Baugh, L Ryan et al. (2009) International Worm Meeting "RNA Polymerase II Accumulates on Promoters of Growth and Development Genes During L1 Arrest."

DeModena, John, Baugh, L Ryan, Sternberg, Paul

[International Worm Meeting, 2009]

Animals cope with fluctuating nutrient availability by altering resource allocation in order to maintain energy homeostasis. For C. elegans life in the wild is characterized by feast or famine, so that organismal fitness presumably depends on developmental responses to nutrient availability. When C. elegans larvae hatch in the absence of food they reversibly arrest development and increase resistance to environmental stress (L1 arrest). Using L1 arrest and recovery as a model, we aim to characterize signaling pathways and gene regulatory mechanisms that maintain energy homeostasis during development. To study nutritional control of larval development, we characterized growth, mRNA expression, and RNA Polymerase II (Pol II) binding genome-wide during L1 arrest and recovery. Growth and gene expression analysis reveal that fed larvae are relatively slow to respond to starvation, while arrested larvae respond rapidly to feeding as if poised for recovery. Chromatin immunoprecipitation of RNA Pol II followed by deep sequencing (ChIP-Seq) shows that while Pol II continues transcribing starvation genes, it accumulates on the promoters of growth and development genes during L1 arrest, as if ''paused'' or otherwise inhibited post-recruitment. Consistent with Pol II promoter accumulation poising arrested larvae for recovery, accumulation decreases in response to feeding, while elongation and mRNA levels increase. Therefore, accumulation of Pol II at promoters can anticipate nutrient-dependent gene expression during development. This work demonstrates the profound influence of energy homeostasis on gene regulation during development, and it suggests that nutritional control of Pol II promoter accumulation may be a general feature of gene regulation involving energy homeostasis.

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

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

[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.

Ryan Baugh et al. (2002) East Coast Worm Meeting "Composition and Dynamics of the C. elegans Early Embryonic Transcriptome"

Gene Brown, Craig P Hunter, Ryan Baugh, Donna Slonim, Andrew Hill

[East Coast Worm Meeting, 2002]

Understanding the system-wide relationship of genotype and phenotype during animal development requires global identification and characterization of embryonic regulatory networks. To obtain temporal profiles of transcript abundance during embryonic development we have isolated RNA from precisely staged C. elegans embryos and then amplified this RNA in order to perform whole-genome microarray analysis. The result is a highly resolved time course that commences with the zygote and extends into mid-gastrulation, spanning the transition from maternal to embryonic control of development and including the presumptive specification of most major cell fates. Transcripts for approximately 6,000 genes are detected at any given time point and nearly half (8,890) of the predicted open reading frames are ever detected. The expression level for the majority of expressed genes (~70%) is temporally modulated. However, rates of synthesis and degradation are matched such that the transcriptome maintains a steady-state frequency distribution. Two complementary approaches are used to group genes by expression pattern: cluster analysis of similarly modulated genes and a classification scheme based on developmental genetic concepts. The resulting gene sets provide a versatile platform for bioinformatic analysis. In summary, these data comprise a comprehensive, quantitative description of mRNA levels during C. elegans early embryogenesis and a baseline for future perturbation experiments intended to delineate specific regulatory network architectures.

Ryan Baugh et al. (2008) C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting "Nutritional Control of Gene Expression During L1 Growth and Arrest"

Paul Sternberg, Ryan Baugh

[C.elegans Aging, Stress, Pathogenesis, and Heterochrony Meeting, 2008]

Upon hatching, L1 larvae must initiate feeding and growth in order to execute post-embryonic development. When larvae hatch in the absence of food they persist in a stress resistant, developmentally arrested state (L1 arrest). We seek to identify the pathways that regulate L1 arrest and to determine how they accomplish developmental arrest and stress resistance by characterizing their effects on gene expression. We characterized mRNA expression genome-wide in a pair of bifurcating time series starting in the late embryo and proceeding through the hatch in the presence and absence of food (E. coli). Known regulators of energy homeostasis are up-regulated during L1 arrest (PI3K, TOR, AMPK, and SIR-2), and expression dynamics suggest that L1 arrest serves as a developmental checkpoint and that arrested L1s are poised for rapid recovery. Steroid hormone metabolism genes and numerous nuclear hormone receptors are up-regulated early in L1 arrest and lipid and fatty acid metabolism genes are up-regulated late. Reporter gene analysis demonstrates nutritional control of transcription and indicates that the response to starvation is anatomically complex while suggesting that the sensory neurons and gut are most affected. This analysis shows that in maintaining homeostasis physiological mechanisms contribute as much to gene regulation as development.

