An expanded GCaMP reporter toolkit for functional imaging in Caenorhabditis elegans.

In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo. In particular, GCaMPs, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters is also well suited for studies requiring fast imaging speeds and low magnification or low-cost platforms.

High-throughput library transgenesis in Caenorhabditis elegans via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS).

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here, we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in Caenorhabditis elegans to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from predefined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using C. elegans, in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

Transgenesis ­ the ability to insert foreign genetic material (known as transgenes) in to the genome of an organism ­ has revolutionized biological research. This approach has made it possible for scientists to study the role of specific genes and to produce animal models which mimic aspects of human diseases. For transgenes to be maintained and passed down to future generations, they must be introduced into germ cells which will go on to form the egg and sperm of the organism. However, despite advances in genetic engineering, this process (called 'specific transgenesis') is still laborious and time-consuming, and limits researchers to working with only a small number of known DNA sequences at a time. In contrast, 'exploratory transgenesis' ­ where dozens of transgenes from a library of DNA sequences are introduced simultaneously into multiple individuals ­ is more efficient and allows for more large-scale experiments. However, this approach can only be done with single-celled organisms like bacteria, and remains virtually impossible in laboratory animals like worms or mice. Stevenson et al. therefore set out to boost the efficiency of exploratory transgenesis in a commonly used laboratory animal, the roundworm Caenorhabditis elegans. To do this, they used the 'library' principle of exploratory transgenesis in order to develop a new resource called TARDIS (short for, Transgenic Arrays Resulting in Diversity of Integrated Sequences). First, Stevenson et al. genetically engineered worms to carry a 'landing site' for foreign DNA. Next, a library of transgenes and a mechanism which cuts pieces of DNA and pastes them into the landing site were introduced into the germ cells of these worms using traditional methods. The worms were then bred to generate a large population of offspring that had inherited this array of foreign DNA sequences. Finally, the 'cut and paste' mechanism was switched on and a random transgene was inserted into the landing site in the genome. This resulted in thousands of worms which each had a unique genetic modification that can be passed on to future generations. These results show for the first time that larger-scale transgenesis experiments are possible in multi-cellular animals. In the future, Stevenson et al. hope that TARDIS can be adapted to different organisms and allow researchers to carry out experiments that were not previously possible.

An expanded GCaMP reporter toolkit for functional imaging in C. elegans.

In living organisms, changes in calcium flux are integral to many different cellular functions and are especially critical for the activity of neurons and myocytes. Genetically encoded calcium indicators (GECIs) have been popular tools for reporting changes in calcium levels in vivo . In particular, GCaMP, derived from GFP, are the most widely used GECIs and have become an invaluable toolkit for neurophysiological studies. Recently, new variants of GCaMP, which offer a greater variety of temporal dynamics and improved brightness, have been developed. However, these variants are not readily available to the Caenorhabditis elegans research community. This work reports a set of GCaMP6 and jGCaMP7 reporters optimized for C. elegans studies. Our toolkit provides reporters with improved dynamic range, varied kinetics, and targeted subcellular localizations. Besides optimized routine uses, this set of reporters are also well-suited for studies requiring fast imaging speeds and low magnification or low-cost platforms.

Graphical-model framework for automated annotation of cell identities in dense cellular images.

Although identifying cell names in dense image stacks is critical in analyzing functional whole-brain data enabling comparison across experiments, unbiased identification is very difficult, and relies heavily on researchers' experiences. Here, we present a probabilistic-graphical-model framework, CRF_ID, based on Conditional Random Fields, for unbiased and automated cell identification. CRF_ID focuses on maximizing intrinsic similarity between shapes. Compared to existing methods, CRF_ID achieves higher accuracy on simulated and ground-truth experimental datasets, and better robustness against challenging noise conditions common in experimental data. CRF_ID can further boost accuracy by building atlases from annotated data in highly computationally efficient manner, and by easily adding new features (e.g. from new strains). We demonstrate cell annotation in Caenorhabditis elegans images across strains, animal orientations, and tasks including gene-expression localization, multi-cellular and whole-brain functional imaging experiments. Together, these successes demonstrate that unbiased cell annotation can facilitate biological discovery, and this approach may be valuable to annotation tasks for other systems.

