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Dave Markwardt et al. (2001) International C. elegans Meeting "A screen for natural targets of NMD using C. elegans cDNA microarrays"

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International C. elegans Meeting,

2001]

mRNA transcripts that contain premature termination codons are degraded in a regulated manner by a specific decay pathway termed mRNA surveillance or nonsense mediated mRNA decay (NMD). mRNA surveillance has been described in all eukaryotes tested including plants, flies, yeast, nematodes, and mammals. Loss-of-function mutations affecting any of seven smg genes eliminates mRNA surveillance in C. elegans . While much progress has been made in defining the cis -acting elements and trans -acting factors necessary for mRNA surveillance, less is know about the mRNAs produced during the normal course of gene expression that serve as substrates of NMD. In order to systematically investigate these natural targets of NMD, I have probed high density microarrays of C. elegans cDNA clones. These arrays, made in Stuart Kim's lab at Stanford University, consist of PCR fragments from 17,871 genes currently annotated in the C. elegans genome (Jiang et al., 2001). We performed nine different hybridizations, each comparing expression profiles of poly(A) + mRNA from smg(-) and smg(+) samples. The microarray screen identified 402 mRNAs whose abundance is 2-fold or greater in smg(-) mutants. Several observations suggest that microarrays are a reliable method for identifying natural targets of mRNA surveillance: 1. Control mRNAs known previously to be natural targets of mRNA surveillance ( rpl-12 and srp-20 ) were consistently elevated on the microarray screen. 2. Both northern blots and quantitative PCR analyses confirm the microarray results. These results indicate that the microarray screen will be useful for the systematic investigation of the C. elegans natural targets of NMD. We are presently analyzing smg(-) affected mRNAs to establish whether they are direct or indirect targets of NMD, and understand why their abundance is increased in smg(-) mutants. We anticipate that analysis of the ~400 candidate genes will identify broad categories of mRNAs produced in wild-type animals yet targeted for degradation by NMD and, possibly, new principles of post-transcriptional regulation of gene expression. Jiang M, Ryu J, Kiraly M, Duke K, Reinke V, Kim SK. Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans . Proc. Nat. Acad. Sci. USA. (2001) 98 (1):218-23.

Marisa Foehr et al. (2007) International Worm Meeting "Dorsoventral patterning of the postembryonic mesoderm requires both LIN-12/Notch and TGF-beta signaling."

Marisa Foehr, Jun Liu

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International Worm Meeting,

2007]

The C. elegans post-embryonic mesodermal lineage arises from a single precursor cell, the M mesoblast, which will diversify to generate distinct dorsal and ventral cell types. The dorsal daughter of M gives rise to a subset of body wall muscles and two non-muscle coelomocytes, whereas the ventral daughter of M gives rise to two sex myoblasts in addition to a subset of body wall muscles. Mutations in the C. elegans Schnurri homolog sma-9 cause ventralization of the M lineage. We have previously shown that SMA-9 antagonizes the Sma/Mab TGF-beta signaling pathway to promote dorsal M lineage fates (Foehr et al., 2006). Interestingly, loss-of-function mutations in the Notch receptor homolog lin-12 cause dorsalization of the M lineage (Greenwald et al., 1983), an exact opposite phenotype of sma-9 mutants. We have found that while LIN-12 protein is present in both the dorsal and ventral M lineage cells, the ligands for LIN-12, LAG-2 and APX-1, are asymmetrically localized in cells adjacent to ventral M-derived cells, and they function redundantly in promoting ventral M lineage fates. To investigate how LIN-12/Notch signaling interacts with SMA-9 and the Sma/Mab TGF-beta pathway in regulating M lineage patterning, we generated double and triple mutant combinations among lin-12, sma-9 and dbl-1 (the ligand for the Sma/Mab TGF-beta pathway) and examined their M lineage phenotypes. Our results suggest that the LIN-12/Notch pathway and the Sma/Mab TGF-beta pathway function independently in regulating dorsoventral patterning of the M lineage, with LIN-12/Notch required for ventral M lineage fates, and SMA-9 antagonism of TGF-beta signaling required for dorsal M lineage fates. Our work provides a model for how combined Notch and TGF-beta signaling regulates the developmental potential of two equipotent cells along the dorsoventral axis.

