L R Baugh et al. (2001) International C. elegans Meeting "7,500 Genes and the Transition from Maternal to Zygotic Control of Development"

Craig P Hunter, L R Baugh, A A Hill, E L Brown

[International C. elegans Meeting, 2001]

An immediate challenge of the post-genomic era is to quantitatively determine the precise temporal and spatial expression patterns of every gene in a model organism during its embryonic development. The relationship between genotype and phenotype can then be predictively modeled in terms of the regulation and consequences of these precise expression patterns. In addition to being quantitative, microarrays have the benefit of making measurements in parallel, thus enabling global observations of the transcriptome during development. The C. elegans embryo is ideally suited for genomic analysis: it is experimentally accessible, well characterized genetically, has a compact genome, and the requirement for specific genes can be directly assessed using RNAi. To measure absolute abundance of transcripts during early development, we developed a sensitive and representative RNA amplification technique that enables quantitative analysis of mRNA levels from as few as 10 embryos. We then collected (in triplicate) pools of 10-15 embryos precisely staged (+/- ~3 min)by morphology at the 4-cell stage and allowed them to develop for proscribed amounts of time. mRNA from each of five time points spanning the 4 to ~102 cell stage was amplified and hybridized to oligonucleotide arrrays generating absolute measurements of transcript abundance for the entire genome. Approximately 7,500 genes are reproducibly detected in the time course, many of which are not represented in EST collections, and about 2,600 of them are significantly modulated over time (ANOVA p<10 -2 ). Transition from maternal to zygotic control of development is evident in the observed degradation of over 1,100 maternal transcripts along with the induction of approximately 1,500 zygotic transcripts. Among these, both simple and complex temporal expression patterns are detected. Intriguingly, reproducibility among replicates and similarity between adjacent timepoints increases towards the end of the time course. This result is consistent with the rate of molecular development decreasing and/or the stability of regulatory networks increasing as embryos approach the 100 cell stage. In either case, the observation suggests convergence onto a regulatory steady state. Statistical, computational and bioinformatic analysis of the data will be presented.

Chen Y et al. (2010) Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI "Insulin Peptides in Nutritional Control of Development in C. elegans"

Chen Y, Kurhanewicz N, Baugh R

[Aging, Metabolism, Stress, Pathogenesis, and Small RNAs, Madison, WI, 2010]

Animals have evolved great ability to adapt to fluctuating nutrient availability by balancing growth and survival. In C. elegans, newly hatched L1 stage larvae remain developmentally arrested until feeding. This phenomenon, called L1 arrest, provides an opportunity to study regulatory mechanisms mediating nutritional control of growth and development. Insulin-like signaling is a key regulator of L1 arrest (Baugh and Sternberg, 2006; Kao et al, 2007). In spite of great interest in the insulin-like pathway given its function in dauer formation, aging and L1 arrest, the function of specific insulin-like peptide ligands is generally not understood. The C. elegans genome encodes 40 insulin-like peptides, and they are thought to function as either agonists or antagonists of the insulin-like receptor DAF-2 and growth (Pierce et al., 2001). We are using a combination of expression and phenotypic analysis to characterize their regulation and function during L1 arrest. Although several insulin-like genes may be broadly redundant, we propose that there is specificity to their function in timing and site of action, and that they comprise regulatory network that responds to nutrient availability and controls growth and development. From our expression data we identify several putative agonists, including ins-4, ins-5, ins-6, and ins-7, which are up-regulated by feeding, and several putative antagonists, including ins-17, ins-18 and ins-24, that are up-regulated by starvation. Transcriptional YFP reporters of them show that the agonists and antagonists are expressed in different subsets of amphid sensory neurons in L1 worms, and emphasize the intestine as a site of nutrient-dependent transcriptional regulation. Moreover, phenotypic analysis of growth rate, starvation survival and early postembryonic cell division on single and multiple deletion mutants will help determine their role in promoting growth and development.

