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1.  Using machine learning to speed up manual image annotation: application to a 3D imaging protocol for measuring single cell gene expression in the developing C. elegans embryo 
BMC Bioinformatics  2010;11:84.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1471-2105-11-84
PMCID: PMC2838868  PMID: 20146825
2.  The Genome Sequence of Caenorhabditis briggsae: A Platform for Comparative Genomics 
PLoS Biology  2003;1(2):e45.
The soil nematodes Caenorhabditis briggsae and Caenorhabditis elegans diverged from a common ancestor roughly 100 million years ago and yet are almost indistinguishable by eye. They have the same chromosome number and genome sizes, and they occupy the same ecological niche. To explore the basis for this striking conservation of structure and function, we have sequenced the C. briggsae genome to a high-quality draft stage and compared it to the finished C. elegans sequence. We predict approximately 19,500 protein-coding genes in the C. briggsae genome, roughly the same as in C. elegans. Of these, 12,200 have clear C. elegans orthologs, a further 6,500 have one or more clearly detectable C. elegans homologs, and approximately 800 C. briggsae genes have no detectable matches in C. elegans. Almost all of the noncoding RNAs (ncRNAs) known are shared between the two species. The two genomes exhibit extensive colinearity, and the rate of divergence appears to be higher in the chromosomal arms than in the centers. Operons, a distinctive feature of C. elegans, are highly conserved in C. briggsae, with the arrangement of genes being preserved in 96% of cases. The difference in size between the C. briggsae (estimated at approximately 104 Mbp) and C. elegans (100.3 Mbp) genomes is almost entirely due to repetitive sequence, which accounts for 22.4% of the C. briggsae genome in contrast to 16.5% of the C. elegans genome. Few, if any, repeat families are shared, suggesting that most were acquired after the two species diverged or are undergoing rapid evolution. Coclustering the C. elegans and C. briggsae proteins reveals 2,169 protein families of two or more members. Most of these are shared between the two species, but some appear to be expanding or contracting, and there seem to be as many as several hundred novel C. briggsae gene families. The C. briggsae draft sequence will greatly improve the annotation of the C. elegans genome. Based on similarity to C. briggsae, we found strong evidence for 1,300 new C. elegans genes. In addition, comparisons of the two genomes will help to understand the evolutionary forces that mold nematode genomes.
With the Caenorhabditis briggsae genome now in hand, C. elegans biologists have a powerful new research tool to refine their knowledge of gene function in C. elegans and to study the path of genome evolution
doi:10.1371/journal.pbio.0000045
PMCID: PMC261899  PMID: 14624247
3.  A novel cell nuclei segmentation method for 3D C. elegans embryonic time-lapse images 
BMC Bioinformatics  2013;14:328.
Background
Recently a series of algorithms have been developed, providing automatic tools for tracing C. elegans embryonic cell lineage. In these algorithms, 3D images collected from a confocal laser scanning microscope were processed, the output of which is cell lineage with cell division history and cell positions with time. However, current image segmentation algorithms suffer from high error rate especially after 350-cell stage because of low signal-noise ratio as well as low resolution along the Z axis (0.5-1 microns). As a result, correction of the errors becomes a huge burden. These errors are mainly produced in the segmentation of nuclei. Thus development of a more accurate image segmentation algorithm will alleviate the hurdle for automated analysis of cell lineage.
Results
This paper presents a new type of nuclei segmentation method embracing an bi-directional prediction procedure, which can greatly reduce the number of false negative errors, the most common errors in the previous segmentation. In this method, we first use a 2D region growing technique together with the level-set method to generate accurate 2D slices. Then a modified gradient method instead of the existing 3D local maximum method is adopted to detect all the 2D slices located in the nuclei center, each of which corresponds to one nucleus. Finally, the bi-directional pred- iction method based on the images before and after the current time point is introduced into the system to predict the nuclei in low quality parts of the images. The result of our method shows a notable improvement in the accuracy rate. For each nucleus, its precise location, volume and gene expression value (gray value) is also obtained, all of which will be useful in further downstream analyses.
Conclusions
The result of this research demonstrates the advantages of the bi-directional prediction method in the nuclei segmentation over that of StarryNite/MatLab StarryNite. Several other modifications adopted in our nuclei segmentation system are also discussed.
doi:10.1186/1471-2105-14-328
PMCID: PMC3903074  PMID: 24252066
4.  AceTree: a tool for visual analysis of Caenorhabditis elegans embryogenesis 
BMC Bioinformatics  2006;7:275.
Background
The invariant lineage of the nematode Caenorhabditis elegans has potential as a powerful tool for the description of mutant phenotypes and gene expression patterns. We previously described procedures for the imaging and automatic extraction of the cell lineage from C. elegans embryos. That method uses time-lapse confocal imaging of a strain expressing histone-GFP fusions and a software package, StarryNite, processes the thousands of images and produces output files that describe the location and lineage relationship of each nucleus at each time point.
Results
We have developed a companion software package, AceTree, which links the images and the annotations using tree representations of the lineage. This facilitates curation and editing of the lineage. AceTree also contains powerful visualization and interpretive tools, such as space filling models and tree-based expression patterning, that can be used to extract biological significance from the data.
Conclusion
By pairing a fast lineaging program written in C with a user interface program written in Java we have produced a powerful software suite for exploring embryonic development.
doi:10.1186/1471-2105-7-275
PMCID: PMC1501046  PMID: 16740163
5.  Evolutionary comparisons reveal a positional switch for spindle pole oscillations in Caenorhabditis embryos 
The Journal of Cell Biology  2013;201(5):653-662.
