Search tips
Search criteria

Results 1-25 (1403607)

Clipboard (0)

Related Articles

1.  A quantitative model of normal C. elegans embryogenesis and its disruption after stress 
Developmental biology  2012;374(1):12-23.
The invariant lineage of Caenorhabditis elegans has powerful potential for quantifying developmental variability in normal and stressed embryos. Previous studies of division timing by automated lineage tracing suggested that variability in cell cycle timing is low in younger embryos, but manual lineage tracing of specific lineages suggested that variability may increase for later divisions. We developed improved automated lineage tracing methods that allow routine lineage tracing through the last round of embryonic cell divisions and we applied these methods to trace the lineage of 18 wild-type embryos. Cell cycle lengths, division axes and cell positions are remarkably consistent among these embryos at all stages, with only slight increases in variability later in development. The resulting quantitative 4-dimensional model of embryogenesis provides a powerful reference dataset to identify defects in mutants or in embryos that have experienced environmental perturbations. We also traced the lineages of embryos imaged at higher temperatures to quantify the decay in developmental robustness under temperature stress. Developmental variability increases modestly at 25°C compared with 22°C and dramatically at 26°C, and we identify homeotic transformations in a subset of embryos grown at 26°C. The deep lineage tracing methods provide a powerful tool for analysis of normal development, gene expression and mutants and we provide a graphical user interface to allow other researchers to explore the average behavior of arbitrary cells in a reference embryo.
PMCID: PMC3548946  PMID: 23220655
robustness; microscopy; cell lineage; image analysis; C. elegans; stress
2.  A Complete Developmental Sequence of a Drosophila Neuronal Lineage as Revealed by Twin-Spot MARCM 
PLoS Biology  2010;8(8):e1000461.
Labeling every neuron in a lineage in the fruit fly olfactory system reveals that every cell is born with a pre-determined cell fate that is invariant and dependent upon neuron birth order
Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.
Author Summary
A brain consists of numerous, potentially individually unique neurons that derive from a limited number of progenitors. It has been shown in various model organisms that specific neurons arise in a lineage made by a repeatedly renewing progenitor at specific times of development. However, except in the worm C. elegans, the stereotype of neural development has never been examined in sufficient detail to account for every single neuron derived from a common progenitor. Here we applied a sophisticated genetic mosaic system to mark single neurons in the adult Drosophila brain and simultaneously reveal in which lineage a targeted neuron had arisen and when along the lineage it was made. We have identified each neuron in a lineage of olfactory projection neurons. There are a remarkable 40 types of neurons within this lineage born over two epochs. Strikingly, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death, such that every neuron type has a unique and invariant cell count. Sequencing an entire neuronal lineage provides definitive evidence for origin-dependent neuron type specification. It further permits a systematic characterization of neuron types for comprehensive circuitry mapping.
PMCID: PMC2927434  PMID: 20808769
3.  Systematic image-driven analysis of the spatial Drosophila embryonic expression landscape 
We created innovative virtual representation for our large scale Drosophila insitu expression dataset. We aligned an elliptically shaped mesh comprised of small triangular regions to the outline of each embryo. Each triangle defines a unique location in the embryo and comparing corresponding triangles allows easy identification of similar expression patterns.The virtual representation was used to organize the expression landscape at stage 4-6. We identified regions with similar expression in the embryo and clustered genes with similar expression patterns.We created algorithms to mine the dataset for adjacent non-overlapping patterns and anti-correlated patterns. We were able to mine the dataset to identify co-expressed and putative interacting genes.Using co-expression we were able to assign putative functions to unknown genes.
Analyzing both temporal and spatial gene expression is essential for understanding development and regulatory networks of multicellular organisms. Interacting genes are commonly expressed in overlapping or adjacent domains. Thus, gene expression patterns can be used to assign putative gene functions and mined to infer candidates for networks.
We have generated a systematic two-dimensional mRNA expression atlas profiling embryonic development of Drosophila melanogaster (Tomancak et al, 2002, 2007). To date, we have collected over 70 000 images for over 6000 genes. To explore spatial relationships between gene expression patterns, we used a novel computational image-processing approach by converting expression patterns from the images into virtual representations (Figure 1). Using a custom-designed automated pipeline, for each image, we segmented and aligned the outline of the embryo to an elliptically shaped mesh, comprised of 311 small triangular regions each defining a unique location within the embryo. By comparing corresponding triangles, we produced a distance score to identify similar patterns. We generated those triangulated images (TIs) for our entire data set at all developmental stages and demonstrated that this representation can be used as for objective computationally defined description for expression in in situ hybridization images from various sources, including images from the literature.
We used the TIs to conduct a comprehensive analysis of the expression landscape. To this end, we created a novel approach to temporally sort and compact TIs to a non-redundant data set suitable for further computational processing. Although generally applicable for all developmental stages, for this study, we focused on developmental stages 4–6. For this stage range, we reduced the initial set of about 5800 TIs to 553 TIs containing 364 genes. Using this filtered data set, to discover how expression subdivides the embryo into regions, we clustered areas with similar expression and demonstrated that expression patterns divide the early embryo into distinct spatial regions resembling a fate map (Figure 3). To discover the range of unique expression patterns, we used affinity propagation clustering (Frey and Dueck, 2007) to group TIs with similar patterns and identified 39 clusters each representing a distinct pattern class. We integrated the remaining genes into the 39 clusters and studied the distribution of expression patterns and the relationships between the clusters.
The clustered expression patterns were used to identify putative positive and negative regulatory interactions. The similar TIs in each cluster not only grouped already known genes with related functions, but previously undescribed genes. A comparative analysis identified subtle differences between the genes within each expression cluster. To investigate these differences, we developed a novel Markov Random Field (MRF) segmentation algorithm to extract patterns. We then extended the MRF algorithm to detect shared expression boundaries, generate similarity measurements, and discriminate even faint/uncertain patterns between two TIs. This enabled us to identify more subtle partial expression pattern overlaps and adjacent non-overlapping patterns. For example, by conducting this analysis on the cluster containing the gene snail, we identified the previously known huckebein, which restricts snail expression (Reuter and Leptin, 1994), and zfh1, which interacts with tinman (Broihier et al, 1998; Su et al, 1999).
By studying the functions of known genes, we assigned putative developmental roles to each of the 39 clusters. Of the 1800 genes investigated, only half of them had previously assigned functions.
Representing expression patterns with geometric meshes facilitates the analysis of a complex process involving thousands of genes. This approach is complementary to the cellular resolution 3D atlas for the Drosophila embryo (Fowlkes et al, 2008). Our method can be used as a rapid, fully automated, high-throughput approach to obtain a map of co-expression, which will serve to select specific genes for detailed multiplex in-situ hybridization and confocal analysis for a fine-grain atlas. Our data are similar to the data in the literature, and research groups studying reporter constructs, mutant animals, or orthologs can easily produce in situ hybridizations. TIs can be readily created and provide representations that are both comparable to each other and our data set. We have demonstrated that our approach can be used for predicting relationships in regulatory and developmental pathways.