Peng Wang et al. (2003) International Worm Meeting "Identification and characterization of the downstream target genes of CeTwist and its partner CeE/DA."

Peng Wang, Ann K Corsi

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International Worm Meeting,

2003]

CeTwist, the product of the hlh-8 gene, is a member of the basic helix-loop-helix (bHLH) family of transcription factors and functions as a heterodimer with CeE/DA, the product of the hlh-2 gene (1). In order to reveal a complete pathway of CeTwist function in C. elegans mesoderm development, it will be necessary to ultimately identify all downstream target genes of hlh-8. In addition, some mutations in human genes with homologs in the CeTwist pathway are thought to be the causes of several syndromes of human craniosynostotic disease. We predict the CeTwist target genes which are found in our work might identify genetic defects of other syndromes of human craniosynostotic disease. Using cDNA microarrays, it is possible to observe global changes in gene expression of nearly every mRNA during development (2). Since hlh-8 is expressed in at most 2% of cells at any given time during development, it may be difficult to obtain meaningful data using DNA microarrays by comparing mRNA from hlh-8 null animals to mRNA from wild-type animals. To circumvent this difficulty, we will overexpress both hlh-8 and hlh-2 from heat shock promoters and compare mRNA isolated from this strain to mRNA isolated from isogenic wild-type animals. We have already built the following strains: experimental group containing pRF4 pHS::hlh-8 pHS::hlh-2 and control group containing pRF4 only. All of the constructs are already integrated onto chromosomes through irradiation. By using RT-PCR to investigate the kinetics of the known target gene arg-1 under heat shock treatment, we have found that a 20 min heat shock treatment at 33oC followed by a 40 min room temperature recovery period is the optimal experimental condition for observing target gene expression. The expectation is that unknown targets will be expressed under these conditions as well. Now we are scaling up to collect enough mRNA from synchronized L2 larvae under our experimental conditions to perform cDNA microarrays. 1. Harfe, B. D., Gomes, A. V., Kenyon, C., Liu, J., Krause, M. and Fire, A. (1998). Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning. Genes Dev 12, 2623-2635; 2. Jiang, M., Ryu, J., Kiraly, M., Reinke, V. and Kim, S. K. (2001) Genome-wide analysis of developmental and sex-regulated gene expression profiles in Caenorhabditis elegans. Proc Natl Acad Sci USA 98, 218-223.

Nirav Amin et al. (2008) Development & Evolution Meeting "Systematic analysis of transcription factors important in C. elegans postembryonic mesodermal development"

Zach Via, Jun Liu, Nirav Amin

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Development & Evolution Meeting,

2008]

The C. elegans postembryonic mesodermal lineage, the M lineage, is a powerful model system to study mesodermal patterning and cell fate specification at single cell resolution. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell undergoes a series of postembryonic cell divisions to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). We and others have previously identified a handful of transcription factors important for the proper development of this lineage. In order to identify additional transcription factors that play a role in the M lineage, we have generated a feeding RNAi library that targets a majority of the predicted transcription factors encoded in the C. elegans genome and conducted an RNAi screen using cell type-specific GFP reporters in the M lineage. From this screen, we identified a novel set of 32 transcription factors that, upon RNAi knockdown, give reproducible phenotypes in the M lineage. Among these 32 transcription factors, four are important for patterning and fate specification of the early M lineage, while the rest appear to play a role in fate decisions in the SM lineage. We have primarily focused on let-381, which encodes a forkhead transcription factor that is essential for C. elegans development. let-381(RNAi) causes a dorsal to ventral fate transformation in the M lineage. We have found that a let-381::gfp translational fusion is expressed in the dorsal M lineage. Previous studies from our lab have shown that SMA-9, the Sma/Mab TGF-beta and LIN-12/Notch signaling pathways are involved in dorsal/ventral patterning of the M lineage. We are currently investigating the relationship between let-381 and these pathways.

