LR Baugh et al. (1999) International C. elegans Meeting "Quantification of Transcriptome Distortion After Amplification Using cDNA Micro-Arrays"

LR Baugh, CP Hunter

[International C. elegans Meeting, 1999]

Complete genomic sequences and micro-array based technologies allow for genome-wide parallel analysis of gene expression. Such transcriptional profiling makes it possible to map clusters of coordinately regulated genes and in turn, in combination with computational and traditional molecular and genetic techniques, to ask questions about the regulation and significance of such clusters. How many determinants define a given cluster? Do those determinants act in cis or trans ? How many clusters are regulated by a given determinant? To what extent do master regulators specify unique fates through conserved sets of target genes? Due to the relatively large amount of Poly A + RNA required to hybridize a single micro-array and thus the virtual impossibility of attaining ample, synchronous starting material, the total message pool, or transcriptome, of single embryos or cells will have to be amplified to address such questions. However, the amplification process should not significantly distort the relative abundance of sequences in the transcriptome. Using a yeast cDNA micro-array, we have quantified the distortion imparted on a transcriptome during different amplification procedures, and we plan to use the results of this analysis in the design of experiments employing a C. elegans cDNA micro-array.

Julia M Claggett et al. (2004) East Coast Worm Meeting "An Investigation into the potential regulatory relationships between components of a network specified by pal-1."

L Ryan Baugh, Julia M Claggett, Craig P Hunter

[East Coast Worm Meeting, 2004]

PAL-1 protein, contributed both maternally and zygotically, is necessary and sufficient to specify and maintain the C blastomere lineage in the C. elegans embryo (Hunter and Kenyon, 1996). A number of this master regulator's targets were identified by microarrays comparing the transcript abundance in wild-type and mutant embryos either lacking or containing extra C blastomeres. Furthermore, we collected these embryos at defined time points, thus additionally providing temporal information. Target genes could then be separated by their transcriptional initiation into four consecutive temporal phases defined by a singular cell cycle beginning with the 2C-cell stage (Baugh et al, 2003). Using reporter YFP constructs for thirteen of the targets and a volume-rendering program, the 3D spatial expression pattern of each target gene was established. On the basis of this spatial information and knowledge of the temporal phase to which each target belongs, we have proposed a set of regulatory relationships between the components. We are currently testing these hypotheses by disrupting potential (capital O, grave accent)upstream(capital O, acute accent) regulators via RNAi and/or mutation and either observing the effect on individual (capital O, grave accent)downstream(capital O, acute accent) reporters or analyzing the effect on transcript abundance using QPCR. We hope that such measurements will give us insight into how the genes within the pal-1 network regulate each other in order to establish and maintain the various cell fates within the C blastomere lineage. Hunter, C.P. and Kenyon, C. (1996). Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cell 87, 217-26. Baugh, L.R., Hill, A.A., Slonim, D.K., Brown, E.L. and Hunter, C.P. (2003). Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900.

Fry, A. et al. (2017) International Worm Meeting "The role of DAF-18/PTEN in nutritional regulation of primordial germ cell quiescence."

Hubbard, E.J., Fry, A.

[International Worm Meeting, 2017]

PTEN is one of the most frequently lost tumor suppressors in human cancers and has been shown to suppress proliferation of stem cells and cancer cells in response to nutritional signals. In a striking parallel, the C. elegans PTEN homolog, DAF-18, suppresses proliferation of primordial germ cells (PGCs) in response to food deprivation. When hatched in the absence of food, C. elegans development is arrested at the first larval stage (L1). During L1 arrest, the primordial germ cells (PGCs) and somatic cells halt proliferation and remain quiescent until food is encountered. DAF-18 is required for this cell cycle arrest of the two PGCs in the absence of food. Previous studies have shown that AKT acts downstream of DAF-18, while TOR signaling is either acting in parallel or partially downstream to regulate PGC quiescence [1]. Additionally, DAF-16/FoxO, which promotes somatic cell quiescence during L1 arrest, is not required for PGC quiescence [2]. Several avenues are open for exploration, including the identification of relevant factors downstream of DAF-18, the tissue in which DAF-18 acts to regulate PGC quiescence, DAF-18-dependent processes within PGCs, and key environmental cues. To examine PGCs in vivo, we have generated a fluorescent germ cell reporter. By tightly synchronizing L1 animals, we show that daf-18 mutant PGCs divide within several hours of hatching in the absence of food, and also divide prematurely in the presence of food. We have also found that zygotic daf-18 is sufficient to restore PGC quiescence in starved L1s. Currently, we are determining the tissue(s) in which DAF-18 acts to regulate PGC quiescence. Additionally, we are investigating the DAF-18-dependence of specific modifications within PGCs that coincide with proliferation. Thus, we are using the powerful system of C. elegans PGC proliferation to gain insight into nutritionally-regulated mechanisms of PTEN control over quiescence. [1] Fukuyama, M., et al.. Biol Open, 2012. [2] Baugh, L.R. and P.W. Sternberg. Curr Biol, 2006.