L Ryan Baugh et al. (2005) International Worm Meeting "Synthetic lethal analysis of posterior embryonic patterning genes identifies conserved genetic interactions"

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

[International Worm Meeting, 2005]

Phenotypic robustness is evidenced when single-gene mutations do not result in an obvious phenotype. One approach to characterizing mechanisms of phenotypic robustness is to identify double mutants where genetic buffering is compromised.Maternal and zygotic activities of the homeodomain protein PAL-1 specify the identity and maintain the development of the multi-potent C-blastomere lineage. We have identified genes whose expression depends either directly or indirectly on PAL-1 function (PAL-1 targets) and shown that they affect specification, differentiation, and morphogenesis of C lineage cells. However, RNAi of most PAL-1 targets does not result in a detectable phenotype. The regulatory network specified by PAL-1 functions autonomously while controlling multiple, discrete developmental processes (i.e., specification and morphogenesis of muscle and epidermal cells), and so we reasoned that the network is organized in a modular fashion and that PAL-1 targets may have overlapping or compensatory functions. To test this hypothesis, we measured synthetic embryonic lethality following RNAi of 22 genes in 15 mutant strains and wild-type. A pair of homologous T-Box transcription factors (tbx-8 and tbx-9) is found to interact in both C. elegans and C. briggsae indicating that their compensatory function is conserved. Furthermore, a muscle module is defined by transitive interactions between the MyoD homolog hlh-1, another basic helix-loop-helix transcription factor, hnd-1, and the MADS-box transcription factor unc-120. Genetic interactions within a homologous set of genes involved in vertebrate myogenesis indicate broad conservation of the muscle module and suggest that other genetic modules identified in C. elegans will be conserved.Technical notes: we determined that soaking worms in two different dsRNA molecules targeting two different genes at once is ineffective and that reducing the concentration of dsRNA for essential genes results in variable RNAi efficiency exacerbated by different genetic backgrounds. We established a statistical model to distinguish specific genetic interactions from independent phenotypic effects, and this model is applicable to other synthetic genetic analyses where phenotype is scored quantitatively and in replicate.

Ryan Baugh et al. (2007) International Worm Meeting "Nutritional control of larval development: DAF-16 is required for transcription of cki-1 and repression of lin-4 during L1 arrest."

Paul Sternberg, Ryan Baugh

[International Worm Meeting, 2007]

C. elegans larvae initiate post-embryonic development only in the presence of food, although they can survive starvation for weeks in a developmentally arrested state (L1 arrest). In contrast to the well-studied dauer arrest, L1 arrest occurs without morphological modification, although larvae in L1 arrest are more resistant to environmental stress than developing larvae. Consistent with its role in dauer formation and aging, we and others have shown that insulin/insulin-like growth factor (IGF) signaling regulates L1 arrest (Baugh and Sternberg, Cur. Biol. 2006; Fukuyama et al, Cur. Biol. 2006; Kao et al, Cell 2007). Our results indicate that daf-2 insulin/IGF receptor mutants have a constitutive L1 arrest phenotype when fed and extended survival of L1 arrest when starved. Conversely, daf-16/FOXO mutants have a defective arrest phenotype, failing to arrest development and dying rapidly when starved. We have shown that DAF-16 is required for transcription of the cyclin-dependent kinase inhibitor cki-1 in lateral epidermal seam cells in response to starvation, accounting for the failure of daf-16 mutants to arrest somatic cell division during L1 arrest. Other developmental events such as cell migration, cell fusion, and expression of the microRNA lin-4, a temporal regulator of post-embryonic development, are also observed in starved daf-16 mutants. These and other results implicate insulin/IGF signaling in the nutritional control of development during the first larval stage in C. elegans, although additional DAF-16-independent pathways must also participate. In addition to performing a genome-wide characterization of mRNA expression during L1 arrest, we are currently investigating the insulin ligands and their sites of action as well as additional candidate signaling pathways that regulate L1 growth and arrest in response to changing environmental conditions.

Musset-Bilal F et al. (1994) Worm Breeder's Gazette "Characterization of the C. elegans Basement Membrane-Associated Protein SPARC"

Schwarzbauer JE, Musset-Bilal F, Ryan CS

[Worm Breeder's Gazette, 1994]

Characterization of the C. elegans Basement Membrane- Associated Frederique Musset-Bilal, Carol S. Ryan, and Jean E. Schwarzbauer Department of Molecular Biology, Princeton University, Princeton NJ 08544

Chen, Yutao et al. (2011) International Worm Meeting "Insulin-like Signaling in Nutritional Control of L1 Development in C. elegans ."