Bergemann, C. et al. (2019) International Worm Meeting "Does mitochondrial toxicant exposure during development cause later life consequences that are inherited by the next generation?"

Baugh, R., Meyer, J., Bergemann, C., Mello, D.

[International Worm Meeting, 2019]

Chemical exposure during development can have dramatic and sustained impacts, especially if the chemicals target the mitochondrial electron transport chain. Altered mitochondria function can contribute to the initiation of metabolic and degenerative diseases. We hypothesized that developmental (pre-conception) exposure to rotenone, a widely-used pesticide and piscicide that inhibits mitochondrial complex I, would result in lifelong and intergenerational effects. For experiments, Caenorhabditis elegans N2 strain (parental generation, P0) were exposed to rotenone (0.03 and 0.5 M) or vehicle (0.25% DMSO; control) in liquid with HB101 bacteria for 52 hours or until they reached the L4 larval stage. Animals were then transferred to OP50-agar plates for 48 hours for chemical depuration, and gravid adults were bleached to obtain synchronized eggs (F1 generation). F1 eggs were grown in liquid for 52 hours in the same conditions as P0 but with no chemical exposure, and then transferred to OP50-agar plates. The P0 generation had a dose-dependent decrease in growth, which was associated to a developmental delay. Since mitochondria metabolism is intrinsically different throughout worm development, we stage matched all treatments so that all assays were performed on mid-L4 worms. Stage-matched P0 animals still had a significant decrease in size; however, rotenone exposures did not significantly affect basal, ATP-linked, or maximal oxygen consumption. Furthermore, we did not observe a difference in mitochondrial copy number in the P0 generation post exposure or in later life. In the F1 generation, growth, respiration, and mitochondrial copy number at the L4 stage showed no difference between offspring from exposed and control P0 animals. Since development is known to be affected by mitochondrial dysfunction, these preliminary data suggest that mitochondria may be impaired in the P0 (exposed) animals but recovers in the next generation based on the parameters analyzed under ideal conditions. However, future experiments will include challenging the F1 generation with rotenone exposure, looking at other phenotypes later in life, and transgenerational effects.

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.

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.

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).

Rand JB et al. (2000) East Coast Worm Meeting "UNC-13 in the C. elegans Nervous System"

Kohn RE, Rand JB, McManus JR, Moulder GL, Duke A, Barstead RJ, Duerr JS

[East Coast Worm Meeting, 2000]

C. elegans unc-13 and its homologues in vertebrates and Drosophila are involved in neurotransmitter release. UNC-13 has several regions homologous to PKC regulatory domains; these domains confer it with calcium, phorbol ester and phospholipid binding properties (Maruyama and Brenner, PNAS 88, 1991). A 5.9kb transcript coding for a 200kDa protein was initially identified (now designated L-R for left and right regions). We have identified two additional types of transcripts. One transcript includes a 1kb novel exon (L-M-R, M for middle region) and another transcript lacks the 5' region included in the other two transcripts (M-R). All three transcripts are identical at the 3' end (R). C. elegans with mutations in the 5' end of the gene (L) alter two types of transcripts (L-R and L-M-R) resulting in an uncoordinated coily phenotype and resistance to the anti-cholinesterease, aldicarb. A 2.7kb deletion near the 3' end (R) (identified by Bob Barstead using PCR analysis) affects all unc-13 transcripts and results in a lethal phenotype. Antibodies recognizing the N-terminal region of UNC-13 (L) label synapses, but not synaptic vesicles, of most or all neurons; many mutations in L and R remove staining with this antibody. Supported by grants from the NIH and OCAST.

Abergel, Z. et al. (2017) International Worm Meeting "Adaptation of the nematode C. elegans to hypoxia and reoxygenation stress reveals an unexpected function of the neuroglobin GLB-5 in innate immunity."

Zuckerman, B., Zelmanovich, V., Abergel, Z., Abergel, R., Gross, E., Smith, Y., Romero, L., Livshits, L.

