Du, Lawrence et al. (2015) International Worm Meeting "Transcriptional control of the C. elegans endoderm master regulator elt-2."

Wu, Allison, Du, Lawrence, Tracy, Sharon, Rifkin, Scott

[International Worm Meeting, 2015]

Cis-regulatory elements are the fundamental control units of gene regulatory networks. They integrate information from transcription factors to organize the expression of a gene in time and space. However, our understanding of how cis-regulatory elements produce a specific spatio-temporal pattern of gene expression is often limited to semi-quantitative descriptions of gene activation. In this study, we determined the quantitative impact of cis-regulatory mutations with single-molecule resolution on the expression levels and variability of elt-2, the key switch for the C. elegans endoderm cell-fate decision and the master regulator of endoderm differentiation. We fused wild-type and mutant versions of the elt-2 promoter to a gfp reporter and inserted these constructs as single copies into a defined location in the C. elegans genome. We then measured early embryonic gfp transcript levels by single-molecule RNA FISH (smFISH). Although the GATA transcription factors that activate elt-2 can bind in vitro to many HGATAR motifs within the elt-2 promoter, mutation of one particular motif had a disproportionate impact on in vivo gene expression. Mutation or deletion of a single highly conserved ACTGATAAG motif at -527 bp upstream of the start codon within the elt-2 promoter abolished the vast majority of embryonic, larval, and adult reporter expression. Absence of this critical motif revealed the presence of low-level, temporally-ectopic, stochastic transcription occurring from secondary HGATAR motifs and proximal promoter regions. Our results indicate that multiple GATA factors converge on a single non-redundant cis-regulatory motif to drive both early embryonic activation as well as later maintenance of gene expression.

Wu, Allison et al. (2011) International Worm Meeting "Comparing gene expression pattern in the skn-1 intestinal developmental network in C. briggsae, C. remanei, and C. elegans to gain insights into the dynamical functional roles of orthologous genes."

Rifkin, Scott, Wu, Allison, Du, Lawrence

[International Worm Meeting, 2011]

Previous studies have shown that, in the C.elegans skn-1 network, the most downstream gene elt-2 determines the onset of gut development while end-3, end-1, and med-1/2 constrain the gene expression variability. Inboth C. briggsae and C. remanei, there has been a large amount of copy number evolution in this network: there are 5 functionally similar med paralogs in C. remanei , 4 med paralogs in C. briggsae and two orthologs of end-3 in C. briggsae. These potentially functionally similar homologs add extra connections to this intestinal specification network. We want to know how and why these redundant genes are preserved, whether these seemingly redundant connections are really redundant, and whether these connections add to or strengthen specific dynamical network properties, such as buffering of noise or environmenal variation or feedback strength. In this study, we use single-molecule fluorescence in situ hybridization (smFISH) to measure the expression pattern of genes in the skn-1 network in these three species, including end-3 orthologs, end-1 and elt-2. By quantitatively measuring the expression dynamics of these genes, we willustrate the dynamical roles these orthologs play in the intestine development in different nematode species and reveal the effects of gene duplication on dynamical network properties.

De Vore, Deanna Michele et al. (2013) International Worm Meeting "Exploring the role of mechanosensory extracellular matrix components in the structure and function of primary cilia."

Knobel, Karla, De Vore, Deanna Michele, Barr, Maureen

[International Worm Meeting, 2013]

PKD2 encodes a transient receptor potential polycystin (TRPP) channel receptor protein found in non-motile, primary cilia of mammalian cells 1. In humans, PKD2 mutations result in Autosomal Dominant Polycystic Kidney Disease (ADPKD). In the nematode Caenorhabditis elegans, the polycystin-2 homolog, PKD-2, localizes to the primary cilia of male-specific sensory neurons (CEMs and RnBs in the head and tail respectively) where it is required for male mating behaviors. Given the ancient and evolutionarily conserved role for polycystin-2 in cilia, I am using C. elegans as a model to identify new genes required for ciliary receptor localization. Our laboratory performed a forward genetic screen for PKD-2::GFP ciliary localization (Cil) defective mutants (Bae et al 2008). The cil-2(my2) mutants exhibit excess unusual PKD-2 accumulation at the ciliary base, cilium proper, and around the distal dendrite. We performed three-factor and deficiency mapping, whole genome sequencing, and single gene rescue experiments and determined that the cil-2(my2) allele is a mutation in the gene, mec-5. MEC-5 is a unique collagen protein found in the extracellular matrix (ECM) surrounding touch receptor neurons (Du et al 1996). In touch receptor neurons (TRNs), MEC-1, MEC-5, and MEC-9 are found in the ECM, play a role in mechanosensation, and may localize degenerin/epithelial sodium channels (DEG/ENaCs) along the TRN processes (Du et al 1996, Emtage et al 2004). Our preliminary data indicates that mec-1 and mec-9 regulate PKD-2::GFP localization in male-specific sensory neurons. We are currently determining how these ECM proteins regulate localization and channel properties of the PKD-2 TRP polycystin channel. These studies will shed light on how sensory receptors like PKD-2 are targeted to cilia, and may advance the understanding and treatment of ADPKD. References: 1) Bae YK et al, Dev Dyn. 2008 Aug;237(8):2021-9. 2) Du H. et al, Neuron. 1996 Jan;16(1):183-94. 3) Emtage L et al, Neuron. 2004 Dec 2;44(5):795-807.

