Sensitive and Precise Quantification of Insulin-Like mRNA Expression in Caenorhabditis elegans

Insulin-like signaling regulates developmental arrest, stress resistance and lifespan in the nematode Caenorhabditis elegans. However, the genome encodes 40 insulin-like peptides, and the regulation and function of individual peptides is largely uncharacterized. We used the nCounter platform to measure mRNA expression of all 40 insulin-like peptides as well as the insulin-like receptor daf-2, its transcriptional effector daf-16, and the daf-16 target gene sod-3. We validated the platform using 53 RNA samples previously characterized by high density oligonucleotide microarray analysis. For this set of genes and the standard nCounter protocol, sensitivity and precision were comparable between the two platforms. We optimized conditions of the nCounter assay by varying the mass of total RNA used for hybridization, thereby increasing sensitivity up to 50-fold and reducing the median coefficient of variation as much as 4-fold. We used deletion mutants to demonstrate specificity of the assay, and we used optimized conditions to assay insulin-like gene expression throughout the C. elegans life cycle. We detected expression for nearly all insulin-like genes and find that they are expressed in a variety of distinct patterns suggesting complexity of regulation and specificity of function. We identified insulin-like genes that are specifically expressed during developmental arrest, larval development, adulthood and embryogenesis. These results demonstrate that the nCounter platform provides a powerful approach to analyzing insulin-like gene expression dynamics, and they suggest hypotheses about the function of individual insulin-like genes

Metazoan Operons Accelerate Recovery from Growth-Arrested States

Existing theories explain why operons are advantageous in prokaryotes, but their occurrence in metazoans is an enigma. Nematode operon genes, typically consisting of growth genes, are significantly upregulated during recovery from growth-arrested states. This expression pattern is anticorrelated to nonoperon genes, consistent with a competition for transcriptional resources. We find that transcriptional resources are initially limiting during recovery and that recovering animals are highly sensitive to any additional decrease in transcriptional resources. We provide evidence that operons become advantageous because, by clustering growth genes into operons, fewer promoters compete for the limited transcriptional machinery, effectively increasing the concentration of transcriptional resources and accelerating recovery. Mathematical modeling reveals how a moderate increase in transcriptional resources can substantially enhance transcription rate and recovery. This design principle occurs in different nematodes and the chordate C. intestinalis. As transition from arrest to rapid growth is shared by many metazoans, operons could have evolved to facilitate these processes

Synthetic lethal analysis of Caenorhabditis elegans posterior embryonic patterning genes identifies conserved genetic interactions

Phenotypic robustness is evidenced when single-gene mutations do not result in an obvious phenotype. It has been suggested that such phenotypic stability results from 'buffering' activities of homologous genes as well as non-homologous genes acting in parallel pathways. One approach to characterizing mechanisms of phenotypic robustness is to identify genetic interactions, specifically, double mutants where buffering is compromised. To identify interactions among genes implicated in posterior patterning of the Caenorhabditis elegans embryo, we measured synthetic lethality following RNA interference of 22 genes in 15 mutant strains. 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

Development of Communication Behaviour: Receiver Ontogeny in Túngara Frogs and a Prospectus for a Behavioural Evolutionary Development

Most studies addressing the development of animal communication have focused on signal production rather than receiver decoding, and similar emphasis has been given to learning over nonlearning. But receivers are an integral part of a communication network, and nonlearned mechanisms appear to be more ubiquitous than learned ones in the communication systems of most animals. Here we review the results of recent experiments and outline future directions for integrative studies on the development of a primarily nonlearned behaviour—recognition of communication signals during ontogeny in a tropical frog. The results suggest that antecedents to adult behaviours might be a common feature of developing organisms. Given the essential role that acoustic communication serves in reproduction for many organisms and that receivers can exert strong influence on the evolution of signals, understanding the evolutionary developmental basis of mate recognition will provide new insights into the evolution of communication systems

The homeodomain protein PAL-1 specifies a lineage-specific regulatory network in the C. elegans embryo

Maternal and zygotic activities of the homeodomain protein PAL-1 specify the identity and maintain the development of the multipotent C blastomere lineage in the C. elegans embryo. To identify PAL-1 regulatory target genes, we used microarrays to compare transcript abundance in wild-type embryos with mutant embryos lacking a C blastomere and to mutant embryos with extra C blastomeres. pal-1-dependent C-lineage expression was verified for select candidate target genes by reporter gene analysis, though many of the target genes are expressed in additional lineages as well. The set of validated target genes includes 12 transcription factors, an uncharacterized wingless ligand and five uncharacterized genes. Phenotypic analysis demonstrates that the identified PAL-1 target genes affect specification, differentiation and morphogenesis of C-lineage cells. In particular, we show that cell fate-specific genes (or tissue identity genes) and a posterior HOX gene are activated in lineage-specific fashion. Transcription of targets is initiated in four temporal phases, which together with their spatial expression patterns leads to a model of the regulatory network specified by PAL-1

Synthetic Lethal Analysis of Caenorhabditis elegans Posterior Embryonic Patterning Genes Identifies Conserved Genetic Interactions

Phenotypic robustness is evidenced when single-gene mutations do not result in an obvious phenotype. It has been suggested that such phenotypic stability results from 'buffering' activities of homologous genes as well as non-homologous genes acting in parallel pathways. One approach to characterizing mechanisms of phenotypic robustness is to identify genetic interactions, specifically, double mutants where buffering is compromised. To identify interactions among genes implicated in posterior patterning of the Caenorhabditis elegans embryo, we measured synthetic lethality following RNA interference of 22 genes in 15 mutant strains. 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.Molecular and Cellular Biolog

Neurohormonal signaling via a sulfotransferase antagonizes insulin-like signaling to regulate a Caenorhabditis elegans stress response.