Discovery of temporal and spatial patterns of gene expression is essential for understanding the regulatory networks and development in multicellular organisms. We analyzed the images from our large-scale spatial expression data set of early Drosophila embryonic development and present a comprehensive computational image analysis of the expression landscape. For this study, we created an innovative virtual representation of embryonic expression patterns using an elliptically shaped mesh grid that allows us to make quantitative comparisons of gene expression using a common frame of reference. Demonstrating the power of our approach, we used gene co-expression to identify distinct expression domains in the early embryo; the result is surprisingly similar to the fate map determined using laser ablation. We also used a clustering strategy to find genes with similar patterns and developed new analysis tools to detect variation within consensus patterns, adjacent non-overlapping patterns, and anti-correlated patterns. Of the 1800 genes investigated, only half had previously assigned functions. The known genes suggest developmental roles for the clusters, and identification of related patterns predicts requirements for co-occurring biological functions.
PMCID: PMC2824522  PMID: 20087342
biological function; embryo; gene expression; in situ hybridization; Markov Random Field
4.  Cell-specific microarray profiling experiments reveal a comprehensive picture of gene expression in the C. elegans nervous system 
Genome Biology  2007;8(7):R135.
A novel strategy for profiling Caenorhabditis elegans cells identifies transcripts highly enriched in either the embryonic or larval C. elegans nervous system, including 19 conserved transcripts of unknown function that are also expressed in the mammalian brain.
With its fully sequenced genome and simple, well-defined nervous system, the nematode Caenorhabditis elegans offers a unique opportunity to correlate gene expression with neuronal differentiation. The lineal origin, cellular morphology and synaptic connectivity of each of the 302 neurons are known. In many instances, specific behaviors can be attributed to particular neurons or circuits. Here we describe microarray-based methods that monitor gene expression in C. elegans neurons and, thereby, link comprehensive profiles of neuronal transcription to key developmental and functional properties of the nervous system.
We employed complementary microarray-based strategies to profile gene expression in the embryonic and larval nervous systems. In the MAPCeL (Microarray Profiling C. elegans cells) method, we used fluorescence activated cell sorting (FACS) to isolate GFP-tagged embryonic neurons for microarray analysis. To profile the larval nervous system, we used the mRNA-tagging technique in which an epitope-labeled mRNA binding protein (FLAG-PAB-1) was transgenically expressed in neurons for immunoprecipitation of cell-specific transcripts. These combined approaches identified approximately 2,500 mRNAs that are highly enriched in either the embryonic or larval C. elegans nervous system. These data are validated in part by the detection of gene classes (for example, transcription factors, ion channels, synaptic vesicle components) with established roles in neuronal development or function. Of particular interest are 19 conserved transcripts of unknown function that are also expressed in the mammalian brain. In addition to utilizing these profiling approaches to define stage-specific gene expression, we also applied the mRNA-tagging method to fingerprint a specific neuron type, the A-class group of cholinergic motor neurons, during early larval development. A comparison of these data to a MAPCeL profile of embryonic A-class motor neurons identified genes with common functions in both types of A-class motor neurons as well as transcripts with roles specific to each motor neuron type.
We describe microarray-based strategies for generating expression profiles of embryonic and larval C. elegans neurons. These methods can be applied to particular neurons at specific developmental stages and, therefore, provide an unprecedented opportunity to obtain spatially and temporally defined snapshots of gene expression in a simple model nervous system.
PMCID: PMC2323220  PMID: 17612406
5.  A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans 
PLoS Genetics  2011;7(6):e1002109.
One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo.
Author Summary
The generation of a neuron from a fertilized egg requires a multi-step cascade of molecules acting from within and outside that cell to direct it towards a neuronal fate, rather than, say, a muscle cell. These cascades are not fully understood. In this study we systematically eliminate the function of almost all genes in the C. elegans genome, one by one, to determine what it takes to build a neuron. We identified 245 genes that affect the development of a specific sensory neuron pair, e.g. the neurons were not generated or the neurons were generated but the terminal fate was not correctly specified. We characterize in more detail the transcription factor hlh-14, which we find is required to generate multiple neurons, and the COMPASS histone methyltransferase complex, which we find to have a surprisingly specific role in the specification of a molecular and functional left-right asymmetry in this sensory neuron pair. Our study represents the first genome-wide analysis of a single neuronal cell fate decision. Further characterization of the genes identified here will enhance our understanding, and thus our capacity for treatment and prevention, of human neurological disorders.
PMCID: PMC3116913  PMID: 21698137
6.  A Negative Regulatory Loop between MicroRNA and Hox Gene Controls Posterior Identities in Caenorhabditis elegans 
PLoS Genetics  2010;6(9):e1001089.
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.
Author Summary
miRNAs are small RNAs found in many multi-cellular species that inhibit gene expression. Many of them play important roles in cancer and cell fate determination, but the function of most miRNAs is uncertain. Using live cell imaging and automated expression analysis, we found a miRNA gene, mir-57, is expressed in a position rather than tissue dependent way. Hox genes also regulate cell fate patterning along anterior-posterior (a-p) axis across different tissues. By investigating interactions between genes of these classes expressed in mir-57 expressing cells, we demonstrated by both genetic analysis and gene expression assays that a negative feedback loop between a posterior Hox gene, nob-1, and mir-57 regulates posterior cell fate determination in C. elegans. On the one hand, the Hox gene is required for normal activation of mir-57 expression, and on the other, the Hox gene functions as a direct target of and is repressed by the miRNA. Given the conservation of the two genes, a negative feedback loop between Hox and miRNA genes might be broadly used across species to regulate cell fate along the a-p axis. Detailed expression analysis may provide a general way to dissect the regulatory role of miRNAs.
PMCID: PMC2932687  PMID: 20824072
7.  Functional Dissection of Caenorhabditis elegans CLK-2/TEL2 Cell Cycle Defects during Embryogenesis and Germline Development 
PLoS Genetics  2009;5(4):e1000451.
CLK-2/TEL2 is essential for viability from yeasts to vertebrates, but its essential functions remain ill defined. CLK-2/TEL2 was initially implicated in telomere length regulation in budding yeast, but work in Caenorhabditis elegans has uncovered a function in DNA damage response signalling. Subsequently, DNA damage signalling defects associated with CLK-2/TEL2 have been confirmed in yeast and human cells. The CLK-2/TEL2 interaction with the ATM and ATR DNA damage sensor kinases and its requirement for their stability led to the proposal that CLK-2/TEL2 mutants might phenocopy ATM and/or ATR depletion. We use C. elegans to dissect developmental and cell cycle related roles of CLK-2. Temperature sensitive (ts) clk-2 mutants accumulate genomic instability and show a delay of embryonic cell cycle timing. This delay partially depends on the worm p53 homolog CEP-1 and is rescued by co-depletion of the DNA replication checkpoint proteins ATL-1 (C. elegans ATR) and CHK-1. In addition, clk-2 ts mutants show a spindle orientation defect in the eight cell stages that lead to major cell fate transitions. clk-2 deletion worms progress through embryogenesis and larval development by maternal rescue but become sterile and halt germ cell cycle progression. Unlike ATL-1 depleted germ cells, clk-2–null germ cells do not accumulate DNA double-strand breaks. Rather, clk-2 mutant germ cells arrest with duplicated centrosomes but without mitotic spindles in an early prophase like stage. This germ cell cycle arrest does not depend on cep-1, the DNA replication, or the spindle checkpoint. Our analysis shows that CLK-2 depletion does not phenocopy PIKK kinase depletion. Rather, we implicate CLK-2 in multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development.