[International Worm Meeting, 2017]

Deprivation of oxygen (hypoxia) followed by reoxygenation (H/R stress) is a major component in several pathological conditions such as vascular inflammation, myocardial ischemia, and stroke. However how animals adapt and recover from H/R stress remains an open question. Previous studies showed that the neuroglobin GLB-5(Haw) is essential for the fast recovery of the nematode Caenorhabditis elegans (C. elegans) from H/R stress. Here, we characterize the changes in neuronal gene expression during the adaptation of worms to hypoxia and recovery from H/R stress. Our analysis shows that innate immunity genes are differentially expressed during both adaptation to hypoxia and recovery from reoxygenation stress. Moreover, we reveal that the prolyl hydroxylase EGL-9, a known regulator of both adaptation to hypoxia and the innate immune response, inhibits the fast recovery from H/R stress through its activity in the O2-sensing neurons AQR, PQR, and URX. Finally, we show that GLB-5(Haw) acts in AQR, PQR, and URX to increase the tolerance of worms to bacterial pathogenesis. Together, our studies suggest that innate immunity and recovery from H/R stress are regulated by overlapping signaling pathways.

A V Bicknell et al. (2002) European Worm Meeting "The C. elegans F47F2.1 gene encodes a cyclic AMP-dependent protein kinase (PK-A) catalytic subunit"

A V Bicknell, M J Fisher, M Tabish, R A Clegg, H H Rees

[European Worm Meeting, 2002]

PK-A mediates all known effects of cyclic AMP on cellular activity in eukaryotes. The holoenzyme is an inactive tetramer of two regulatory (R) subunits and two catalytic (C) subunits. Following binding of cyclic AMP to the R subunits, dissociation of active C-subunits occurs. In mammals, , and isoforms of C-subunit, encoded by different genes, have been identified.

Angelo RG et al. (1997) International C. elegans Meeting "DISCOVERY OF A POTENTIAL ANCHOR/SCAFFOLD PROTEIN FOR C. ELEGANS PROTEIN KINASE A (PKA)"

Angelo RG, Rubin CS

[International C. elegans Meeting, 1997]

C. elegans PKA is composed exclusively of catalytic and regulatory (R) subunits encoded by the kin-1 and kin-2 genes. Since C. elegans lacks other PKA isoforms, this enzyme must disseminate signals carried by cAMP to all cell compartments. Approximately 60% of total R (PKA) is in the particulate fraction of disrupted C. elegans, indicating that protein/protein interactions may diversify PKA signaling by anchoring the kinase at specific intracellular locations. Co-localization of PKA with upstream activators and/or downstream effectors can create target sites for cAMP action. To understand the mechanism of PKA localization in C. elegans, a cDNA expression library was screened with recombinant radiolabeled-R (0.5 nM) to obtain high-affinity binding proteins. A cDNA encoding a novel, 143 kDa A kinase anchor protein (AKAP1) was retrieved and sequenced. The corresponding gene (rap-1) is located in LGII and contains 17 exons. AKAP1 is an acidic protein (pI=4.4) that is unrelated to previously characterized proteins. C. elegans R is avidly bound by both soluble and immobilized fragments of AKAP1. Competition binding studies indicate that C. elegans R is a preferred ligand, whereas mammalian RII and RI isoforms are only weakly sequestered. Scatchard analysis yielded a Kd value of ~10 nM for the R/AKAP1 complex. Deletion mutagenesis, coupled with protein expression in E. coli and in vitro assays, demonstrated that residues 235 to 255 govern high-affinity binding of R by AKAP1. A hydrophobic surface generated by amino acids with branched aliphatic side chains may be a key determinant of R binding activity. Site directed mutagenesis will pinpoint essential residues involved in PKA binding. Studies aimed at identifying the AKAP1 binding site on R are also in progress. High-affinity anti-AKAP1 IgGs are being used to determine (a) whether AKAP1 expression is developmentally-regulated and cell- specific and (b) the relationship between R (PKA) and AKAP1 in vivo.