Keith A Boroevich et al. (2004) West Coast Worm Meeting "Identification of Regulatory Elements in Caenorhabditis elegans using Orthology Biased Gibbs Sampling"

Steven JM Jones, David L Baillie, Keith A Boroevich

[West Coast Worm Meeting, 2004]

Much attention has recently been placed on the problem of identification of gene regulatory sequences. Two main computational approaches exist: 1) identification of sequences that share similarity to known regulatory elements, and 2) de novo identification of common motifs in a set of co-regulated genes. Orthology Biased Gibbs Sampling (OrBS) applies a modification of the Gibbs Sampler put forth by Lawrence, Altschult et al . 1 in a attempt to solve this problem. Additional features include analysis of negative strand, multiple (or no) occurrences of a motif in any given sequence and identification of multiple unique motifs, all of which have appeared in previous incarnations of the Gibbs Sampler. The unique feature of OrBS is the use of comparative genomics as an informative prior and in the calculation of motif probability. OrBS will be applied to the identification of regulatory elements C. elegans . Clustering of spatially co-regulated genes as defined by the C. elegans Gene Expression Project (see Johnsen et al . poster). This data is more amenable to regulatory element detection than the more common microarray data because the sequence in which a cis -acting element may reside is strictly defined. Interspecies sequence conservation will be identified through alignment of the C. elegans gene promoter region to the promoter region of the C. briggsae ortholog using LAGAN 2 . Predicted regulatory elements will be verified in vivo using site directed mutagenesis and promoter::GFP fusions. References: 1 Lawrence CE, Altschul SF, Boguski MS, Liu JS, Neuwald AF, Wootton JC. (1993). Science 262: 208-214. 2 Brudno M, Do C, Cooper G, Kim MF, Davydov E, Green ED, Sidow A, Batzoglou S. (2003). Genome Research 13: 721-731.

Xu, Yichi et al. (2017) International Worm Meeting "Systematic decoding of cell movement in C. elegans early embryogenesis."

Xu, Yichi, Bao, Zhirong

[International Worm Meeting, 2017]

Morphogenesis is the fundamental process in biology that describes the formation of a tissue or organism. It encompasses multiple layers of controlling upon molecular basis (Turing, 1952) and cellular basis. Taking advantage of the invariant cell lineage in C. elegans (Sulston, 1983), we asked how cells move to correct destinations. Previous studies proposed cell movement is guided by local cell-cell interaction (Schnabel, 2006), which were supported by evidences in early-stage embryos and limited types of mutants. Here, we hypothesized that, upon cell-cell interaction, differential affinities between neighboring cells drive their next moves. With our collection of all cell tracks up to 350-cell stage in hundreds of wild-type and perturbed embryos (Du, 2015), we devised a computational model to optimize cellular configuration over time and infer cell-cell affinities for individual embryos. The comparison between wild-type and perturbed embryos demonstrated those affinities were specified by cell fate rather than cell lineage. This study provided a basis to systematically elucidate how affinity network can serve as a new paradigm to positional information and orchestration in complex tissues.