Author Summary
PI3K-related protein kinases (PIKKs) ATM and ATR are essential upstream components of DNA damage signalling pathways, while TOR-1 acts as a nutrient sensor. CLK-2/TEL2 is a conserved gene initially implicated in budding yeast telomere length regulation and uncovered in the same genetic screen as the yeast TEL1 ATM like kinase. CLK-2/TEL2 was first implicated in DNA damage response signalling by C. elegans genetics, a function confirmed in yeast and human cells. In addition, CLK-2/TEL2 is essential for cellular and organismal survival from yeasts to vertebrates, but the essential phenotypes were not defined. A direct interaction between CLK-2/TEL2 and all PI3K-related protein kinases and the reduction of PIKK protein levels upon CLK-2/TEL2 depletion lead to the widely discussed notion that CLK-2/TEL2 mutants might phenocopy PIKK depletion phenotypes. We take advantage of embryonic lineage analysis and germline cytology to dissect developmental and cell cycle related functions of CLK-2. CLK-2 depletion does not phenocopy PIKK kinase depletion. We rather link CLK-2 to multiple developmental and cell cycle related processes and show that CLK-2 and ATR have antagonising functions during early C. elegans embryonic development. Furthermore, we implicate CLK-2 in a distinct cell lineage decision and show that its depletion leads to a novel germline cell cycle arrest phenotype.
PMCID: PMC2660272  PMID: 19360121
8.  Myogenic Conversion and Transcriptional Profiling of Embryonic Blastomeres in C. elegans 
Methods (San Diego, Calif.)  2011;56(1):50-54.
Myogenesis has proven to be a powerful paradigm for understanding cell fate specification and differentiation in many model organisms. This includes the nematode C. elegans for which the genetic, cellular, and molecular tools have allowed an in-depth understanding of muscle development. One tool not yet available in C. elegans is a robust, pure and prolific cell culture system to study myogenesis. As an alternative, this chapter describes a method by which the cell fates of early, uncommitted blastomeres in the embryo are converted to a myogenic lineage. This technique permits the nearly synchronous induction of myogenesis in vivo with the potential to generate a nearly homogeneous population of cells. Coupled with the RNA isolation and cDNA amplification methods that are also described, one can now profile gene expression throughout myogenesis using any platform of choice (e.g. expression arrays, next generation sequencing). Although limited by the artificial nature of this developing mass of muscle inside the eggshell, blastomere conversion and transcriptional profiling is a very powerful tool to investigate changes in gene expression associated with myogenesis in C. elegans that is applicable to many different cell types. When coupled with next generation sequencing, the method has the potential to yield a very high-resolution map of changes in gene expression throughout myogenesis.
PMCID: PMC3278551  PMID: 22019720
Myogenesis; C. elegans; Blastomere Conversion; Cell Fate; HLH-1; Transcription
9.  De Novo Inference of Systems-Level Mechanistic Models of Development from Live-Imaging-Based Phenotype Analysis 
Cell  2014;156(0):359-372.
Elucidation of complex phenotypes for mechanistic insights presents a significant challenge in systems biology. We report a strategy to automatically infer mechanistic models of cell fate differentiation based on live-imaging data. We use cell lineage tracing and combinations of tissue-specific marker expression to assay progenitor cell fate and detect fate changes upon genetic perturbation. Based on the cellular phenotypes, we further construct a model for how fate differentiation progresses in progenitor cells and predict cell-specific gene modules and cell-to-cell signaling events that regulate the series of fate choices. We validate our approach in C. elegans embryogenesis by perturbing 20 genes in over 300 embryos. The result not only recapitulates current knowledge but also provides insights into gene function and regulated fate choice, including an unexpected self-renewal. Our study provides a powerful approach for automated and quantitative interpretation of complex in vivo information.
PMCID: PMC3998820  PMID: 24439388
10.  Spindle assembly checkpoint genes reveal distinct as well as overlapping expression that implicates MDF-2/Mad2 in postembryonic seam cell proliferation in Caenorhabditis elegans 
BMC Cell Biology  2010;11:71.
The spindle assembly checkpoint (SAC) delays anaphase onset by inhibiting the activity of the anaphase promoting complex/cyclosome (APC/C) until all of the kinetochores have properly attached to the spindle. The importance of SAC genes for genome stability is well established; however, the roles these genes play, during postembryonic development of a multicellular organism, remain largely unexplored.
We have used GFP fusions of 5' upstream intergenic regulatory sequences to assay spatiotemporal expression patterns of eight conserved genes implicated in the spindle assembly checkpoint function in Caenorhabditis elegans. We have shown that regulatory sequences for all of the SAC genes drive ubiquitous GFP expression during early embryonic development. However, postembryonic spatial analysis revealed distinct, tissue-specific expression of SAC genes with striking co-expression in seam cells, as well as in the gut. Additionally, we show that the absence of MDF-2/Mad2 (one of the checkpoint genes) leads to aberrant number and alignment of seam cell nuclei, defects mainly attributed to abnormal postembryonic cell proliferation. Furthermore, we show that these defects are completely rescued by fzy-1(h1983)/CDC20, suggesting that regulation of the APC/CCDC20 by the SAC component MDF-2 is important for proper postembryonic cell proliferation.
Our results indicate that SAC genes display different tissue-specific expression patterns during postembryonic development in C. elegans with significant co-expression in hypodermal seam cells and gut cells, suggesting that these genes have distinct as well as overlapping roles in postembryonic development that may or may not be related to their established roles in mitosis. Furthermore, we provide evidence, by monitoring seam cell lineage, that one of the checkpoint genes is required for proper postembryonic cell proliferation. Importantly, our research provides the first evidence that postembryonic cell division is more sensitive to SAC loss, in particular MDF-2 loss, than embryonic cell division.
PMCID: PMC2955571  PMID: 20858267
11.  Automated cellular annotation for high-resolution images of adult Caenorhabditis elegans 
Bioinformatics  2013;29(13):i18-i26.
Motivation: Advances in high-resolution microscopy have recently made possible the analysis of gene expression at the level of individual cells. The fixed lineage of cells in the adult worm Caenorhabditis elegans makes this organism an ideal model for studying complex biological processes like development and aging. However, annotating individual cells in images of adult C.elegans typically requires expertise and significant manual effort. Automation of this task is therefore critical to enabling high-resolution studies of a large number of genes.