In one-cell stage embryos of different worm species, a conserved positional switch controls the onset of mitotic spindle oscillations, whereas the maximum amplitude of oscillations is determined by the time spent in the oscillating phase.
During the first embryonic division in Caenorhabditis elegans, the mitotic spindle is pulled toward the posterior pole of the cell and undergoes vigorous transverse oscillations. We identified variations in spindle trajectories by analyzing the outwardly similar one-cell stage embryo of its close relative Caenorhabditis briggsae. Compared with C. elegans, C. briggsae embryos exhibit an anterior shifting of nuclei in prophase and reduced anaphase spindle oscillations. By combining physical perturbations and mutant analysis in both species, we show that differences can be explained by interspecies changes in the regulation of the cortical Gα–GPR–LIN-5 complex. However, we found that in both species (1) a conserved positional switch controls the onset of spindle oscillations, (2) GPR posterior localization may set this positional switch, and (3) the maximum amplitude of spindle oscillations is determined by the time spent in the oscillating phase. By investigating microevolution of a subcellular process, we identify new mechanisms that are instrumental to decipher spindle positioning.
doi:10.1083/jcb.201210110
PMCID: PMC3664713  PMID: 23690175
6.  Caenorhabditis briggsae Recombinant Inbred Line Genotypes Reveal Inter-Strain Incompatibility and the Evolution of Recombination 
PLoS Genetics  2011;7(7):e1002174.
The nematode Caenorhabditis briggsae is an emerging model organism that allows evolutionary comparisons with C. elegans and exploration of its own unique biological attributes. To produce a high-resolution C. briggsae recombination map, recombinant inbred lines were generated from reciprocal crosses between two strains and genotyped at over 1,000 loci. A second set of recombinant inbred lines involving a third strain was also genotyped at lower resolution. The resulting recombination maps exhibit discrete domains of high and low recombination, as in C. elegans, indicating these are a general feature of Caenorhabditis species. The proportion of a chromosome's physical size occupied by the central, low-recombination domain is highly correlated between species. However, the C. briggsae intra-species comparison reveals striking variation in the distribution of recombination between domains. Hybrid lines made with the more divergent pair of strains also exhibit pervasive marker transmission ratio distortion, evidence of selection acting on hybrid genotypes. The strongest effect, on chromosome III, is explained by a developmental delay phenotype exhibited by some hybrid F2 animals. In addition, on chromosomes IV and V, cross direction-specific biases towards one parental genotype suggest the existence of cytonuclear epistatic interactions. These interactions are discussed in relation to surprising mitochondrial genome polymorphism in C. briggsae, evidence that the two strains diverged in allopatry, the potential for local adaptation, and the evolution of Dobzhansky-Muller incompatibilities. The genetic and genomic resources resulting from this work will support future efforts to understand inter-strain divergence as well as facilitate studies of gene function, natural variation, and the evolution of recombination in Caenorhabditis nematodes.
Author Summary
The nematode Caenorhabditis briggsae is increasingly used for comparisons with its more famous relative, C. elegans. To improve genomic resources for C. briggsae, we created two sets of inbred lines derived from crosses between diverged C. briggsae strains. High-throughput genotyping of these has improved the resolution of the recombination map and genome assembly. It also allows detailed comparisons of recombination both within and between species. Unexpectedly, we found that alleles from one parental strain were much more likely to be fixed on three of the six chromosomes in one of the sets of lines. One of these biases is caused by a pronounced developmental delay in F2 progeny that is seen in both reciprocal crosses, whereas the other two manifest in only one of the two cross directions. This indicates that the parental strains have diverged in both nuclear and nuclear-cytoplasmic interactions, either because of local adaptation or restricted gene flow across much of the genome.
doi:10.1371/journal.pgen.1002174
PMCID: PMC3136444  PMID: 21779179
7.  Analysis of C. elegans NR2E nuclear receptors defines three conserved clades and ligand-independent functions 
Background
The nuclear receptors (NRs) are an important class of transcription factors that are conserved across animal phyla. Canonical NRs consist of a DNA-binding domain (DBD) and ligand-binding domain (LBD). While most animals have 20–40 NRs, nematodes of the genus Caenorhabditis have experienced a spectacular proliferation and divergence of NR genes. The LBDs of evolutionarily-conserved Caenorhabditis NRs have diverged sharply from their Drosophila and vertebrate orthologs, while the DBDs have been strongly conserved. The NR2E family of NRs play critical roles in development, especially in the nervous system. In this study, we explore the phylogenetics and function of the NR2E family of Caenorhabditis elegans, using an in vivo assay to test LBD function.
Results
Phylogenetic analysis reveals that the NR2E family of NRs consists of three broadly-conserved clades of orthologous NRs. In C. elegans, these clades are defined by nhr-67, fax-1 and nhr-239. The vertebrate orthologs of nhr-67 and fax-1 are Tlx and PNR, respectively. While the nhr-239 clade includes orthologs in insects (Hr83), an echinoderm, and a hemichordate, the gene appears to have been lost from vertebrate lineages. The C. elegans and C. briggsae nhr-239 genes have an apparently-truncated and highly-diverged LBD region. An additional C. elegans NR2E gene, nhr-111, appears to be a recently-evolved paralog of fax-1; it is present in C. elegans, but not C. briggsae or other animals with completely-sequenced genomes. Analysis of the relatively unstudied nhr-111 and nhr-239 genes demonstrates that they are both expressed—nhr-111 very broadly and nhr-239 in a small subset of neurons. Analysis of the FAX-1 LBD in an in vivo assay revealed that it is not required for at least some developmental functions.