Yuan Jiang et al. (2004) East Coast Worm Meeting "MLS-2, an HMX class homeodomain protein essential for mesodermal patterning and cell fate specification"

Jun Kelly Liu, Yuan Jiang

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East Coast Worm Meeting,

2004]

We are interested in understanding mesodermal patterning and fate specification by studying the C. elegans postembryonic mesodermal lineage, the M lineage. The M lineage is derived from a single precursor cell, the M mesoblast, and gives rise to six cell types: striated bodywall muscles (BWMs), nonmuscle coelomocytes (CCs), and four classes of non-striated sex muscles which are descendants of the sex myoblasts (SMs). We are studying the function of the mls-2 (mesodermal lineage specification) gene in M lineage patterning and fate specification. The mls-2(cc615) mutation causes randomization of division planes in the M lineage, and subsequent fate transformation of CCs and BWMs to SMs. In addition, cc615mutants have defects in SM migration and show some larval and adult lethality. We have cloned the wild type mls-2 gene (C39E6.4). mls-2 encodes a homeodomain protein that belongs to the HMX family of homeodomain proteins that are also present in sea urchin, Drosophila and vertebrates., We examined the expression pattern of mls-2 using both functional mls-2::gfp fusion construct and affinity purified anti-MLS-2 antibodies. We found that the MLS-2 protein is localized in nuclei of early M lineage cells and a subset of head neurons. Furthermore, mls-2 expression in the M lineage and the head neurons appears to require distinct cis-acting elements. Overexpression of mls-2 in the early M lineage where mls-2 is normally expressed caused a variety of defects in the M lineage. Forced expression of mls-2 in the later M lineage such as in SMs where mls-2 is not normally expressed resulted in extra rounds of divisions of SMs. This suggests that mls-2 may have multiple roles in the M lineage and that MLS-2 protein level is critical for the correct patterning of the M lineage. The M lineage defects of mls-2(cc615) are very similar to those of mab-5, hlh-1, egl-27 and egl-20 mutants. We are currently carrying out molecular and genetic epistasis experiments to investigate the relationship between mls-2 and those factors.

Nirav Amin et al. (2008) Development & Evolution Meeting "The Six2 homeodomain protein CEH-34 functions downstream of Wnt signaling for proper fate specification in the postembryonic mesoderm"

Jun Liu, Sung-Eun Lim, Tania Jaber, Nirav Amin

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Development & Evolution Meeting,

2008]

We are using the C. elegans postembryonic mesodermal lineage, the M lineage, as a model to study mesodermal patterning and cell fate specification. The M lineage arises from a single pluripotent cell, the M mesoblast, during embryogenesis. In hermaphrodites, the M cell first undergoes a dorsal-ventral division and then a left-right division to produce four cells located in four quadrants of the L1 larva. These four cells subsequently undergo two to three more rounds of cell divisions along the anterior-posterior axis to produce 18 cells: 14 body wall muscles (BWMs), 2 coelomocytes (CCs), and 2 sex myoblasts (SMs). In particular, M.dlp and M.drp each give rise to an anterior CC and a posterior BWM. In an RNAi screen for transcription factors important for proper M lineage development, we identified the Six2 homeodomain factor ceh-34 for its requirement in the proper specification of M-derived CC fates. ceh-34(RNAi) results in a fate transformation of the anterior CC to its posterior sister cell, the BWM. We have generated translational ceh-34::gfp fusions and found that in the M lineage, ceh-34 is specifically expressed in the CC precursor cells. To understand the mechanisms underlying the asymmetric expression pattern of ceh-34, we determined the roles of the Wnt/beta-catenin asymmetry pathway in the M lineage because this pathway has been shown to regulate other asymmetric cell fates along the anterior-posterior axis. We have found that during both of the anterior-posterior cell divisions in the early M lineage, the TCF homolog POP-1 is enriched in the anterior daughters while the beta-catenin homolog SYS-1 is enriched in the posterior daughters. Furthermore, sys-1(q544) loss-of-function mutants have extra M lineage-derived CCs, while pop-1(RNAi) leads to a loss of M-derived CCs. We showed that ceh-34(RNAi) is epistatic to sys-1(q544) mutations and that ceh-34::gfp is ectopically expressed in the M lineage in sys-1(RNAi) animals but reduced in pop-1(RNAi) animals. These observations suggest that ceh-34 functions downstream of pop-1 and sys-1 in the M lineage. We are currently testing if POP-1 and SYS-1 directly regulate the asymmetric expression of ceh-34 in M lineage development.