Results: In this article, we describe an automated method for annotating a subset of 154 cells (including various muscle, intestinal and hypodermal cells) in high-resolution images of adult C.elegans. We formulate the task of labeling cells within an image as a combinatorial optimization problem, where the goal is to minimize a scoring function that compares cells in a test input image with cells from a training atlas of manually annotated worms according to various spatial and morphological characteristics. We propose an approach for solving this problem based on reduction to minimum-cost maximum-flow and apply a cross-entropy–based learning algorithm to tune the weights of our scoring function. We achieve 84% median accuracy across a set of 154 cell labels in this highly variable system. These results demonstrate the feasibility of the automatic annotation of microscopy-based images in adult C.elegans.
PMCID: PMC3694659  PMID: 23812982
12.  The embryonic muscle transcriptome of Caenorhabditis elegans 
Genome Biology  2007;8(9):R188.
Fluorescence activated cell sorting and microarray profiling were used to identify 1,312 expressed genes that are enriched in myo-3::GFP-positive muscle cells of Caenorhabditis elegans.
The force generating mechanism of muscle is evolutionarily ancient; the fundamental structural and functional components of the sarcomere are common to motile animals throughout phylogeny. Recent evidence suggests that the transcription factors that regulate muscle development are also conserved. Thus, a comprehensive description of muscle gene expression in a simple model organism should define a basic muscle transcriptome that is also found in animals with more complex body plans. To this end, we applied microarray profiling of Caenorhabtidis elegans cells (MAPCeL) to muscle cell populations extracted from developing C. elegans embryos.
We used fluorescence-activated cell sorting to isolate myo-3::green fluorescent protein (GFP) positive muscle cells, and their cultured derivatives, from dissociated early C. elegans embryos. Microarray analysis identified 7,070 expressed genes, 1,312 of which are enriched in the myo-3::GFP positive cell population relative to the average embryonic cell. The muscle enriched gene set was validated by comparisons with known muscle markers, independently derived expression data, and GFP reporters in transgenic strains. These results confirm the utility of MAPCeL for cell type specific expression profiling and reveal that 60% of these transcripts have human homologs.
This study provides a comprehensive description of gene expression in developing C. elegans embryonic muscle cells. The finding that more than half of these muscle enriched transcripts encode proteins with human homologs suggests that mutant analysis of these genes in C. elegans could reveal evolutionarily conserved models of muscle gene function, with ready application to human muscle pathologies.
PMCID: PMC2375026  PMID: 17848203
13.  The Caenorhabditis elegans Synthetic Multivulva Genes Prevent Ras Pathway Activation by Tightly Repressing Global Ectopic Expression of lin-3 EGF 
PLoS Genetics  2011;7(12):e1002418.
The Caenorhabditis elegans class A and B synthetic multivulva (synMuv) genes redundantly antagonize an EGF/Ras pathway to prevent ectopic vulval induction. We identify a class A synMuv mutation in the promoter of the lin-3 EGF gene, establishing that lin-3 is the key biological target of the class A synMuv genes in vulval development and that the repressive activities of the class A and B synMuv pathways are integrated at the level of lin-3 expression. Using FISH with single mRNA molecule resolution, we find that lin-3 EGF expression is tightly restricted to only a few tissues in wild-type animals, including the germline. In synMuv double mutants, lin-3 EGF is ectopically expressed at low levels throughout the animal. Our findings reveal that the widespread ectopic expression of a growth factor mRNA at concentrations much lower than that in the normal domain of expression can abnormally activate the Ras pathway and alter cell fates. These results suggest hypotheses for the mechanistic basis of the functional redundancy between the tumor-suppressor-like class A and B synMuv genes: the class A synMuv genes either directly or indirectly specifically repress ectopic lin-3 expression; while the class B synMuv genes might function similarly, but alternatively might act to repress lin-3 as a consequence of their role in preventing cells from adopting a germline-like fate. Analogous genes in mammals might function as tumor suppressors by preventing broad ectopic expression of EGF-like ligands.
Author Summary
Extracellular signals that drive cells to divide must be carefully restricted so that only the correct cells receive those signals. Failure to properly control the expression of signaling molecules can lead to aberrant development and cancer. Studies of vulval development in the nematode Caenorhabditis elegans have helped define various multi-step signaling pathways involved in cancer. Here we report that two groups of proteins that control the EGF/Ras/MAP kinase pathway of vulval development act by tightly repressing the spatial expression of the gene lin-3, which encodes an EGF-like signaling molecule. Using a technique that detects single mRNA molecules, we show that inactivation of these proteins causes a low ectopic expression of lin-3 in many cells. In response, the EGF/Ras/MAP kinase pathway is activated in cells normally not exposed to the lin-3 signal, and vulval development is abnormal. This process is analogous to the cancerous growth that occurs in humans when mutations cause both tumor cells and the microenvironment surrounding the tumor cells to ectopically express factors that drive cellular proliferation. We propose that mammalian genes analogous to those that repress lin-3 expression in C. elegans vulval development act as tumor suppressors by preventing broad ectopic expression of EGF-like ligands.
PMCID: PMC3248470  PMID: 22242000
14.  A High-Fidelity Cell Lineage Tracing Method for Obtaining Systematic Spatiotemporal Gene Expression Patterns in Caenorhabditis elegans 
G3: Genes|Genomes|Genetics  2013;3(5):851-863.
Advances in microscopy and fluorescent reporters have allowed us to detect the onset of gene expression on a cell-by-cell basis in a systematic fashion. This information, however, is often encoded in large repositories of images, and developing ways to extract this spatiotemporal expression data is a difficult problem that often uses complex domain-specific methods for each individual data set. We present a more unified approach that incorporates general previous information into a hierarchical probabilistic model to extract spatiotemporal gene expression from 4D confocal microscopy images of developing Caenorhabditis elegans embryos. This approach reduces the overall error rate of our automated lineage tracing pipeline by 3.8-fold, allowing us to routinely follow the C. elegans lineage to later stages of development, where individual neuronal subspecification becomes apparent. Unlike previous methods that often use custom approaches that are organism specific, our method uses generalized linear models and extensions of standard reversible jump Markov chain Monte Carlo methods that can be readily extended to other organisms for a variety of biological inference problems relating to cell fate specification. This modeling approach is flexible and provides tractable avenues for incorporating additional previous information into the model for similar difficult high-fidelity/low error tolerance image analysis problems for systematically applied genomic experiments.
PMCID: PMC3656732  PMID: 23550142
C. elegans; cell fate; gene expression; image analysis; lineage
15.  LIN-42, the Caenorhabditis elegans PERIOD homolog, Negatively Regulates MicroRNA Transcription 
PLoS Genetics  2014;10(7):e1004486.