Conclusions
Our analysis supports three conserved clades of NR2E receptors, only two of which are represented in vertebrates, indicating three ancestral NR2E genes in the urbilateria. The lack of a requirement for a FAX-1 LBD suggests that the relatively high level of sequence divergence for Caenorhabditis LBDs reflects relaxed selection on the primary sequence as opposed to divergent positive selection. This observation is consistent with a model in which divergence of some Caenorhabditis LBDs is allowed, at least in part, by the absence of a ligand requirement.
doi:10.1186/1471-2148-12-81
PMCID: PMC3517510  PMID: 22690911
8.  High Rate of Large Deletions in Caenorhabditis briggsae Mitochondrial Genome Mutation Processes 
Mitochondrial DNA (mtDNA) mutations underlie a variety of human genetic disorders and are associated with the aging process. mtDNA polymorphisms are widely used in a variety of evolutionary applications. Although mtDNA mutation spectra are known to differ between distantly related model organisms, the extent to which mtDNA mutation processes vary between more closely related species and within species remains enigmatic. We analyzed mtDNA divergence in two sets of 250-generation Caenorhabditis briggsae mutation-accumulation (MA) lines, each derived from a different natural isolate progenitor: strain HK104 from Okayama, Japan, and strain PB800 from Ohio, United States. Both sets of C. briggsae MA lines accumulated numerous large heteroplasmic mtDNA deletions, whereas only one similar event was observed in a previous analysis of Caenorhabditis elegans MA line mtDNA. Homopolymer length change mutations were frequent in both sets of C. briggsae MA lines and occurred in both intergenic and protein-coding gene regions. The spectrum of C. briggsae mtDNA base substitution mutations differed from the spectrum previously observed in C. elegans. In C. briggsae, the HK104 MA lines experienced many different base substitution types, whereas the PB800 lines displayed only C:G → T:A transitions, although the difference was not significant. Over half of the mtDNA base substitutions detected in the C. briggsae MA lines were in a heteroplasmic state, whereas all those previously characterized in C. elegans MA line mtDNA were fixed changes, indicating a narrower mtDNA bottleneck in C. elegans as compared with C. briggsae. Our results show that C. briggsae mtDNA is highly susceptible to large deletions and that the mitochondrial mutation process varies between Caenorhabditis nematode species.
doi:10.1093/gbe/evp055
PMCID: PMC2839355  PMID: 20333220
bottleneck; heteroplasmy; mutation-accumulation line; nematode
9.  Comparison of C. elegans and C. briggsae Genome Sequences Reveals Extensive Conservation of Chromosome Organization and Synteny 
PLoS Biology  2007;5(7):e167.
To determine whether the distinctive features of Caenorhabditis elegans chromosomal organization are shared with the C. briggsae genome, we constructed a single nucleotide polymorphism–based genetic map to order and orient the whole genome shotgun assembly along the six C. briggsae chromosomes. Although these species are of the same genus, their most recent common ancestor existed 80–110 million years ago, and thus they are more evolutionarily distant than, for example, human and mouse. We found that, like C. elegans chromosomes, C. briggsae chromosomes exhibit high levels of recombination on the arms along with higher repeat density, a higher fraction of intronic sequence, and a lower fraction of exonic sequence compared with chromosome centers. Despite extensive intrachromosomal rearrangements, 1:1 orthologs tend to remain in the same region of the chromosome, and colinear blocks of orthologs tend to be longer in chromosome centers compared with arms. More strikingly, the two species show an almost complete conservation of synteny, with 1:1 orthologs present on a single chromosome in one species also found on a single chromosome in the other. The conservation of both chromosomal organization and synteny between these two distantly related species suggests roles for chromosome organization in the fitness of an organism that are only poorly understood presently.
Author Summary
The importance of chromosomal organization in the fitness of a species is only poorly understood. The publication of the C. elegans genome sequence in 1998 revealed features of higher level organization that suggested its chromosomes were organized into distinct domains. Chromosome arms were accumulating changes more rapidly than the centers of chromosomes. In this paper, we have compared the organization of the nematode C. briggsae genome with that of C. elegans. By building a genetic map based on DNA variations between two strains of C. briggsae, and by using that map to organize the draft genome sequence of C. briggsae published in 2003, we found the following: (1) Intrachromosomal rearrangements are frequent within and even between arms but are less common within central regions and between arms and centers. (2) Genes have remained overwhelmingly on the same chromosomes. (3) The distinctive features that distinguish C. elegans arms from centers also are seen in C. briggsae chromosomes. The conservation of these features between these two species, despite the approximately 100 million years since their most recent common ancestor, provides clear evidence of the selective advantages of the domain architecture of chromosomes. The continuing association of genes on the same chromosomes suggests that this may also be advantageous.
The conservation of both chromosomal organization and synteny between two distantly related species suggests roles for chromosome organization in the fitness of an organism.
doi:10.1371/journal.pbio.0050167
PMCID: PMC1914384  PMID: 17608563
10.  fog-2 and the Evolution of Self-Fertile Hermaphroditism in Caenorhabditis 
PLoS Biology  2004;3(1):e6.