Webster, A.K. et al. (2019) International Worm Meeting "Transgenerational effects of extended dauer diapause on starvation survival and gene expression plasticity in C. elegans."

Hibshman, J.D., Chitrakar, R., Jordan, J.M., Webster, A.K., Baugh, L.R.

[International Worm Meeting, 2019]

Phenotypic plasticity is facilitated by epigenetic regulation, and remnants of such regulation may persist after plasticity-inducing cues are gone. However, the relationship between plasticity and transgenerational epigenetic memory is not understood. Dauer diapause in Caenorhabditis elegans provides an opportunity to determine how a plastic response to the early-life environment affects traits later in life and in subsequent generations. We report that, after extended diapause, postdauer worms initially exhibit reduced reproductive success and greater interindividual variation. In contrast, F3 progeny of postdauers display increased starvation resistance and lifespan, revealing potentially adaptive transgenerational effects. Transgenerational effects are dependent on the duration of diapause, indicating an effect of extended starvation. In agreement, RNA-seq demonstrates a transgenerational effect on nutrient-responsive genes. Further, postdauer F3 progeny exhibit reduced gene expression plasticity, suggesting a trade-off between plasticity and epigenetic memory. This work reveals complex effects of nutrient stress over different time scales in an animal that evolved to thrive in feast and famine.

Webster, A.K. et al. (2019) International Worm Meeting "Population selection and sequencing of C. elegans wild isolates identifies a region on chromosome III affecting starvation resistance."

Hibshman, J.D., Cook, D.E., Jordan, J.M., Baugh, L.R., Hung, A., Moore, B.T., Andersen, E.C., Tanny, R.E., Webster, A.K., Kaplan, R.E.W., Guzman, R.

[International Worm Meeting, 2019]

To understand the genetic basis of complex traits, it is important to be able to efficiently phenotype many genetically distinct individuals. In the nematode Caenorhabditis elegans, individuals have been isolated from diverse populations around the globe and whole-genome sequenced. As a result, hundreds of wild strains with known genome sequences can be used for genome-wide association studies (GWAS). However, phenotypic analysis of these strains can be laborious, particularly for quantitative traits requiring multiple measurements per strain. Starvation resistance is likely a fitness-proximal trait for nematodes, and it is related to metabolic disease risk in humans. However, natural variation in C. elegans starvation resistance has not been characterized, and precise measurement of the trait is time-intensive. Here, we developed a population selection and sequencing-based approach to phenotype starvation resistance in a pool of 97 wild strains. We used restriction site-associated DNA sequencing (RAD-seq) to infer the frequency of each strain among survivors in a mixed culture over time during starvation. We used manual starvation survival assays to validate the trait, confirming that strains that increased in frequency over time are starvation-resistant relative to strains that decreased in frequency. These results document natural variation in starvation resistance. Further, we found that variation in starvation resistance is significantly associated with variation at a region on chromosome III. Using a near-isogenic line (NIL), we showed the importance of this genomic interval for starvation resistance. This study demonstrates the feasibility of using population selection and sequencing in an animal model for phenotypic analyses of quantitative traits, reveals natural variation of starvation resistance in C. elegans, and identifies a genomic region that contributes to such variation.

Joanne C Wen et al. (2003) International Worm Meeting "Functional Characterization of Early Embryonic PAL-1 Targets"

Craig P Hunter, Joanne C Wen, L Ryan Baugh

[International Worm Meeting, 2003]

The activation and maintenance of C lineage specification occurs through maternal and zygotic PAL-1 activity, respectively (Hunter & Kenyon, 1996; Edgar et al, 2001). A set of targets of this master regulatory transcription factor were identified by transcript profiling embryos with perturbed PAL-1 activity (see abstract by Baugh et al). To functionally characterize PAL-1 targets, we have used RNAi to assess the lethality and terminal phenotypes following loss of function. To identify interactions between targets, we are performing epistasis analysis both by scoring synthetic lethality and by examining the effect of RNAi against one target on the expression of reporters for other targets. Our hope is that such functional characterization of a key set of PAL-1 targets will generate the data necessary to begin modeling the PAL-1 regulatory network.

Fradin, H. et al. (2015) International Worm Meeting "Genome sequence of a parthenogen closely related to Caenorhabditis reveals fusion of all ancestral chromosomes."

Fitch, D.H.A., Fradin, H., Baugh, L.R., Gunsalus, K., Zegar, C.M., Lucas, J., Piano, F., Gutwein, M.R., Kovtun, M., Corcoran, D.L., Kiontke, K.