Duggan A et al. (1997) International C. elegans Meeting "CLONING OF THE EGL-46 GENE"

Duggan A, Chalfie M

[International C. elegans Meeting, 1997]

The egl-46 gene is required for the correct differentiation of the HSN neurons, a pair of motor neurons necessary for egg-laying (1,2). In addition, an extra pair of touch receptor-like cells is present in egl-46 mutant animals (3). This finding led to the hypothesis that egl-46 represses touch cell fate in these cells. We have cloned the egl-46 gene using transformation rescue and we have confirmed the assignment by identifying the mutations present in the four known egl-46 alleles. A 5.5kb fragment of CO9E6 partially rescues the Egl phenotype of egl-46 animals. The egl-46 gene (K11G9.4) encodes a C2H2 zinc finger protein that contains three putative zinc fingers. We have confirmed the exon-intron structure of egl-46 by sequencing a cDNA and by 5' RACE analysis. egl-46::lacZ and egl-46::GFP reporter constructs are expressed in a complex pattern which includes cells in the head, tail and ventral cord. We are now trying to determine the identity of the staining cells. Future work will address the mechanism by which the egl-46 gene controls neuronal fate and/or differentiation. (1) Desai, C., Garriga, G., McIntire, S.L. and H.R.Horvitz (1988) Nature 336, 638-646. (2) Desai, C. and H.R.Horvitz (1989) Genetics 121, 703-721. (3) Mitani, S., Du, H., Hall, D.H., Driscoll, M. and M. Chalfie (1993) Development 119, 773-783.

Mavji N Patel et al. (2001) International C. elegans Meeting "Evolution of germ-line signals that regulate growth and ageing in nematodes"

Armand M Leroi, Constantina Karageorgi, Christopher G Knight, Mavji N Patel

[International C. elegans Meeting, 2001]

One of the least understood aspects of animal development is how growth and body size are regulated 1,2 . Here we show that a signal from the germ-line represses growth in the nematode Caenorhabditis elegans . Laser-microbeam ablation of cells that give rise to the germ-line causes adults to become giant. Ablation of these cells in self-sterile mutant worms also causes gigantism suggesting that the germ-line represses growth because it is the source of a growth-antagonizing signal rather than a sink of resources required for reproduction. The C. elegans germ-line also emits a signal that represses longevity 3 . This longevity-repressing signal requires the activity of DAF-16, a forkhead/winged-helix transcription factor 3 , but we find that that the growth-repressing signal does not. The growth-repressing signal also does not require the activity of DBL-1, a TGF-beta-related protein that promotes growth in worms 4,5 . By ablating the germ-line precursors of other species of free-living nematodes we found that both the growth-repressing and longevity-repressing signals are evolutionarily variable. Some species have both, others have just one or the other. We suggest that variation in germ-line signaling contributes to body size and life-history diversity in the nematodes. 1. I, Conlon, M. Raff, Cell 96, 235 (1999). 2. S. J. Day, P. A. Lawrence, Development 127, 2977 (2000). 3. H. Hsin, C. Kenyon, Nature 399 , 362 (1999). 4. K. Morita, K. L. Chow, N. Ueno, Development 126, 1337 (1999). 5. Y. Suzuki et al ., Development 126, 241 (1999).

James L Lissemore et al. (2003) International Worm Meeting "Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene."

Laura L Lackner, George D Fedoriw, James L Lissemore

[International Worm Meeting, 2003]

PCR is a powerful and common molecular biology technique with many applications. We have developed an exercise for an undergraduate molecular biology lab course using PCR to detect deletions in the unc-93 gene of C. elegans. unc-93 encodes a putative transmembrane protein muscle protein of unknown function. Wildtype and three different unc-93 deletion mutant strains (carrying alleles lr12, lr28, and lr81) were grown, harvested, and frozen at -20 C by the instructor. Students isolate genomic DNA from each strain using a genomic DNA isolation kit from Gentra Systems, Inc. (Minneapolis, MN). We have used this kit for several years and it seems to be almost foolproof in the hands of undergraduates. Following proteinase K and RNase A treatment, proteins in the worm extract are precipitated by high salt and the genomic DNA is recovered by isopropanol precipitation. PCR reactions using two sets of primers (A and B) from two different regions of the unc-93 gene are carried out on the genomic DNA from wildtype and mutant strains and the results analyzed by agarose gel electrophoresis. Primer pair A yields a 789 bp fragment, while primer pair B yields a 728 bp fragment. The A primers detect a 173 bp deletion in lr28 and a 78 bp deletion in lr81 while the B primers detect a 517 bp deletion in lr12. The use of wildtype DNA and primers for amplifying two different regions of the unc-93 gene provide internal controls. Five student groups carried out this exercise in a molecular biology lab course during the Spring 2003 semester and their results will be presented. We gratefully acknowledge the assistance of Dr. Beth DeStasio, Biology Dept., Lawrence University, Appleton, WI, who provided the unc-93 strains used in this exercise.