During C. elegans development, microRNAs (miRNAs) function as molecular switches that define temporal gene expression and cell lineage patterns in a dosage-dependent manner. It is critical, therefore, that the expression of miRNAs be tightly regulated so that target mRNA expression is properly controlled. The molecular mechanisms that function to optimize or control miRNA levels during development are unknown. Here we find that mutations in lin-42, the C. elegans homolog of the circadian-related period gene, suppress multiple dosage-dependent miRNA phenotypes including those involved in developmental timing and neuronal cell fate determination. Analysis of mature miRNA levels in lin-42 mutants indicates that lin-42 functions to attenuate miRNA expression. Through the analysis of transcriptional reporters, we show that the upstream cis-acting regulatory regions of several miRNA genes are sufficient to promote highly dynamic transcription that is coupled to the molting cycles of post-embryonic development. Immunoprecipitation of LIN-42 complexes indicates that LIN-42 binds the putative cis-regulatory regions of both non-coding and protein-coding genes and likely plays a role in regulating their transcription. Consistent with this hypothesis, analysis of miRNA transcriptional reporters in lin-42 mutants indicates that lin-42 regulates miRNA transcription. Surprisingly, strong loss-of-function mutations in lin-42 do not abolish the oscillatory expression patterns of lin-4 and let-7 transcription but lead to increased expression of these genes. We propose that lin-42 functions to negatively regulate the transcriptional output of multiple miRNAs and mRNAs and therefore coordinates the expression levels of genes that dictate temporal cell fate with other regulatory programs that promote rhythmic gene expression.
Author Summary
MicroRNAs play pervasive roles in controlling gene expression throughout animal development. Given that individual microRNAs are predicted to regulate hundreds of mRNAs and that most mRNA transcripts are microRNA targets, it is essential that the expression levels of microRNAs be tightly regulated. With the goal of unveiling factors that regulate the expression of microRNAs that control developmental timing, we identified lin-42, the C. elegans homolog of the human and Drosophila period gene implicated in circadian gene regulation, as a negative regulator of microRNA expression. By analyzing the transcriptional expression patterns of representative microRNAs, we found that the transcription of many microRNAs is normally highly dynamic and coupled aspects of post-embryonic growth and behavior. We suggest that lin-42 functions to modulate the transcriptional output of temporally-regulated microRNAs and mRNAs in order to maintain optimal expression of these genes throughout development.
PMCID: PMC4102445  PMID: 25032706
16.  Plectus - a stepping stone in embryonic cell lineage evolution of nematodes 
EvoDevo  2012;3:13.
Recent studies have challenged the widespread view that the pattern of embryogenesis found in Caenorhabditis elegans (clade 9) is characteristic of nematodes in general. To understand this still largely unexplored landscape of developmental events, we set out to examine more distantly related nematodes in detail for temporospatial differences in pattern formation and cell specification. Members of the genus Plectus (clade 6) seem to be suitable candidates to show variety, with certain idiosyncratic features during early development and the convenient availability of cultivatable species.
The study was conducted using 4-D lineage analysis, 3-D modeling of developing embryos and laser-induced ablation of individual blastomeres.
Detailed cell lineage studies of several Plectus species reveal that pattern formation and cell fate assignment differ markedly from C. elegans. Descendants of the first somatic founder cell S1 (AB) - but not the progeny of other founder cells - demonstrate extremely variable spatial arrangements illustrating that here distinct early cell-cell interactions between invariant partners, as found in C. elegans, cannot take place. Different from C. elegans, in Plectus alternative positional variations among early S1 blastomeres resulting in a ‘situs inversus’ pattern, nevertheless give rise to adults with normal left-right asymmetries. In addition, laser ablations of early blastomeres uncover inductions between variable cell partners.
Our results suggest that embryonic cell specification in Plectus is not correlated with cell lineage but with position. With this peculiarity, Plectus appears to occupy an intermediate position between basal nematodes displaying a variable early development and the C. elegans-like invariant pattern. We suggest that indeterminate pattern formation associated with late, position-dependent fate assignment represents a plesiomorphic character among nematodes predominant in certain basal clades but lost in derived clades. Thus, the behavior of S1 cells in Plectus can be considered an evolutionary relict in a transition phase between two different developmental strategies.
PMCID: PMC3464786  PMID: 22748136
Nematode; embryogenesis; cell lineage; cell specification; evolution; developmental system drift; Plectus; C. elegans
17.  Coordination of Cell Proliferation and Cell Fate Determination by CES-1 Snail 
PLoS Genetics  2013;9(10):e1003884.
The coordination of cell proliferation and cell fate determination is critical during development but the mechanisms through which this is accomplished are unclear. We present evidence that the Snail-related transcription factor CES-1 of Caenorhabditis elegans coordinates these processes in a specific cell lineage. CES-1 can cause loss of cell polarity in the NSM neuroblast. By repressing the transcription of the BH3-only gene egl-1, CES-1 can also suppress apoptosis in the daughters of the NSM neuroblasts. We now demonstrate that CES-1 also affects cell cycle progression in this lineage. Specifically, we found that CES-1 can repress the transcription of the cdc-25.2 gene, which encodes a Cdc25-like phosphatase, thereby enhancing the block in NSM neuroblast division caused by the partial loss of cya-1, which encodes Cyclin A. Our results indicate that CDC-25.2 and CYA-1 control specific cell divisions and that the over-expression of the ces-1 gene leads to incorrect regulation of this functional ‘module’. Finally, we provide evidence that dnj-11 MIDA1 not only regulate CES-1 activity in the context of cell polarity and apoptosis but also in the context of cell cycle progression. In mammals, the over-expression of Snail-related genes has been implicated in tumorigenesis. Our findings support the notion that the oncogenic potential of Snail-related transcription factors lies in their capability to, simultaneously, affect cell cycle progression, cell polarity and apoptosis and, hence, the coordination of cell proliferation and cell fate determination.
Author Summary
Animal development is a complex process and requires the coordination in space and time of various processes. These processes include the controlled production of cells, also referred to as ‘cell proliferation’, and the adoption by cells of specific fates, also referred to as ‘cell fate determination’. The observation that uncontrolled cell proliferation and cell fate determination contribute to conditions such as cancer, demonstrates that a precise coordination of these processes is not only important for development but for the prevention of disease throughout life. Snail-related transcription factors have previously been shown to be involved in the regulation of cell proliferation and cell fate determination. For example, the Caenorhabditis elegans Snail-related protein CES-1 affects cell fate determination in a specific cell lineage, the NSM (neurosecretory motorneuron) lineage. We now present evidence that CES-1 also controls cell proliferation in this lineage. Within a short period of time, CES-1 therefore coordinates cell proliferation and cell fate determination in one and the same lineage. Based on this finding, we propose that CES-1 is an important coordinator that is involved in the precise control - in space (NSM lineage) and time (<150 min) - of processes that are critical for animal development.
PMCID: PMC3814331  PMID: 24204299
18.  DNA Methylation Restricts Lineage-specific Functions of Transcription Factor Gata4 during Embryonic Stem Cell Differentiation 
PLoS Genetics  2013;9(6):e1003574.