Somatic and germline sex determination pathways have diverged significantly in animals, making comparisons between taxa difficult. To overcome this difficulty, we compared the genes in the germline sex determination pathways of Caenorhabditis elegans and C. briggsae, two Caenorhabditis species with similar reproductive systems and sequenced genomes. We demonstrate that C. briggsae has orthologs of all known C. elegans sex determination genes with one exception: fog-2. Hermaphroditic nematodes are essentially females that produce sperm early in life, which they use for self fertilization. In C. elegans, this brief period of spermatogenesis requires FOG-2 and the RNA-binding protein GLD-1, which together repress translation of the tra-2 mRNA. FOG-2 is part of a large C. elegans FOG-2-related protein family defined by the presence of an F-box and Duf38/FOG-2 homogy domain. A fog-2-related gene family is also present in C. briggsae, however, the branch containing fog-2 appears to have arisen relatively recently in C. elegans, post-speciation. The C-terminus of FOG-2 is rapidly evolving, is required for GLD-1 interaction, and is likely critical for the role of FOG-2 in sex determination. In addition, C. briggsae gld-1 appears to play the opposite role in sex determination (promoting the female fate) while maintaining conserved roles in meiotic progression during oogenesis. Our data indicate that the regulation of the hermaphrodite germline sex determination pathway at the level of FOG-2/GLD-1/tra-2 mRNA is fundamentally different between C. elegans and C. briggsae, providing functional evidence in support of the independent evolution of self-fertile hermaphroditism. We speculate on the convergent evolution of hermaphroditism in Caenorhabditis based on the plasticity of the C. elegans germline sex determination cascade, in which multiple mutant paths yield self fertility.
A comparison of sex determination genes in C. elegans and C. briggsae provides evidence in support of the convergent evolution of self-fertile hermaphroditism in the Caenorhabditis clade
doi:10.1371/journal.pbio.0030006
PMCID: PMC539060  PMID: 15630478
11.  Evolution of embryonic development in nematodes 
EvoDevo  2011;2:18.
Background
Nematodes can be subdivided into basal Enoplea (clades 1 and 2) and more derived Chromadorea (clades 3 to 12). Embryogenesis of Caenorhabditis elegans (clade 9) has been analyzed in most detail. Their establishment of polarity and asymmetric cleavage requires the differential localization of PAR proteins. Earlier studies on selected other nematodes revealed that embryonic development of nematodes is more diverse than the essentially invariant development of C. elegans and the classic study object Ascaris had suggested. To obtain a more detailed picture of variations and evolutionary trends we compared embryonic cell lineages and pattern formation in embryos of all 12 nematode clades.
Methods
The study was conducted using 4-D microscopy and 3-D modeling of developing embryos.
Results
We found dramatic differences compared to C. elegans in Enoplea but also considerable variations among Chromadorea. We discovered 'Polarity Organizing Centers' (POCs) that orient cleavage spindles along the anterior-posterior axis in distinct cells over consecutive cell generations. The resulting lineally arranged blastomeres represent a starting point for the establishment of bilateral symmetry within individual lineages. We can discern six different early cleavage types and suggest that these variations are due to modifications in the activity of the POCs in conjunction with changes in the distribution of PAR proteins. In addition, our studies indicate that lineage complexity advanced considerably during evolution, that is we observe trends towards an increase of somatic founder cells, from monoclonal to polyclonal lineages and from a variable (position-dependent) to an invariable (lineage-dependent) way of cell fate specification. In contrast to the early phase of embryogenesis, the second half ('morphogenesis') appears similar in all studied nematodes. Comparison of early cleavage between the basal nematode Tobrilus stefanskii and the tardigrade Hypsibius dujardini revealed surprising similarities indicating that the presence of POCs is not restricted to nematode embryos.
Conclusions
The pattern of cleavage, spatial arrangement and differentiation of cells diverged dramatically during the history of the phylum Nematoda without corresponding changes in the phenotype. While in all studied representatives the same distinctive developmental steps need to be taken, cell behavior leading to these is not conserved.
doi:10.1186/2041-9139-2-18
PMCID: PMC3195109  PMID: 21929824
nematode; embryogenesis; cell lineage; polarity; symmetry formation; cell specification; evolution; Tobrilus; Prionchulus; C. elegans
12.  The Function of a Spindle Checkpoint Gene bub-1 in C. elegans Development 
PLoS ONE  2009;4(6):e5912.
Background
The serine/threonine kinase BUB1 (Budding Uninhibited by Benzimidazole 1) was originally identified in yeast as a checkpoint protein, based on its mutant's incapacity of delaying the cell cycle in response to loss of microtubules. Our understanding of its function is primarily from studies carried out in yeast S. cerevisiae. It has been shown that it is a component of the mitotic spindle checkpoint and regulates the separation of sister chromatids through its downstream molecules. However, its roles in multi-cellular organisms remain unclear.
Methods and Findings
In nematode C. elegans, rapid cell divisions primarily occur in embryos and in germline of postembryonic larvae and adults. In addition, a select set of cells undergo a few rounds of cell division postembryonically. One common phenotype associated with impaired cell division is described as Stu (Sterile and Uncoordinated) [1], [2]. We conducted a genetic screen for zygotic mutants that displayed Stu phenotype in C. elegans. We isolated seven Stu mutants that fell into five complementation groups. We report here that two mutations, FanWang5 (fw5) and FanWang8 (fw8) affect the bub-1 gene, a homolog of yeast BUB1. Both mutant alleles of fw5 and fw8 exhibited variable behavioral defects, including developmental arrest, uncoordination and sterility. The number of postembryonically born neurons in the ventral cord decreased and their axon morphology was abnormal. Also, the decrease of neurons in the ventral cord phenotype could not be suppressed by a caspase-3 loss-of-function mutant. In addition, bub-1(fw5 and fw8) mutants showed widespread effects on postembryonic development in many cell lineages. We found that bub-1 functioned maternally in several developmental lineages at the embryonic stage in C. elegans. Studies in yeast have shown that BUB1 functions as a spindle checkpoint protein by regulating the anaphase promoting complex/cyclosome (APC/C). We performed double mutant analysis and observed that bub-1 genetically interacted with several downstream genes, including fzy-1/CDC20, mat-2/APC1 and emb-27/APC6.