A Multicellular Network Mechanism for Temperature-Robust Food Sensing.

Responsiveness to external cues is a hallmark of biological systems. In complex environments, it is crucial for organisms to remain responsive to specific inputs even as other internal or external factors fluctuate. Here, we show how the nematode Caenorhabditis elegans can discriminate between different food levels to modulate its lifespan despite temperature perturbations. This end-to-end robustness from environment to physiology is mediated by food-sensing neurons that communicate via transforming growth factor ß (TGF-ß) and serotonin signals to form a multicellular gene network. Specific regulations in this network change sign with temperature to maintain similar food responsiveness in the lifespan output. In contrast to robustness of stereotyped outputs, our findings uncover a more complex robustness process involving the higher order function of discrimination in food responsiveness. This process involves rewiring a multicellular network to compensate for temperature and provides a basis for understanding gene-environment interactions. Together, our findings unveil sensory computations that integrate environmental cues to govern physiology.

An automated platform to monitor long-term behavior and healthspan in Caenorhabditis elegans under precise environmental control.

Health and longevity in all organisms are strongly influenced by the environment. To fully understand how environmental factors interact with genetic and stochastic factors to modulate the aging process, it is crucial to precisely control environmental conditions for long-term studies. In the commonly used model organism Caenorhabditis elegans, existing assays for healthspan and lifespan have inherent limitations, making it difficult to perform large-scale longitudinal aging studies under precise environmental control. To address these constraints, we developed the Health and Lifespan Testing Hub (HeALTH), an automated, microfluidic-based system for robust longitudinal behavioral monitoring. Our system provides long-term (i.e. entire lifespan) spatiotemporal environmental control. We demonstrate healthspan and lifespan studies under a variety of genetic and environmental perturbations while observing how individuality plays a role in the aging process. This system is generalizable beyond aging research, particularly for short- or long-term behavioral assays, and could be adapted for other model systems.

smFISH in chips: a microfluidic-based pipeline to quantify in situ gene expression in whole organisms.

Gene expression and genetic regulatory networks in multi-cellular organisms control complex physiological processes ranging from cellular differentiation to development to aging. Traditional methods to investigate gene expression relationships rely on using bulk, pooled-population assays (e.g. RNA-sequencing and RT-PCR) to compare gene expression levels in hypo- or hyper-morphic mutant animals (e.g. gain-of-function or knockout). This approach is limited, especially in complex gene networks, as these genetic mutations may affect the expressions of related genes in unforseen ways. In contrast, we developed a microfluidic-based pipeline to discover gene relationships in a single genetic background. The microfluidic device provides efficient reagent exchange and the ability to track individual animals. By automating a robust microfluidic reagent exchange strategy, we adapted and validated single molecule fluorescent in situ hybridization (smFISH) on-chip and combined this technology with live-imaging of fluorescent transcriptional reporters. Together, this multi-level information enabled us to quantify a gene expression relationship with single-animal resolution. While this microfluidic-based pipeline is optimized for live-imaging and smFISH C. elegans studies, the strategy is highly-adaptable to other biological models as well as combining other live and end-point biological assays, such as behavior-based toxicology screening and immunohistochemistry.

Digging deeper: methodologies for high-content phenotyping in Caenorhabditis elegans.

Deep phenotyping is an emerging conceptual paradigm and experimental approach aimed at measuring and linking many aspects of a phenotype to understand its underlying biology. To date, deep phenotyping has been applied mostly in cultured cells and used less in multicellular organisms. However, in the past decade, it has increasingly been recognized that deep phenotyping could lead to a better understanding of how genetics, environment and stochasticity affect the development, physiology and behavior of an organism. The nematode Caenorhabditis elegans is an invaluable model system for studying how genes affect a phenotypic trait, and new technologies have taken advantage of the worm's physical attributes to increase the throughput and informational content of experiments. Coupling of these technical advancements with computational and analytical tools has enabled a boom in deep-phenotyping studies of C. elegans. In this Review, we highlight how these new technologies and tools are digging into the biological origins of complex, multidimensional phenotypes.