DNA methylation changes dynamically during development and is essential for embryogenesis in mammals. However, how DNA methylation affects developmental gene expression and cell differentiation remains elusive. During embryogenesis, many key transcription factors are used repeatedly, triggering different outcomes depending on the cell type and developmental stage. Here, we report that DNA methylation modulates transcription-factor output in the context of cell differentiation. Using a drug-inducible Gata4 system and a mouse embryonic stem (ES) cell model of mesoderm differentiation, we examined the cellular response to Gata4 in ES and mesoderm cells. The activation of Gata4 in ES cells is known to drive their differentiation to endoderm. We show that the differentiation of wild-type ES cells into mesoderm blocks their Gata4-induced endoderm differentiation, while mesoderm cells derived from ES cells that are deficient in the DNA methyltransferases Dnmt3a and Dnmt3b can retain their response to Gata4, allowing lineage conversion from mesoderm cells to endoderm. Transcriptome analysis of the cells' response to Gata4 over time revealed groups of endoderm and mesoderm developmental genes whose expression was induced by Gata4 only when DNA methylation was lost, suggesting that DNA methylation restricts the ability of these genes to respond to Gata4, rather than controlling their transcription per se. Gata4-binding-site profiles and DNA methylation analyses suggested that DNA methylation modulates the Gata4 response through diverse mechanisms. Our data indicate that epigenetic regulation by DNA methylation functions as a heritable safeguard to prevent transcription factors from activating inappropriate downstream genes, thereby contributing to the restriction of the differentiation potential of somatic cells.
Author Summary
Animal bodies are constructed from many different specialized cell types that are generated during embryogenesis from a single fertilized egg, and acquire their specific characteristics through a series of differentiation steps. After being committed to a specific cell type, it is generally difficult for differentiated cells to convert to other cell types, at least partly because the cells maintain some memory or mark of their developmental history. Such cellular memory is mediated by “epigenetic” mechanisms, which function to stabilize the cell state. DNA methylation, a chemical modification of genomic cytosine residues, is one such mechanism. Genomic DNA methylation patterns in early embryonic cells are established in a cell-type-dependent manner, and these specific patterns are propagated through cell divisions in a clonal manner. However, our understanding of how DNA methylation controls cell differentiation and developmental gene regulation is limited. In this study, using an in vitro model of differentiation, we obtained evidence that DNA methylation modulates the cell's response to DNA-binding transcription factors in a cell-type-dependent manner. These findings extend our understanding of how cellular traits are stabilized within specific lineages during development, and may contribute to advances in cellular engineering.
PMCID: PMC3694845  PMID: 23825962
19.  Dynamic Chromatin Organization during Foregut Development Mediated by the Organ Selector Gene PHA-4/FoxA 
PLoS Genetics  2010;6(8):e1001060.
Central regulators of cell fate, or selector genes, establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different sets of target genes at various times and in diverse cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. We have used the Nuclear Spot Assay and GFP reporters to examine PHA-4 interactions with target promoters in living embryos and with single cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen of candidate nuclear factors identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx, but emr-1 did not modulate PHA-4 binding in the intestine. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, may facilitate promoter access and productive transcription. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells, preventing target gene expression in that organ. PHA-4 binding within the pharynx is limited by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.
Author Summary
Central regulators of cell fate establish the identity of cells by direct regulation of large cohorts of genes. In Caenorhabditis elegans, foregut (or pharynx) identity relies on the FoxA transcription factor PHA-4, which activates different target genes in different cellular environments. An outstanding question is how PHA-4 distinguishes between target genes for appropriate transcriptional control. Here we examine PHA-4 interactions with target promoters in living embryos and with single-cell resolution. While PHA-4 was found throughout the digestive tract, binding and activation of pharyngeally expressed promoters was restricted to a subset of pharyngeal cells and excluded from the intestine. An RNAi screen identified emerin (emr-1) as a negative regulator of PHA-4 binding within the pharynx. Upon promoter association, PHA-4 induced large-scale chromatin de-compaction, which, we hypothesize, facilitates promoter access. Our results reveal two tiers of PHA-4 regulation. PHA-4 binding is prohibited in intestinal cells and is limited in the pharynx by the nuclear lamina component EMR-1/emerin. The data suggest that association of PHA-4 with its targets is a regulated step that contributes to promoter selectivity during organ formation. We speculate that global re-organization of chromatin architecture upon PHA-4 binding promotes competence of pharyngeal gene transcription and, by extension, foregut development.
PMCID: PMC2920861  PMID: 20714352
20.  Chromosome-Biased Binding and Gene Regulation by the Caenorhabditis elegans DRM Complex 
PLoS Genetics  2011;7(5):e1002074.
DRM is a conserved transcription factor complex that includes E2F/DP and pRB family proteins and plays important roles in development and cancer. Here we describe new aspects of DRM binding and function revealed through genome-wide analyses of the Caenorhabditis elegans DRM subunit LIN-54. We show that LIN-54 DNA-binding activity recruits DRM to promoters enriched for adjacent putative E2F/DP and LIN-54 binding sites, suggesting that these two DNA–binding moieties together direct DRM to its target genes. Chromatin immunoprecipitation and gene expression profiling reveals conserved roles for DRM in regulating genes involved in cell division, development, and reproduction. We find that LIN-54 promotes expression of reproduction genes in the germline, but prevents ectopic activation of germline-specific genes in embryonic soma. Strikingly, C. elegans DRM does not act uniformly throughout the genome: the DRM recruitment motif, DRM binding, and DRM-regulated embryonic genes are all under-represented on the X chromosome. However, germline genes down-regulated in lin-54 mutants are over-represented on the X chromosome. We discuss models for how loss of autosome-bound DRM may enhance germline X chromosome silencing. We propose that autosome-enriched binding of DRM arose in C. elegans as a consequence of germline X chromosome silencing and the evolutionary redistribution of germline-expressed and essential target genes to autosomes. Sex chromosome gene regulation may thus have profound evolutionary effects on genome organization and transcriptional regulatory networks.
Author Summary
X chromosomes differ in number between the sexes and differ from autosomes in their associated proteins and gene regulatory properties. In C. elegans both X chromosomes are partially silenced in hermaphrodite germlines. Germline-expressed and essential genes are autosome-enriched and are thought to have fled the X chromosome during evolution because silencing these genes would result in sterility or lethality. We discovered that the C. elegans DRM complex, which controls transcription of genes implicated in development and cancer, avoids the X chromosome. We first describe how DNA–binding components of the DRM complex together recognize DNA sequences upstream of its target genes, and we describe that DRM controls different target genes in the germline versus the soma. We show that the DRM binding motif, the genes bound by DRM, and the embryonic genes regulated by DRM are all under-represented on the X chromosome. Interestingly, compromising DRM function in the germline enhances X chromosome silencing, and we discuss how autosome-bound DRM might regulate X-linked genes in trans. We propose that autosome-enriched binding of DRM co-evolved with the redistribution of its germline-expressed and essential target genes to autosomes. Our data highlight how X chromosome gene regulation may impact both the genomic distribution of gene sets and their transcriptional regulators.
PMCID: PMC3093354  PMID: 21589891
21.  Both the Caspase CSP-1 and a Caspase-Independent Pathway Promote Programmed Cell Death in Parallel to the Canonical Pathway for Apoptosis in Caenorhabditis elegans 
PLoS Genetics  2013;9(3):e1003341.