Conclusions
Our results demonstrate a conserved role of bub-1 in cell-cycle regulation and reveal that C. elegans bub-1 is required both maternally and zygotically. Further, our genetic analysis is consistent with that the function of bub-1 in C. elegans is likely similar to its yeast and mammalian homologs.
doi:10.1371/journal.pone.0005912
PMCID: PMC2691579  PMID: 19526056
13.  Function and evolution of the serotonin-synthetic bas-1 gene and other aromatic amino acid decarboxylase genes in Caenorhabditis 
Background
Aromatic L-amino acid decarboxylase (AADC) enzymes catalyze the synthesis of biogenic amines, including the neurotransmitters serotonin and dopamine, throughout the animal kingdom. These neurotransmitters typically perform important functions in both the nervous system and other tissues, as illustrated by the debilitating conditions that arise from their deficiency. Studying the regulation and evolution of AADC genes is therefore desirable to further our understanding of how nervous systems function and evolve.
Results
In the nematode C. elegans, the bas-1 gene is required for both serotonin and dopamine synthesis, and maps genetically near two AADC-homologous sequences. We show by transformation rescue and sequencing of mutant alleles that bas-1 encodes an AADC enzyme. Expression of a reporter construct in transgenics suggests that the bas-1 gene is expressed, as expected, in identified serotonergic and dopaminergic neurons. The bas-1 gene is one of six AADC-like sequences in the C. elegans genome, including a duplicate that is immediately downstream of the bas-1 gene. Some of the six AADC genes are quite similar to known serotonin- and dopamine-synthetic AADC's from other organisms whereas others are divergent, suggesting previously unidentified functions. In comparing the AADC genes of C. elegans with those of the congeneric C. briggsae, we find only four orthologous AADC genes in C. briggsae. Two C. elegans AADC genes – those most similar to bas-1 – are missing from C. briggsae. Phylogenetic analysis indicates that one or both of these bas-1-like genes were present in the common ancestor of C. elegans and C. briggsae, and were retained in the C. elegans line, but lost in the C. briggsae line. Further analysis of the two bas-1-like genes in C. elegans suggests that they are unlikely to encode functional enzymes, and may be expressed pseudogenes.
Conclusions
The bas-1 gene of C. elegans encodes a serotonin- and dopamine-synthetic AADC enzyme. Two C. elegans AADC-homologous genes that are closely related to bas-1 are missing from the congeneric C. briggsae; one or more these genes was present in the common ancestor of C. elegans and C. briggsae. Despite their persistence in C. elegans, evidence suggests the bas-1-like genes do not encode functional AADC proteins. The presence of the genes in C. elegans raises questions about how many 'predicted genes' in sequenced genomes are functional, and how duplicate genes are retained or lost during evolution. This is another example of unexpected retention of duplicate genes in eukaryotic genomes.
doi:10.1186/1471-2148-4-24
PMCID: PMC514703  PMID: 15287963
14.  Bias and Evolution of the Mutationally Accessible Phenotypic Space in a Developmental System 
PLoS Genetics  2010;6(3):e1000877.
Genetic and developmental architecture may bias the mutationally available phenotypic spectrum. Although such asymmetries in the introduction of variation may influence possible evolutionary trajectories, we lack quantitative characterization of biases in mutationally inducible phenotypic variation, their genotype-dependence, and their underlying molecular and developmental causes. Here we quantify the mutationally accessible phenotypic spectrum of the vulval developmental system using mutation accumulation (MA) lines derived from four wild isolates of the nematodes Caenorhabditis elegans and C. briggsae. The results confirm that on average, spontaneous mutations degrade developmental precision, with MA lines showing a low, yet consistently increased, proportion of developmental defects and variants. This result indicates strong purifying selection acting to maintain an invariant vulval phenotype. Both developmental system and genotype significantly bias the spectrum of mutationally inducible phenotypic variants. First, irrespective of genotype, there is a developmental bias, such that certain phenotypic variants are commonly induced by MA, while others are very rarely or never induced. Second, we found that both the degree and spectrum of mutationally accessible phenotypic variation are genotype-dependent. Overall, C. briggsae MA lines exhibited a two-fold higher decline in precision than the C. elegans MA lines. Moreover, the propensity to generate specific developmental variants depended on the genetic background. We show that such genotype-specific developmental biases are likely due to cryptic quantitative variation in activities of underlying molecular cascades. This analysis allowed us to identify the mutationally most sensitive elements of the vulval developmental system, which may indicate axes of potential evolutionary variation. Consistent with this scenario, we found that evolutionary trends in the vulval system concern the phenotypic characters that are most easily affected by mutation. This study provides an empirical assessment of developmental bias and the evolution of mutationally accessible phenotypes and supports the notion that such bias may influence the directions of evolutionary change.