James L Lissemore et al. (2001) International C. elegans Meeting "Using PCR in an undergraduate lab course to detect deletions in the unc-93 gene"

James L Lissemore, George D Fedoriw, Laura L Lackner

[International C. elegans Meeting, 2001]

PCR is a powerful and common molecular biology technique with many applications. We have developed an exercise for an undergraduate molecular biology lab course using PCR to detect deletions in the unc-93 gene of C. elegans. unc-93 encodes a putative transmembrane protein muscle protein of unknown function. Wildtype and three different unc-93 deletion mutant strains (carrying alleles lr12, lr28, and lr81) were grown, harvested, and frozen at -20 C by the instructor. Students isolate genomic DNA from each strain using a genomic DNA isolation kit from Gentra Systems, Inc. (Minneapolis, MN). We have used this kit for several years and it seems to be almost foolproof in the hands of undergraduates. Following proteinase K and RNase A treatment, proteins in the worm extract are precipitated by high salt and the genomic DNA is recovered by isopropanol precipitation. PCR reactions using two sets of primers (A and B) from two different regions of the unc-93 gene are carried out on the genomic DNA from wildtype and mutant strains and the results analyzed by agarose gel electrophoresis. Primer pair A yields a 789 bp fragment, while primer pair B yields a 728 bp fragment. The A primers detect a 173 bp deletion in lr28 and a 78 bp deletion in lr81 while the B primers detect a 517 bp deletion in lr12. The use of wildtype DNA and primers for amplifying two different regions of the unc-93 gene provide internal controls. Five student groups carried out this exercise in a molecular biology lab course during the Spring 2001 semester and their results will be presented. We gratefully acknowledge the assistance of Dr. Beth DeStasio, Biology Dept., Lawrence University, Appleton, WI, who provided the unc-93 strains and primers used in this exercise.

Donavan TS Cheng et al. (2007) International Worm Meeting "A NON-ALIGNMENT GIBBS-SAMPLING BASED METHOD FOR SEQUENCE ELEMENT DETECTION USING PHYLOGENETIC CONSERVATION."

Donavan TS Cheng, Michael A Beer

[International Worm Meeting, 2007]

Current attempts to infer regulatory networks from genome-wide expression data require motif detection algorithms to identify transcription factor (TF) binding sites in the regulatory regions of co-expressed genes. These algorithms typically use either over-represention or phylogenetic conservation to predict TF binding sites. While these approaches have been successful in yeast [1,2,5], in larger genomes motif detection is significantly more challenging, and will likely require comparative genomics. Several recent algorithms have begun to combine these two sources of information to predict functional sites more accurately. [1,2] However, many rely on an initial global alignment of orthologous regulatory regions as a preprocessing step. Unfortunately, TFs generally recognize sites that are short and degenerate, and global alignment may be overly restrictive for the detection of such sites. We have developed a non-alignment based Gibbs sampling [3,4] algorithm that searches for over-represented sequence elements, conserved across orthologous regulatory regions in related species. Motif composition is represented probabilistically by a position weight matrix and a posterior probability score is used to assess the significance of the motif found. The algorithm employs a stochastic search to converge to optima and unlike other alignment-based techniques, does not require a prior estimate of the evolutionary relationship between species sampled. We compare the performance of our algorithm on synthetic data to that of existing algorithms that employ either over-representation, phylogenetic conservation, or both. We further validate our motif predictions on in vivo binding data in yeast. [5] We present novel motif predictions from sets of coexpressed genes C. elegans and the related sequenced nematode species C. briggsae, and C. remanei. 1. Siddharthan R, Siggia ED, van Nimwegen E. PLOS Comp. Biol. 1, e67 (2005). 2. Sinha S, Blanchette M, Tompa M. BMC Bioinf. 5, 170 (2004). 3. Roth FP, Hughes JD, Estep PW, Church, GM. Nat. Biotech. 16, 939-945 (1998). 4. Neuwald AF, Liu JS, Lawrence CE. Prot. Sci. 4, 1618-1632 (1995). 5. Harbison CT, Gordon DB, Lee TI, Rinaldi NJ, et al. Nature 431, 99-104 (2004).