechanism might be broadly used across species. The strategy used here to explore mir-57 function provides a path to dissect the regulatory relationship between genes.
[Show abstract][Hide abstract] ABSTRACT: Author Summary
The C. elegans transcription factor PHA-4 is a member of the highly conserved FOXA family of transcription factors. These factors act as master regulators of organ development by controlling how genes are turned off and on as tissues are formed. Additionally they regulate genes in response to nutrient levels and control both longevity and survival of the organism. However, the extent to which these factors control similar or distinct gene targets for each of these functions is unknown. For this reason, we have used the technique of chromatin immunoprecipitation followed by deep sequencing (ChIP–Seq), to define the target binding sites of PHA-4 on a genome-wide scale, when it is either functioning as an organ identity regulator or in response to environmental stress. Our data clearly demonstrate distinct sets of biologically relevant target genes for the transcription factor PHA-4 under these two different conditions. Not only have we defined PHA-4 targets, but we established an experimental ChIP–Seq pipeline to facilitate the identification of binding sites for many transcription factors in the future.
[Show abstract][Hide abstract] ABSTRACT: Image analysis is an essential component in many biological experiments that study gene expression, cell cycle progression, and protein localization. A protocol for tracking the expression of individual C. elegans genes was developed that collects image samples of a developing embryo by 3-D time lapse microscopy. In this protocol, a program called StarryNite performs the automatic recognition of fluorescently labeled cells and traces their lineage. However, due to the amount of noise present in the data and due to the challenges introduced by increasing number of cells in later stages of development, this program is not error free. In the current version, the error correction (i.e., editing) is performed manually using a graphical interface tool named AceTree, which is specifically developed for this task. For a single experiment, this manual annotation task takes several hours.
In this paper, we reduce the time required to correct errors made by StarryNite. We target one of the most frequent error types (movements annotated as divisions) and train a support vector machine (SVM) classifier to decide whether a division call made by StarryNite is correct or not. We show, via cross-validation experiments on several benchmark data sets, that the SVM successfully identifies this type of error significantly. A new version of StarryNite that includes the trained SVM classifier is available at http://starrynite.sourceforge.net.
We demonstrate the utility of a machine learning approach to error annotation for StarryNite. In the process, we also provide some general methodologies for developing and validating a classifier with respect to a given pattern recognition task.
[Show abstract][Hide abstract] ABSTRACT: Transcription factors are key components of regulatory networks that control development, as well as the response to environmental stimuli. We have established an experimental pipeline in Caenorhabditis elegans that permits global identification of the binding sites for transcription factors using chromatin immunoprecipitation and deep sequencing. We describe and validate this strategy, and apply it to the transcription factor PHA-4, which plays critical roles in organ development and other cellular processes. We identified thousands of binding sites for PHA-4 during formation of the embryonic pharynx, and also found a role for this factor during the starvation response. Many binding sites were found to shift dramatically between embryos and starved larvae, from developmentally regulated genes to genes involved in metabolism. These results indicate distinct roles for this regulator in two different biological processes and demonstrate the versatility of transcription factors in mediating diverse biological roles.
[Show abstract][Hide abstract] ABSTRACT: Comparative studies of Caenorhabditis briggsae and C. elegans have provided insights into gene function and developmental control in both organisms. C. elegans is a well developed model organism with a variety of molecular and genetic tools to study gene functions. In contrast, there are only very limited tools available for its closest relative, C. briggsae. To take advantage of the full potential of this comparative approach, we have developed several genetic and molecular tools to facilitate functional analysis in C. briggsae. First, we designed and implemented an SNP-based oligonucleotide microarray for rapid mapping of genetic mutants in C. briggsae. Second, we generated a mutagenized frozen library to permit the isolation of targeted deletions and used the library to recover a deletion mutant of cbr-unc-119 for use as a transgenic marker. Third, we used the cbr-unc-119 mutant in ballistic transformation and generated fluorescently labeled strains that allow automated lineaging and cellular resolution expression analysis. Finally, we demonstrated the potential of automated lineaging by profiling expression of egl-5, hlh-1, and pha-4 at cellular resolution and by detailed phenotyping of the perturbations on the Wnt signaling pathway. These additions to the experimental toolkit for C. briggsae should greatly increase its utility in comparative studies with C. elegans. With the emerging sequence of nematode species more closely related to C. briggsae, these tools may open novel avenues of experimentation in C. briggsae itself.
[Show abstract][Hide abstract] ABSTRACT: The C. elegans cell lineage provides a unique opportunity to look at how cell lineage affects patterns of gene expression. We developed an automatic cell lineage analyzer that converts high-resolution images of worms into a data table showing fluorescence expression with single-cell resolution. We generated expression profiles of 93 genes in 363 specific cells from L1 stage larvae and found that cells with identical fates can be formed by different gene regulatory pathways. Molecular signatures identified repeating cell fate modules within the cell lineage and enabled the generation of a molecular differentiation map that reveals points in the cell lineage when developmental fates of daughter cells begin to diverge. These results demonstrate insights that become possible using computational approaches to analyze quantitative expression from many genes in parallel using a digital gene expression atlas.
[Show abstract][Hide abstract] ABSTRACT: Glia are essential components of nervous systems. However, genetic programs promoting glia development and regulating glia-neuron interactions have not been extensively explored. Here we describe transcriptional programs required for development and function of the C. elegans cephalic sheath (CEPsh) glia. We demonstrate ventral- and dorsal-restricted roles for the mls-2/Nkx/Hmx and vab-3/Pax6/Pax7 genes, respectively, in CEPsh glia differentiation and expression of the genes hlh-17/Olig and ptr-10/Patched-related. Using mls-2 and vab-3 mutants, as well as CEPsh glia-ablated animals, we show that CEPsh glia are important for sensory dendrite extension, axon guidance/branching within the nerve ring, and nerve ring assembly. We demonstrate that UNC-6/Netrin, expressed in ventral CEPsh glia, mediates glia-dependent axon guidance. Our results suggest possible similarities between CEPsh glia development and oligodendrocyte development in vertebrates, and demonstrate that C. elegans provides a unique environment for studying glial functions in vivo.
Development 08/2008; 135(13):2263-75. DOI:10.1242/dev.019547 · 6.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As a fundamental process of development, cell proliferation must be coordinated with other processes such as fate differentiation. Through statistical analysis of individual cell cycle lengths of the first 8 out of 10 rounds of embryonic cell division in Caenorhabditis elegans, we identified synchronous and invariantly ordered divisions that are tightly associated with fate differentiation. Our results suggest a three-tier model for fate control of cell cycle pace: the primary control of cell cycle pace is established by lineage and the founder cell fate, then fine-tuned by tissue and organ differentiation within each lineage, then further modified by individualization of cells as they acquire unique morphological and physiological roles in the variant body plan. We then set out to identify the pace-setting mechanisms in different fates. Our results suggest that ubiquitin-mediated degradation of CDC-25.1 is a rate-determining step for the E (gut) and P(3) (muscle and germline) lineages but not others, even though CDC-25.1 and its apparent decay have been detected in all lineages. Our results demonstrate the power of C. elegans embryogenesis as a model to dissect the interaction between differentiation and proliferation, and an effective approach combining genetic and statistical analysis at single-cell resolution.