Author Summary
Random mutation does not generate random phenotypic variation because genetic and developmental architecture may constrain and bias the mutationally inducible phenotypic spectrum. Understanding such biases in the introduction of phenotypic variation is thus essential to reveal which phenotypes can ultimately be explored and selected through evolution. Here we used lines which had accumulated spontaneous random mutation over 250 generations starting from four distinct wild isolates of the nematode species C. briggsae and C. elegans, to study how a developmental system—vulval cell fate patterning—responds to mutational perturbations. We show that developmental defects and variants increase upon mutation accumulation in lines derived from all four isolates. However, some mutationally induced phenotypic variants occur more frequently than others, and the degree and spectrum of developmental variation further differed between isolates. These results illustrate how the phenotypic spectrum induced by random mutation can be biased due to both developmental system features and variation in the genetic background. Moreover, the mutationally most sensitive phenotypic characters are the ones that show most evolutionary variation among closely related species. These observations show how random mutation translates into a biased, limited range of phenotypes—a phenomenon likely impacting possible trajectories of phenotypic evolution.
doi:10.1371/journal.pgen.1000877
PMCID: PMC2837400  PMID: 20300655
15.  Plectus - a stepping stone in embryonic cell lineage evolution of nematodes 
EvoDevo  2012;3:13.
Background
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.
Methods
The study was conducted using 4-D lineage analysis, 3-D modeling of developing embryos and laser-induced ablation of individual blastomeres.
Results
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.
Conclusions
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.
doi:10.1186/2041-9139-3-13
PMCID: PMC3464786  PMID: 22748136
Nematode; embryogenesis; cell lineage; cell specification; evolution; developmental system drift; Plectus; C. elegans
16.  The Caenorhabditis globin gene family reveals extensive nematode-specific radiation and diversification 
Background
Globin isoforms with variant properties and functions have been found in the pseudocoel, body wall and cuticle of various nematode species and even in the eyespots of the insect-parasite Mermis nigrescens. In fact, much higher levels of complexity exist, as shown by recent whole genome analysis studies. In silico analysis of the genome of Caenorhabditis elegans revealed an unexpectedly high number of globin genes featuring a remarkable diversity in gene structure, amino acid sequence and expression profiles.
Results
In the present study we have analyzed whole genomic data from C. briggsae, C. remanei, Pristionchus pacificus and Brugia malayi and EST data from several other nematode species to study the evolutionary history of the nematode globin gene family. We find a high level of conservation of the C. elegans globin complement, with even distantly related nematodes harboring orthologs to many Caenorhabditis globins. Bayesian phylogenetic analysis resolves all nematode globins into two distinct globin classes. Analysis of the globin intron-exon structures suggests extensive loss of ancestral introns and gain of new positions in deep nematode ancestors, and mainly loss in the Caenorhabditis lineage. We also show that the Caenorhabditis globin genes are expressed in distinct, mostly non-overlapping, sets of cells and that they are all under strong purifying selection.
Conclusion
Our results enable reconstruction of the evolutionary history of the globin gene family in the nematode phylum. A duplication of an ancestral globin gene occurred before the divergence of the Platyhelminthes and the Nematoda and one of the duplicated genes radiated further in the nematode phylum before the split of the Spirurina and Rhabditina and was followed by further radiation in the lineage leading to Caenorhabditis. The resulting globin genes were subject to processes of subfunctionalization and diversification leading to cell-specific expression patterns. Strong purifying selection subsequently dampened further evolution and facilitated fixation of the duplicated genes in the genome.
doi:10.1186/1471-2148-8-279
PMCID: PMC2576238  PMID: 18844991
17.  Coevolution within and between Regulatory Loci Can Preserve Promoter Function Despite Evolutionary Rate Acceleration 
PLoS Genetics  2012;8(9):e1002961.
Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the unc-47 promoters of Caenorhabditis elegans and C. briggsae persists despite divergence within a cis-regulatory element and between this element and the trans-regulatory environment. Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.
Author Summary
Some phenotypes, including gene expression patterns, are conserved between distantly related species. However, the molecular bases of those phenotypes are not necessarily conserved. Instead, regulatory DNA sequences and the proteins with which they interact can change over time with balanced effects, preserving expression patterns and concealing regulatory divergence. Coevolution between interacting molecules makes gene regulation highly species-specific, and it can be detected when the cis-regulatory DNA of one species is used to drive expression in another species. In this way, we identified regions of the C. elegans and C. briggsae unc-47 promoters that have coevolved with the lineage-specific trans-regulatory environments of these organisms. The C. briggsae promoter experienced accelerated sequence change relative to related species. All of this evolution occurred without changing the expression pattern driven by the promoter in its endogenous environment.
doi:10.1371/journal.pgen.1002961
PMCID: PMC3447958  PMID: 23028368
18.  Comparative RNAi Screens in C. elegans and C. briggsae Reveal the Impact of Developmental System Drift on Gene Function 
PLoS Genetics  2014;10(2):e1004077.
Although two related species may have extremely similar phenotypes, the genetic networks underpinning this conserved biology may have diverged substantially since they last shared a common ancestor. This is termed Developmental System Drift (DSD) and reflects the plasticity of genetic networks. One consequence of DSD is that some orthologous genes will have evolved different in vivo functions in two such phenotypically similar, related species and will therefore have different loss of function phenotypes. Here we report an RNAi screen in C. elegans and C. briggsae to identify such cases. We screened 1333 genes in both species and identified 91 orthologues that have different RNAi phenotypes. Intriguingly, we find that recently evolved genes of unknown function have the fastest evolving in vivo functions and, in several cases, we identify the molecular events driving these changes. We thus find that DSD has a major impact on the evolution of gene function and we anticipate that the C. briggsae RNAi library reported here will drive future studies on comparative functional genomics screens in these nematodes.