Insulin and insulin-like signaling regulates a broad spectrum of growth and metabolic responses to a variety of internal and environmental stimuli. For example, the inhibition of insulin-like signaling in C. elegans mediates its response to both osmotic stress and starvation. We report that in response to osmotic stress the cytosolic sulfotransferase SSU-1 antagonizes insulin-like signaling and promotes developmental arrest. Both SSU-1 and the DAF-16 FOXO transcription factor, which is activated when insulin signaling is low, are needed to drive specific responses to reduced insulin-like signaling. We demonstrate that SSU-1 functions in a single pair of sensory neurons to control intercellular signaling via the nuclear hormone receptor NHR-1 and promote both the specific transcriptional response to osmotic stress and altered lysophosphatidylcholine metabolism. Our results show the requirement of a sulfotransferase-nuclear hormone receptor neurohormonal signaling pathway for some but not all consequences of reduced insulin-like signaling

Nutritional control of mRNA isoform expression during developmental arrest and recovery in C. elegans

Nutrient availability profoundly influences gene expression. Many animal genes encode multiple transcript isoforms, yet the effect of nutrient availability on transcript isoform expression has not been studied in genome-wide fashion. When Caenorhabditis elegans larvae hatch without food, they arrest development in the first larval stage (L1 arrest). Starved larvae can survive L1 arrest for weeks, but growth and post-embryonic development are rapidly initiated in response to feeding. We used RNA-seq to characterize the transcriptome during L1 arrest and over time after feeding. Twenty-seven percent of detectable protein-coding genes were differentially expressed during recovery from L1 arrest, with the majority of changes initiating within the first hour, demonstrating widespread, acute effects of nutrient availability on gene expression. We used two independent approaches to track expression of individual exons and mRNA isoforms, and we connected changes in expression to functional consequences by mining a variety of databases. These two approaches identified an overlapping set of genes with alternative isoform expression, and they converged on common functional patterns. Genes affecting mRNA splicing and translation are regulated by alternative isoform expression, revealing post-transcriptional consequences of nutrient availability on gene regulation. We also found that phosphorylation sites are often alternatively expressed, revealing a common mode by which alternative isoform expression modifies protein function and signal transduction. Our results detail rich changes in C. elegans gene expression as larvae initiate growth and post-embryonic development, and they provide an excellent resource for ongoing investigation of transcriptional regulation and developmental physiology

First Measurement of the Clustering Evolution of Photometrically-Classified Quasars

We present new measurements of the quasar autocorrelation from a sample of \~80,000 photometrically-classified quasars taken from SDSS DR1. We find a best-fit model of $\omega(\theta) = (0.066\pm^{0.026}_{0.024})\theta^{-(0.98\pm0.15)}$ for the angular autocorrelation, consistent with estimates from spectroscopic quasar surveys. We show that only models with little or no evolution in the clustering of quasars in comoving coordinates since z~1.4 can recover a scale-length consistent with local galaxies and Active Galactic Nuclei (AGNs). A model with little evolution of quasar clustering in comoving coordinates is best explained in the current cosmological paradigm by rapid evolution in quasar bias. We show that quasar biasing must have changed from b_Q~3 at a (photometric) redshift of z=2.2 to b_Q~1.2-1.3 by z=0.75. Such a rapid increase with redshift in biasing implies that quasars at z~2 cannot be the progenitors of modern L* objects, rather they must now reside in dense environments, such as clusters. Similarly, the duration of the UVX quasar phase must be short enough to explain why local UVX quasars reside in essentially unbiased structures. Our estimates of b_Q are in good agreement with recent spectroscopic results, which demonstrate the implied evolution in b_Q is consistent with quasars inhabiting halos of similar mass at every redshift. Treating quasar clustering as a function of both redshift and luminosity, we find no evidence for luminosity dependence in quasar clustering, and that redshift evolution thus affects quasar clustering more than changes in quasars' luminosity. We provide a new method for quantifying stellar contamination in photometrically-classified quasar catalogs via the correlation function.Comment: 34 pages, 10 figures, 1 table, Accepted to ApJ after: (i) Minor textual changes; (ii) extra points added to Fig.

Pairing of Competitive and Topologically Distinct Regulatory Modules Enhances Patterned Gene Expression

Biological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time series, whole-genome microarray analyses to systematically perturb and characterize components of a Caenorhabditis elegans lineage-specific transcriptional regulatory network. These data are supported by selected reporter gene analyses and comprehensive yeast one-hybrid and promoter sequence analyses. Based on these results, we define and characterize two modules composed of muscle- and epidermal-specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross-inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage. The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches.Molecular and Cellular Biolog