Kurhanewicz, Nicole, Baugh, Ryan, Chen, Yutao

[International Worm Meeting, 2011]

In the wild animals must coordinate development with fluctuating nutrient availability. Insulin-like signaling is conserved among metazoan, and it functions to buffer the physiological effects of nutrient fluctuation. In C. elegans, newly hatched L1 larvae remain developmentally arrested until feeding. This phenomenon, called L1 arrest or L1 diapause, 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 characterized. The C. elegans genome encodes 40 insulin-like peptides. Although redundancy of these genes is likely, some of them have been shown to have specific phenotypes (Lin et al. 2001, Li et al., 2003, Murphy et al., 2007, Patel et al. 2008, Michaelson et al. 2010,). In addition, over-expression analysis suggests that insulin-like peptides function as either agonists or antagonists of the insulin-like receptor DAF-2, promoting development or arrest, respectively (Pierce et al., 2001). With an mRNA expression analysis platform called nCounter by Nanostring, we comprehensively characterized expression dynamics for all 40 insulin-like genes in response to feeding and starvation. From these expression data we identified several insulin-like genes up-regulated by feeding and several up-regulated by starvation, and we hypothesize that they function as agonists and antagonists, respectively. Destabilized YFP reporter gene analysis confirms nutritional control of transcription, reveals the intestine as the primary site of regulation, and suggests that the putative agonists and antagonists are expressed in different subsets of neurons in L1 worms. We are currently using genetic analysis to study the function of putative agonists during recovery from L1 arrest. By combining expression and phenotypic analysis, we aim to determine the timing and site of action for the insulin-like peptides governing L1 development, dissecting the organismal regulatory network they comprise.

Chen, Yutao et al. (2013) International Worm Meeting "Nutritional Control of Insulin-Like Peptide Expression during L1 Arrest and Recovery."

Baugh, Ryan, Chen, Yutao

[International Worm Meeting, 2013]

Animals must coordinate development with fluctuating nutrient availability. Nutrient availability governs post-embryonic development in C. elegans: larvae that hatch in the absence of food do not initiate post-embryonic development but enter "L1 arrest" (or "L1 diapause") and can survive starvation for weeks. Insulin-like signaling is a key regulator of L1 arrest and recovery. However, the C.elegans genome encodes 40 putative insulin-like peptides (ILPs), and there is evidence that they can function to promote development ("agonists") or arrest ("antagonists"). We used the nCounter platform to measure high-resolution mRNA expression dynamics for all 40 ILPs in response to feeding and fasting in recently hatched L1 larvae. Expression of 23 of the ILPs is significantly affected by nutrient availability. We classified several ILPs as candidate agonists or antagonists based on up-regulation in response to feeding or fasting, respectively. 10 candidate agonists (daf-28, ins-3, ins-4, ins-5, ins-6, ins-7, ins-9, ins-26, ins-33 and ins-35) were selected for further analysis. Phenotypic analysis of single or several multiple deletion mutants has not revealed detectable effects on L1 growth or development. Destabilized YFP reporter gene analysis shows that ten putative agonists are expressed in the intestine and various neurons. 6 out of 10 are expressed in the chemosensory neuron ASI and interneuron PVT. ASI is known to regulate dauer formation and lifespan but PVT has not been shown to be directly involved in feeding or stress response. Quantitative image analysis confirms nutritional control of transcription and reveals the intestine as the primary site of transcriptional regulation. Up-regulation in response to feeding in ASI is also evident in 3 candidates. We are currently testing if intestinal expression is regulated autonomously by ingestion of food or non-autonomously by neuronal signals. In summary, our expression analysis reveals the dynamics and sites of ILP expression in response to nutrient availability, providing insight into how post-embryonic development is governed by insulin-like signaling.

Zaslaver, Alon et al. (2009) International Worm Meeting "Metazoan operons accelerate transcription and recovery rates."

Zaslaver, Alon, Baugh, Ryan, Sternberg, Paul

[International Worm Meeting, 2009]

Existing theories efficiently explain why operons are advantageous in prokaryotes, but their emergence in metazoans is still an enigma. We present a combination of genomic meta-analysis, experiment and theory to explain how operons could be adaptive during metazoan evolution. Focusing first on C. elegans, we show that operon genes, typically consisted of growth genes, are significantly up-regulated during recovery from multiple growth arrested states, and that this expression pattern is anti-correlated to the expression pattern of non-operon genes. In addition, we find that transcriptional resources are initially limited during arrest recovery, and that recovering worms are extremely sensitive to any additional limitation in transcriptional resources. By clustering growth genes into operons, fewer promoters compete for limited transcriptional machinery, effectively increasing the concentration of transcriptional resources and accelerating growth during recovery. A simple mathematical model of transcription dynamics reveals how a moderate increase in transcriptional resources can lead to a substantial enhancement in transcription rate and recovery. We find evidence for this design principle in different nematodes (e.g., Pristionchus pacificus and Brugia malayi) as well as in the chordate Ciona intestinalis. As recovery from a growth arrested state into a fast growing state is a physiological feature shared by many metazoans, operons could evolve as an evolutionary solution to facilitate these processes.