Author Summary
Although two related species may appear similar, the genetic pathways that underpin this shared biology may have drifted and changed. This phenomenon is known as Developmental System Drift (DSD). One consequence of DSD is that equivalent genes may play different roles in phenotypically similar, related species but there have been no systematic studies to examine this. How often do genes have different functions in similar species? Are certain genes more likely to change functions? Finally, what are the molecular changes that drive this? Here, we compare the effects of reducing the levels of over 1300 different genes in two species of nematode worm. These worms are very similar— they live in the same ecological niche, and have near-identical development and behavior. We find that over 25% of conserved genes have different functions in these two species, showing that DSD has a major impact on how gene function evolves. Intriguingly, we find that genes that have arisen recently are most likely to change functions and that this is often driven by changes in their expression. This is the first systematic comparison of loss of function phenotypes in related species and sheds light on how genetic pathways rewire during DSD.
doi:10.1371/journal.pgen.1004077
PMCID: PMC3916228  PMID: 24516395
19.  Quantitative Analysis of Cytokinesis In Situ during C. elegans Postembryonic Development 
PLoS ONE  2014;9(10):e110689.
The physical separation of a cell into two daughter cells during cytokinesis requires cell-intrinsic shape changes driven by a contractile ring. However, in vivo, cells interact with their environment, which includes other cells. How cytokinesis occurs in tissues is not well understood. Here, we studied cytokinesis in an intact animal during tissue biogenesis. We used high-resolution microscopy and quantitative analysis to study the three rounds of division of the C. elegans vulval precursor cells (VPCs). The VPCs are cut in half longitudinally with each division. Contractile ring breadth, but not the speed of ring closure, scales with cell length. Furrowing speed instead scales with division plane dimensions, and scaling is consistent between the VPCs and C. elegans blastomeres. We compared our VPC cytokinesis kinetics data with measurements from the C. elegans zygote and HeLa and Drosophila S2 cells. Both the speed dynamics and asymmetry of ring closure are qualitatively conserved among cell types. Unlike in the C. elegans zygote but similar to other epithelial cells, Anillin is required for proper ring closure speed but not asymmetry in the VPCs. We present evidence that tissue organization impacts the dynamics of cytokinesis by comparing our results on the VPCs with the cells of the somatic gonad. In sum, this work establishes somatic lineages in post-embryonic C. elegans development as cell biological models for the study of cytokinesis in situ.
doi:10.1371/journal.pone.0110689
PMCID: PMC4203819  PMID: 25329167
20.  Pristionchus pacificus, a nematode with only three juvenile stages, displays major heterochronic changes relative to Caenorhabditis elegans. 
The nematode Pristionchus pacificus (Diplogastridae) has been described as a satellite organism for a functional comparative approach to the model organism Caenorhabditis elegans because genetic, molecular, and cell-biological tools can be used in a similar way in both species. Here we show that P. pacificus has three juvenile stages, instead of the usual four found in other nematodes. Embryogenesis is lengthened and many developmental events that take place during the first juvenile stage in C. elegans occur during late embryogenesis in P. pacificus. Video imaging and transmission electron microscopy revealed no embryonic moult. The timing of later developmental events relative to the moults differs between P. pacificus and C. elegans. In addition, the post-embryonic blast-cell divisions display a specific change in timing between the two species, resulting in heterochrony between different cell lineages, such as vulval and gonadal lineages. Developmental events appear to come into register during the last larval stage. Thus, differences in developmental timing between P. pacificus and C. elegans represent a deep heterochronic change. We designate the three juvenile stages of P. pacificus as J1 to J3. Comparison with other species of the family Diplogastridae indicates that this pattern represents an apomorphic character for the monophylum Diplogastridae.
PMCID: PMC1690183  PMID: 10501036
21.  Mesodermal expression of the C. elegans HMX homolog mls-2 requires the PBC homolog CEH-20 
Mechanisms of development  2008;125(5-6):451-461.
Metazoan development proceeds primarily through the regulated expression of genes encoding transcription factors and components of cell signaling pathways. One way to decipher the complex developmental programs is to assemble the underlying gene regulatory networks by dissecting the cis-regulatory modules that direct temporal-spatial expression of developmental genes and identify corresponding trans-regulatory factors. Here, we focus on the regulation of a HMX homoebox gene called mls-2, which functions at the intersection of a network that regulates cleavage orientation, cell proliferation and fate specification in the C. elegans postembryonic mesoderm. In addition to its transient expression in the postembryonic mesodermal lineage, the M lineage, mls-2 expression is detected in a subset of embryonic cells, in three pairs of head neurons and transiently in the somatic gonad. Through mutational analysis of the mls-2 promoter, we identified two elements (E1 and E2) involved in regulating the temporal-spatial expression of mls-2. In particular, we showed that one of the elements (E1) required for mls-2 expression in the M lineage contains two critical putative PBC-Hox binding sites that are evolutionarily conserved in C. briggsae and C. remanei. Furthermore, the C. elegans PBC homolog CEH-20 is required for mls-2 expression in the M lineage. Our data suggests that mls-2 might be a direct target of CEH-20 in the M lineage and that the regulation of CEH-20 on mls-2 is likely Hox-independent.
doi:10.1016/j.mod.2008.01.009
PMCID: PMC2441446  PMID: 18316179
C. elegans; mesoderm; M lineage; mls-2; ceh-20; homeodomain; PBC; HMX; Hox; PBC-Hox binding site; promoter dissection
22.  Poly-G/poly-C tracts in the genomes of Caenorhabditis 
BMC Genomics  2007;8:403.