Anita U Rao et al. (2007) International Worm Meeting "Genetic Dissection of Heme Pathways in C. elegans."

Brian Le, Iqbal Hamza, Anita U Rao

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International Worm Meeting,

2007]

Heme serves as a cofactor for a number of proteins involved in key metabolic processes. In eukaryotes, heme synthesis occurs in the mitochondria by an evolutionarily conserved multi-step pathway. Hemes are hydrophobic and thus insoluble in the aqueous environment of the cell. Moreover, free heme is cytotoxic because of peroxidase activity. We therefore hypothesize that intracellular pathways exist for trafficking of heme from the site of synthesis in the mitochondria to various cellular destinations. However, identification of these heme transport pathways has been difficult because heme synthesis is regulated by multiple effectors and is tightly coordinated with apo-protein synthesis. We have previously shown that C. elegans and related helminths do not make heme albeit requiring exogenous heme for normal metabolic processes. Importantly, C. elegans show a biphasic response for heme; worms are growth-arrested at 1.5 mM and at 800 mM heme. These results suggest that although worms are obligate heme auxotrophs they are likely to have all the pathways for heme utilization beyond the point of heme synthesis. To identify pathways for heme transport in C. elegans, we exploited their biphasic response to heme by screening for mutants that could survive heme toxicity. We screened 300,000 haploid genomes and isolated 13 mutants at 800 mM heme in liquid axenic medium. Based on the mutants growth profile in medium containing low and high heme, we categorized the mutants into three broad phenoclusters: class A, class B and class C. Class A mutants grew robustly under low and high (800 and 1000mM) heme, Class B mutants grew exceptionally well under low heme, moderately well at 800 mM heme, and not at all at 1000 mM heme, and Class C mutants grow moderately well under high heme (800mM), but exhibit normal growth under low heme. The mutants were further sub-clustered by using gallium protoporphyrin (GaPP), a toxic heme analog. Complementation analyses revealed that these 13 mutants fall into five complementation groups. Genetic mapping by recombination localized the mutants from each complementation group to chromosome III. We are now producing a high resolution map to define a genetic interval and pinpoint the exact nature of the molecular lesion in these mutants.

J Liu et al. (1999) International C. elegans Meeting "Hox factors and other components in patterning of embryonic and postembryonic myogenic lineages"

J Liu, AZ Fire

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International C. elegans Meeting,

1999]

Bodywall muscles are derived from four of the embryonic founder cells as well as the postembryonic M blast cell. We are studying patterning of both the embryonic and postembryonic muscles in order to understand how myogenic cell fates are specified during development. As an entry point to study muscle patterning during embryonic development, we characterized enhancer elements from the hlh-1 regulatory region that are able to drive reporter expression in muscle precursors of the MS, D and C lineages. Analyses of these elements suggested regulatory roles of the Hox genes and the hlh-1 gene itself. These analyses also suggested regulatory mechanisms involving unknown bZIP, bHLH and novel transcription factors. We are currently in the process of genetically identifying these factors. The postembryonic M lineage provides an alternative model to study myogenic fate specification. The M lineage gives rise to 14 bodywall muscles, 2 coelomocytes and 16 sex muscles. A number of genes had previously been identified to function in patterning the M lineage, including the Hox gene mab-5 and the bHLH transcription factor twist (1, 2, 3). The role of the Hox factors in patterning the M lineage has been something of a mystery: mab-5 is expressed during the entire M lineage, but mab-5 mutants cause only limited lineage transformation. We will describe a series of experiments indicating a more central role for the Hox genes in activating twist and specifying the M lineage. We found that lin-39 mab-5 double mutants fail to activate twist and completely lack products of the M lineage. Expression (either ectopic or in a normal pattern) of either Hox gene is sufficient to activate twist expression. However, twist activation is not sufficient for specification of the M lineage. Current efforts are directed towards identification of other factors involved in specifying the M lineage. 1. Kenyon, C. (1986), Cell 46: 477-487 2. Harfe B. D., Vaz Gomes A., Kenyon C., Liu J., Krause M., Fire A. (1998) Genes & Dev. 12: 2623-2635 3. A. Corsi, S. Kostas, E. Jorgensen, A. Fire, and M. Krause (poster)