[International Worm Meeting, 2015]

Parthenogenesis is an asexual mode of reproduction in which an embryo develops from an unfertilized egg. The transition from sexual reproduction to parthenogenesis occurred repeatedly in nematodes. An excellent model for understanding the evolution of parthenogenesis is provided by Diploscapter sp. 1, which belongs to the asexual lineage closest to C. elegans. We report a D. sp. 1 genome sequence of 165 Mb assembled de novo using Illumina short-insert libraries and larger PacBio reads. We find 28,028 protein-coding genes using RNA-seq transcriptomic data and in silico gene predictions. Validation of the completeness of the genome and gene content using a set of conserved eukaryotic genes and independently assembled transcriptome data shows that the assembly is of high quality. Microscopy reveals a single pair of homologous chromosomes in D. sp. 1 and in two other close species in which males seem to be absent. This was previously described in the parthenogenetic D. coronatus and is rare in eukaryotes. The closest gonochoristic relatives of Diploscapter have six or seven chromosomes. We find that scaffolds in the D. sp. 1 genome each have strong homology to a specific Caenorhabditis chromosome, consistent with the high macro-synteny generally observed in nematodes. As we note no large-scale loss of genes in D. sp. 1, we hypothesize that the whole set of ancestral chromosomes fused. We find evidence of this fusion event in our assembly and we can hypothesize in which spatial order the ancestral chromosomes fused. C. elegans mutant strains with telomere maintenance defects often display chromosome fusions. Two genes involved in telomere maintenance-mrt-1 and pot-1-are not detected in D. sp. 1. This suggests a model for the evolution of a fused chromosome in D. sp. 1 through altered telomere maintenance. Our genome sequences collapse into two divergent haplotypes. However, 10 % are homozygous and found on regions homologous to C. elegans chromosomes I and X. Microscopy and genomic features support a model of oocyte maturation with only one meiotic division that maintains diploidy and heterozygosity. Our study shows that evolution from sexual to asexual reproduction can be accompanied by dramatic changes in genome structure. It establishes D. sp. 1 and its relatives as an outstanding lineage for studying transition to parthenogenesis.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Romain Levayer et al. (2006) Development & Evolution Meeting "Identification of new substrates for the DYRK kinase MBK-2"

Geraldine Seydoux, Michael Stitzel, Romain Levayer

[Development & Evolution Meeting, 2006]

In C. elegans, the transition from oocyte-to-embryo requires degradation of oocyte proteins, including the oocyte maturation protein OMA-1 and the katanin subunit MEI-1. The DYRK kinase MBK-2 has been identified as a key regulator of these degradation events (Pellettieri et al., 2003). MBK-2 phosphorylates OMA-1 and MEI-1 on PEST sequences to mark these proteins for degradation (Nishi and Lin 2005, Stitzel et al. 2006, Shirayama et al., 2006). Perdurance of OMA-1 and MEI-1 explains some, but not all, of the phenotypes of mbk-2 mutants, suggesting that MBK-2 has additional substrates (Pellettieri et al., 2003). Here we describe a bioinformatic approach to identify additional MBK-2 substrates, based on properties common to OMA-1 and MEI-1. We screened a list of 4168 candidate genes, identified as "Strictly Maternal" or "Maternal Degradation" in an early embryo transcriptome analysis (Baugh et al. 2003), for: DYRK kinase consensus site PEST degradation motifs overlapping the DYRK site Embryonic lethal/sterile phenotype as reported in Wormbase Evolutionary conservation of the DYRK site Following these criteria, we identified 69 potential MBK-2 substrates. We are currently testing selected candidates for 1) phosphorylation by recombinant MBK-2 in vitro, and 2) MBK-2-dependent degradation using GFP reporters in vivo. We will report our results at the meeting.

L Ryan Baugh et al. (2003) International Worm Meeting "Characterization of the Embryonic Regulatory Network Specified by PAL-1"

Craig P Hunter, Joanne C Wen, Donna K Slonim, Andrew A Hill, Eugene L Brown, Kate Hill-Harfe, L Ryan Baugh

[International Worm Meeting, 2003]

Maternal PAL-1 specifies the C lineage by activating a lineage-specific transcriptional program (Hunter & Kenyon 1996). Zygotic PAL-1 maintains this program while the C lineage is patterned to give rise to multiple cell fates and lineage-specific morphogenetic behaviors (Edgar et al. 2001). To understand how master regulators control and integrate diverse developmental processes, we have used microarrays to identify PAL-1 regulated genes specifically expressed in the C lineage. We are now using transcriptional reporters, RNAi, bioinformatics, and numerical simulation to characterize the spatial and temporal expression, activity and regulation of these genes. We first generated transcript profiles of high temporal resolution from the 4 to 190-cell stage in wild type (Baugh et al, 2003). To identify genes regulated by PAL-1 activity we then profiled mutants that either lack a C lineage or that have multiple somatic lineages specified as C. Hundreds of candidate genes have been identified and ordered by their time of activation. Candidates encoding putative transcription factors and signaling molecules have been confirmed as PAL-1 target genes by scoring reporters following RNAi of pal-1 and its repressor mex-3. We are now using RNAi to functionally characterize the validated set, and we are mining the genome sequence for motifs that may correspond to transcription factor binding sites. It is our goal to determine the structure of the regulatory network specified by PAL-1 by determining the full set of regulatory relationships among these genes.