Caspases are cysteine proteases that can drive apoptosis in metazoans and have critical functions in the elimination of cells during development, the maintenance of tissue homeostasis, and responses to cellular damage. Although a growing body of research suggests that programmed cell death can occur in the absence of caspases, mammalian studies of caspase-independent apoptosis are confounded by the existence of at least seven caspase homologs that can function redundantly to promote cell death. Caspase-independent programmed cell death is also thought to occur in the invertebrate nematode Caenorhabditis elegans. The C. elegans genome contains four caspase genes (ced-3, csp-1, csp-2, and csp-3), of which only ced-3 has been demonstrated to promote apoptosis. Here, we show that CSP-1 is a pro-apoptotic caspase that promotes programmed cell death in a subset of cells fated to die during C. elegans embryogenesis. csp-1 is expressed robustly in late pachytene nuclei of the germline and is required maternally for its role in embryonic programmed cell deaths. Unlike CED-3, CSP-1 is not regulated by the APAF-1 homolog CED-4 or the BCL-2 homolog CED-9, revealing that csp-1 functions independently of the canonical genetic pathway for apoptosis. Previously we demonstrated that embryos lacking all four caspases can eliminate cells through an extrusion mechanism and that these cells are apoptotic. Extruded cells differ from cells that normally undergo programmed cell death not only by being extruded but also by not being engulfed by neighboring cells. In this study, we identify in csp-3; csp-1; csp-2 ced-3 quadruple mutants apoptotic cell corpses that fully resemble wild-type cell corpses: these caspase-deficient cell corpses are morphologically apoptotic, are not extruded, and are internalized by engulfing cells. We conclude that both caspase-dependent and caspase-independent pathways promote apoptotic programmed cell death and the phagocytosis of cell corpses in parallel to the canonical apoptosis pathway involving CED-3 activation.
Author Summary
Caspases are cysteine proteases that in many cases drive apoptosis, an evolutionarily conserved and highly stereotyped form of cellular suicide with functions in animal development and tissue maintenance. The dysregulation of apoptosis can contribute to diseases as diverse as cancer, autoimmunity, and neurodegeneration. Caspases are often thought to be required for, or even to define, apoptosis. Although there is evidence that apoptosis can occur in the absence of caspase activity, caspase-independence can be difficult to prove, as most animals have multiple caspases. The nematode Caenorhabditis elegans has four caspases, CED-3, CSP-1, CSP-2, and CSP-3. CED-3 has a well-established role in apoptosis, but less is known about the functions of the CSP caspases. In this study, we show that CSP-1 promotes apoptosis in the developing C. elegans embryo and that CSP-1 is regulated differently than its homolog CED-3. Furthermore, we show that apoptosis and the engulfment of dying cells can occur in mutants lacking all four caspases, proving that neither apoptosis nor cell-corpse engulfment require caspase function and that caspase-independent activities can contribute to apoptosis of some cells during animal development.
PMCID: PMC3591282  PMID: 23505386
22.  A regulatory network modeled from wild-type gene expression data guides functional predictions in Caenorhabditis elegans development 
BMC Systems Biology  2012;6:77.
Complex gene regulatory networks underlie many cellular and developmental processes. While a variety of experimental approaches can be used to discover how genes interact, few biological systems have been systematically evaluated to the extent required for an experimental definition of the underlying network. Therefore, the development of computational methods that can use limited experimental data to define and model a gene regulatory network would provide a useful tool to evaluate many important but incompletely understood biological processes. Such methods can assist in extracting all relevant information from data that are available, identify unexpected regulatory relationships and prioritize future experiments.
To facilitate the analysis of gene regulatory networks, we have developed a computational modeling pipeline method that complements traditional evaluation of experimental data. For a proof-of-concept example, we have focused on the gene regulatory network in the nematode C. elegans that mediates the developmental choice between mesodermal (muscle) and ectodermal (skin) cell fates in the embryonic C lineage. We have used gene expression data to build two models: a knowledge-driven model based on gene expression changes following gene perturbation experiments, and a data-driven mathematical model derived from time-course gene expression data recovered from wild-type animals. We show that both models can identify a rich set of network gene interactions. Importantly, the mathematical model built only from wild-type data can predict interactions demonstrated by the perturbation experiments better than chance, and better than an existing knowledge-driven model built from the same data set. The mathematical model also provides new biological insight, including a dissection of zygotic from maternal functions of a key transcriptional regulator, PAL-1, and identification of non-redundant activities of the T-box genes tbx-8 and tbx-9.
This work provides a strong example for a mathematical modeling approach that solely uses wild-type data to predict an underlying gene regulatory network. The modeling approach complements traditional methods of data analysis, suggesting non-intuitive network relationships and guiding future experiments.
PMCID: PMC3463499  PMID: 22734688
23.  Expression Profiling of MAP Kinase–Mediated Meiotic Progression in Caenorhabditis elegans 
PLoS Genetics  2006;2(11):e174.
The LET-60 (Ras)/LIN-45 (Raf)/MPK-1 (MAP kinase) signaling pathway plays a key role in the development of multiple tissues in Caenorhabditis elegans. For the most part, the identities of the downstream genes that act as the ultimate effectors of MPK-1 signaling have remained elusive. A unique allele of mpk-1, ga111, displays a reversible, temperature-sensitive, tissue-specific defect in progression through meiotic prophase I. We performed gene expression profiling on mpk-1(ga111) animals to identify candidate downstream effectors of MPK-1 signaling in the germ line. This analysis delineated a cohort of genes whose expression requires MPK-1 signaling in germ cells in the pachytene stage of meiosis I. RNA in situ hybridization analysis shows that these genes are expressed in the germ line in an MPK-1-dependent manner and have a spatial expression pattern consistent with the location of activated MPK-1. We found that one MPK-1 signaling-responsive gene encoding a C2H2 zinc finger protein plays a role in meiotic chromosome segregation downstream of MPK-1. Additionally, discovery of genes responsive to MPK-1 signaling permitted us to order MPK-1 signaling relative to several events occurring in pachytene, including EFL-1/DPL-1 gene regulation and X chromosome reactivation. This study highlights the utility of applying global gene expression methods to investigate genes downstream of commonly used signaling pathways in vivo.
In many tissues in developing organisms, signaling pathways interpret extracellular cues that change how genes are expressed inside the nucleus and thus direct the appropriate developmental choice. Identification of the genes that are responsive to signaling pathways is critical for understanding how these pathways can promote the correct cell fate. Additionally, understanding the relationships between different regulatory pathways will also help to decipher the network of gene expression that underlies development. The nematode Caenorhabditis elegans has many signaling pathways that are highly similar to those acting in mammals. In particular, the Ras/Raf/MAP kinase signaling pathway acts in many tissues in C. elegans to direct a diverse set of cell fates. Here, we identify a set of genes whose expression alters in response to Ras/Raf/MAP kinase signaling in the germ line during meiosis. We show that this set of genes is primarily expressed in the germ line and that at least one of these genes is important for proper germ cell fate downstream of Ras/Raf/MAP kinase signaling. We also find that the Ras/Raf/MAP kinase signaling pathway functions independently of a second regulatory pathway, the E2F pathway, that acts at a similar time during germ cell development.