Yuan Jiang et al. (2006) Development & Evolution Meeting "CEH-20 regulates the expression of mls-2 in the postembryonic mesoderm"

Yuan Jiang, Jun Liu

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Development & Evolution Meeting,

2006]

mls-2 encodes a HMX homeodomain protein that plays critical roles in the C. elegans postembryonic mesodermal lineage, the M lineage. mls-2 is expressed in the M lineage as well as a few other cell types, such as several pairs of head neurons including ASK and AIM. To uncover mechanisms involved in regulating the expression of mls-2, we generated a series of promoter deletions and identified an M lineage enhancer (position -2221 to -2076) that is required for mls-2 expression in the M lineage. We further identified several essential elements within this M enhancer. Among these elements are two putative Exd/Abd-B binding sites that are conserved in C. briggsae. We have found that mls-2 expression in the M lineage is dependent on the C.elegans Exd/Pbx homolog CEH-20. We are currently testing whether CEH-20 directly binds to the mls-2 promoter and whether CEH-20 regulates mls-2 expression through acting together with the Abd-B class Hox proteins, EGL-5/PHP-3/NOB-1, and the Hth/Meis homolog, UNC-62.

Spica, Patrick et al. (2015) International Worm Meeting "The Effects of Wrapping NGM Culture Plates with Parafilm M® on the Growth and Development of Caenorhabditis elegans."

Scanga, Sara, Spica, Patrick, Shinn-Thomas, Jessica, Klempic, Emra

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International Worm Meeting,

2015]

Parafilm M® is a thin thermoplastic used to seal a variety of containers in scientific laboratories. It is commonly used to seal Nematode Growth Media (NGM) culture plates to prevent microbial contamination and media dehydration. However, the effects on C. elegans of wrapping culture plates with Parafilm M® during experiments are unknown. Parafilm M® may limit gas exchange between the external and culture environment, potentially affecting the biology and life history of C. elegans, including its larval growth rate, viability, fecundity, lifespan, and behavior. In particular, wrapping culture plates with Parafilm M® may produce a hypoxic (low oxygen) environment compared to plates with no Parafilm M® (normoxic). Anoxic (no oxygen) and hypoxic conditions have been shown to change the metabolism, development, and longevity of C. elegans.Our research aims to determine the effects of wrapping NGM culture plates with Parafilm M® on C. elegans. We hypothesized that worms cultured on plates wrapped in Parafilm M® would exhibit a slower rate of larval growth and increased mortality compared to worms grown in normoxic conditions. Synchronized L1 worms were individually transferred to culture plates and incubated within an anoxic environment, hypoxic environment, normoxic environment, or wrapped one time with Parafilm M®. BD GasPaks™ were used to create anoxic and hypoxic environments, and normoxic culture conditions consisted of unsealed plates. Larval growth rate and mortality were measured 5 times over 48 hours. We found no significant difference in the growth rate between worms cultured in normoxic conditions and on plates wrapped with Parafilm M®. However, the growth rate between worms cultured on plates wrapped with Parafilm M® and worms in anoxic and hypoxic conditions was significantly higher. Mortality was significantly higher in worms cultured in anoxic conditions, but was not significantly different among the other three environmental conditions. Our data suggest that wrapping C. elegans culture plates one time with Parafilm M® does not affect the larval growth rate or viability. Future studies will focus on additional biological and life history metrics, such as fecundity and lifespan, to verify that wrapping with Parafilm M® has no unexpected effects on the outcomes of C. elegans studies.

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