Background
In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts.
Results
In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggsae. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants.
Conclusion
The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.
doi:10.1186/1471-2164-8-403
PMCID: PMC2211496  PMID: 17986356
23.  Cloning of Caenorhabditis U2AF65: an alternatively spliced RNA containing a novel exon. 
Molecular and Cellular Biology  1997;17(2):946-953.
The U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF, is an essential splicing factor required for recognition of the polypyrimidine tract and subsequent U2 snRNP assembly at the branch point. Because Caenorhabditis elegans introns lack both polypyrimidine tract and branch point consensus sequences but have a very highly conserved UUUUCAG/R consensus at their 3' splice sites, we hypothesized that U2AF might serve to recognize this sequence and thus promote intron recognition in C. elegans. Here we report the cloning of the gene for the large subunit of U2AF, uaf-1. Three classes of cDNA were identified. In the most abundant class the open reading frame is similar to that for the U2AF65 from mammals and flies. The remaining two classes result from an alternative splicing event in which an exon containing an in-frame stop codon is inserted near the beginning of the second RNA recognition motif. However, this alternative mRNA is apparently not translated. Interestingly, the inserted exon contains 10 matches to the 3' splice site consensus. To determine whether this feature is conserved, we sequenced uaf-1 from the related nematode Caenorhabditis briggsae. It is composed of six exons, including an alternatively spliced third exon interrupting the gene at the same location as in C. elegans. uaf-1 is contained in an operon with the rab-18 gene in both species. Although the alternative exons from the two species are not highly conserved and would not encode related polypeptides, the C. briggsae alternative exon has 18 matches to the 3' splice site consensus. We hypothesize that the array of 3' splice site-like sequences in the pre-mRNA and alternatively spliced exon may have a regulatory role. The alternatively spliced RNA accumulates at high levels following starvation, suggesting that this RNA may represent an adaption for reducing U2AF65 levels when pre-mRNA levels are low.
PMCID: PMC231820  PMID: 9001248
24.  OSM-11 Facilitates LIN-12 Notch Signaling during Caenorhabditis elegans Vulval Development  
PLoS Biology  2008;6(8):e196.
Notch signaling is critical for cell fate decisions during development. Caenorhabditis elegans and vertebrate Notch ligands are more diverse than classical Drosophila Notch ligands, suggesting possible functional complexities. Here, we describe a developmental role in Notch signaling for OSM-11, which has been previously implicated in defecation and osmotic resistance in C. elegans. We find that complete loss of OSM-11 causes defects in vulval precursor cell (VPC) fate specification during vulval development consistent with decreased Notch signaling. OSM-11 is a secreted, diffusible protein that, like previously described C. elegans Delta, Serrate, and LAG-2 (DSL) ligands, can interact with the lineage defective-12 (LIN-12) Notch receptor extracellular domain. Additionally, OSM-11 and similar C. elegans proteins share a common motif with Notch ligands from other species in a sequence defined here as the Delta and OSM-11 (DOS) motif. osm-11 loss-of-function defects in vulval development are exacerbated by loss of other DOS-motif genes or by loss of the Notch ligand DSL-1, suggesting that DOS-motif and DSL proteins act together to activate Notch signaling in vivo. The mammalian DOS-motif protein Deltalike1 (DLK1) can substitute for OSM-11 in C. elegans development, suggesting that DOS-motif function is conserved across species. We hypothesize that C. elegans OSM-11 and homologous proteins act as coactivators for Notch receptors, allowing precise regulation of Notch receptor signaling in developmental programs in both vertebrates and invertebrates.
Author Summary
The classic view of Notch receptor activation involves receptor binding to transmembrane Notch ligands that contain a conserved DSL (Delta, Serrate, and LAG-2) domain. Here, we find that the Caenorhabditis elegans OSM-11 protein is a novel ligand of the well-characterized Notch signal transduction pathway and plays a role in cell fate specification during development. OSM-11 is a secreted, diffusible protein whose loss decreases Notch signaling in vivo. OSM-11 and related C. elegans proteins do not contain a DSL domain, but contain a conserved motif we have named DOS (Delta and OSM-11) that is also found in the extracellular domain of known Notch ligands in organisms other than C. elegans. The functional mammalian homolog of OSM-11 is the secreted protein Deltalike1 (Dlk1), also known as Preadipocyte Factor 1 (PREF1), which plays a poorly defined role in Notch signaling regulating obesity and other developmental decisions. This suggests that Notch ligands are split into two complementary coligand families that act together to regulate Notch signaling in developmental contexts. In addition to regulating development, DOS ligands play roles in osmotic stress and C. elegans behavior, suggesting previously unsuspected roles for Notch signaling across species.
The C. elegans OSM-11 protein acts with DSL ligands to activate Notch signaling in cell fate specification and defines a conserved family of potential Notch co-ligands.
doi:10.1371/journal.pbio.0060196
PMCID: PMC2504490  PMID: 18700817
25.  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.
doi:10.1016/j.ydbio.2012.11.034
PMCID: PMC3548946  PMID: 23220655
robustness; microscopy; cell lineage; image analysis; C. elegans; stress

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