PMCID: PMC1635537  PMID: 17096596
24.  Gender-Associated Genes in Filarial Nematodes Are Important for Reproduction and Potential Intervention Targets 
A better understanding of reproductive processes in parasitic nematodes may lead to development of new anthelmintics and control strategies for combating disabling and disfiguring neglected tropical diseases such as lymphatic filariasis and onchocerciasis. Transcriptomatic analysis has provided important new insights into mechanisms of reproduction and development in other invertebrates. We have performed the first genome-wide analysis of gender-associated (GA) gene expression in a filarial nematode to improve understanding of key reproductive processes in these parasites.
Methodology/Principal Findings
The Version 2 Filarial Microarray with 18,104 elements representing ∼85% of the filarial genome was used to identify GA gene transcripts in adult Brugia malayi worms. Approximately 19% of 14,293 genes were identified as GA genes. Many GA genes have potential Caenorhabditis elegans homologues annotated as germline-, oogenesis-, spermatogenesis-, and early embryogenesis- enriched. The potential C. elegans homologues of the filarial GA genes have a higher frequency of severe RNAi phenotypes (such as lethal and sterility) than other C. elegans genes. Molecular functions and biological processes associated with GA genes were gender-segregated. Peptidase, ligase, transferase, regulator activity for kinase and transcription, and rRNA and lipid binding were associated with female GA genes. In contrast, catalytic activity from kinase, ATP, and carbohydrate binding were associated with male GA genes. Cell cycle, transcription, translation, and biological regulation were increased in females, whereas metabolic processes of phosphate and carbohydrate metabolism, energy generation, and cell communication were increased in males. Significantly enriched pathways in females were associated with cell growth and protein synthesis, whereas metabolic pathways such as pentose phosphate and energy production pathways were enriched in males. There were also striking gender differences in environmental information processing and cell communication pathways. Many proteins encoded by GA genes are secreted by Brugia malayi, and these encode immunomodulatory molecules such as antioxidants and host cytokine mimics. Expression of many GA genes has been recently reported to be suppressed by tetracycline, which blocks reproduction in female Brugia malayi. Our localization of GA transcripts in filarial reproductive organs supports the hypothesis that these genes encode proteins involved in reproduction.
Genome-wide expression profiling coupled with a robust bioinformatics analysis has greatly expanded our understanding of the molecular biology of reproduction in filarial nematodes. This study has highlighted key molecules and pathways associated with reproductive and other biological processes and identified numerous potential candidates for rational drug design to target reproductive processes.
Author Summary
Lymphatic filariasis is a neglected tropical disease that is caused by thread-like parasitic worms that live and reproduce in lymphatic vessels of the human host. There are no vaccines to prevent filariasis, and available drugs are not effective against all stages of the parasite. In addition, recent reports suggest that the filarial nematodes may be developing resistance to key medications. Therefore, there is an urgent need to identify new drug targets in filarial worms. The purpose of this study was to perform a genome-wide analysis of gender-associated gene transcription to improve understanding of key reproductive processes in filarial nematodes. Our results indicate that thousands of genes are differentially expressed in male and female adult worms. Many of those genes are involved in specific reproductive processes such as embryogenesis and spermatogenesis. In addition, expression of some of those genes is suppressed by tetracycline, a drug that leads to sterilization of adult female worms in many filarial species. Thus, gender-associated genes represent priority targets for design of vaccines and drugs that interfere with reproduction of filarial nematodes. Additional work with this type of integrated systems biology approach should lead to important new tools for controlling filarial diseases.
PMCID: PMC3026763  PMID: 21283610
25.  Identification of 526 Conserved Metazoan Genetic Innovations Exposes a New Role for Cofactor E-like in Neuronal Microtubule Homeostasis 
PLoS Genetics  2013;9(10):e1003804.
The evolution of metazoans from their choanoflagellate-like unicellular ancestor coincided with the acquisition of novel biological functions to support a multicellular lifestyle, and eventually, the unique cellular and physiological demands of differentiated cell types such as those forming the nervous, muscle and immune systems. In an effort to understand the molecular underpinnings of such metazoan innovations, we carried out a comparative genomics analysis for genes found exclusively in, and widely conserved across, metazoans. Using this approach, we identified a set of 526 core metazoan-specific genes (the ‘metazoanome’), approximately 10% of which are largely uncharacterized, 16% of which are associated with known human disease, and 66% of which are conserved in Trichoplax adhaerens, a basal metazoan lacking neurons and other specialized cell types. Global analyses of previously-characterized core metazoan genes suggest a prevalent property, namely that they act as partially redundant modifiers of ancient eukaryotic pathways. Our data also highlights the importance of exaptation of pre-existing genetic tools during metazoan evolution. Expression studies in C. elegans revealed that many metazoan-specific genes, including tubulin folding cofactor E-like (TBCEL/coel-1), are expressed in neurons. We used C. elegans COEL-1 as a representative to experimentally validate the metazoan-specific character of our dataset. We show that coel-1 disruption results in developmental hypersensitivity to the microtubule drug paclitaxel/taxol, and that overexpression of coel-1 has broad effects during embryonic development and perturbs specialized microtubules in the touch receptor neurons (TRNs). In addition, coel-1 influences the migration, neurite outgrowth and mechanosensory function of the TRNs, and functionally interacts with components of the tubulin acetylation/deacetylation pathway. Together, our findings unveil a conserved molecular toolbox fundamental to metazoan biology that contains a number of neuronally expressed and disease-related genes, and reveal a key role for TBCEL/coel-1 in regulating microtubule function during metazoan development and neuronal differentiation.
Author Summary
The evolution of multicellular animals (metazoans) from their single-celled ancestor required new molecular tools to create and coordinate the various biological functions involved in a communal, or multicellular, lifestyle. This would eventually include the unique cellular and physiological demands of specialized tissues like the nervous system. To identify and understand the genetic bases of such unique metazoan traits, we used a comparative genomics approach to identify 526 metazoan-specific genes which have been evolutionarily conserved throughout the diversification of the animal kingdom. Interestingly, we found that some of those genes are still completely uncharacterized or poorly studied. We used the metazoan model organism C. elegans to examine the expression of some poorly characterized metazoan-specific genes and found that many, including one encoding tubulin folding cofactor E-like (TBCEL; C. elegans COEL-1), are expressed in cells of the nervous system. Using COEL-1 as an example to understand the metazoan-specific character of our dataset, our studies reveal a new role for this protein in regulating the stability of the microtubule cytoskeleton during development, and function of the touch receptor neurons. In summary, our findings help define a conserved molecular toolbox important for metazoan biology, and uncover an important role for COEL-1/TBCEL during development and in the nervous system of the metazoan C. elegans.
PMCID: PMC3789837  PMID: 24098140

Results 1-25 (1403607)