Quantification of Information Encoded by Gene Expression Levels During Lifespan Modulation Under Broad-range Dietary Restriction in C. elegans.

Sensory systems allow animals to detect, process, and respond to their environment. Food abundance is an environmental cue that has profound effects on animal physiology and behavior. Recently, we showed that modulation of longevity in the nematode Caenorhabditis elegans by food abundance is more complex than previously recognized. The responsiveness of the lifespan to changes in food level is determined by specific genes that act by controlling information processing within a neural circuit. Our framework combines genetic analysis, high-throughput quantitative imaging and information theory. Here, we describe how these techniques can be used to characterize any gene that has a physiological relevance to broad-range dietary restriction. Specifically, this workflow is designed to reveal how a gene of interest regulates lifespan under broad-range dietary restriction; then to establish how the expression of the gene varies with food level; and finally, to provide an unbiased quantification of the amount of information conveyed by gene expression about food abundance in the environment. When several genes are examined simultaneously under the context of a neural circuit, this workflow can uncover the coding strategy employed by the circuit.

Genetic control of encoding strategy in a food-sensing neural circuit.

Neuroendocrine circuits encode environmental information via changes in gene expression and other biochemical activities to regulate physiological responses. Previously, we showed that daf-7 TGFß and tph-1 tryptophan hydroxylase expression in specific neurons encode food abundance to modulate lifespan in Caenorhabditis elegans, and uncovered cross- and self-regulation among these genes (Entchev et al., 2015). Here, we now extend these findings by showing that these interactions between daf-7 and tph-1 regulate redundancy and synergy among neurons in food encoding through coordinated control of circuit-level signal and noise properties. Our analysis further shows that daf-7 and tph-1 contribute to most of the food-responsiveness in the modulation of lifespan. We applied a computational model to capture the general coding features of this system. This model agrees with our previous genetic analysis and highlights the consequences of redundancy and synergy during information transmission, suggesting a rationale for the regulation of these information processing features.

A gene-expression-based neural code for food abundance that modulates lifespan.

How the nervous system internally represents environmental food availability is poorly understood. Here, we show that quantitative information about food abundance is encoded by combinatorial neuron-specific gene-expression of conserved TGFß and serotonin pathway components in Caenorhabditis elegans. Crosstalk and auto-regulation between these pathways alters the shape, dynamic range, and population variance of the gene-expression responses of daf-7 (TGFß) and tph-1 (tryptophan hydroxylase) to food availability. These intricate regulatory features provide distinct mechanisms for TGFß and serotonin signaling to tune the accuracy of this multi-neuron code: daf-7 primarily regulates gene-expression variability, while tph-1 primarily regulates the dynamic range of gene-expression responses. This code is functional because daf-7 and tph-1 mutations bidirectionally attenuate food level-dependent changes in lifespan. Our results reveal a neural code for food abundance and demonstrate that gene expression serves as an additional layer of information processing in the nervous system to control long-term physiology.

The quality of economic studies of cancer pharmacogenomics: a quantitative appraisal of the evidence.

This study evaluated the quality of health economic studies of cancer pharmacogenomics (PGx). A systematic search of the literature for economic studies of PGx was conducted in four common cancers. Evaluation of study quality was carried out using the quality of health economic studies instrument. Thirty-nine articles met our eligibility criteria and were selected and accepted for further statistical analyses. The majority of articles (85%) were studies focusing on breast cancer. The overall weighted mean quality score was 85.10, with a range from 21 to 100. Eighty-seven percent of articles were categorized as good quality, whereas some 10 and 3% were categorized as moderate and poor quality, respectively. The quality of economic studies of cancer PGx is generally good but varied widely. We identified several attributes that are predictive of quality. Our findings may be useful for oncologists, health economists and decision makers interested in evaluating studies involving PGx.

Genetic identification of HSD-1, a conserved steroidogenic enzyme that directs larval development in Caenorhabditis elegans.

In C. elegans, steroid hormones function in conjunction with insulin/IGF-1-like signaling in promoting reproductive development over entry into the diapausal dauer stage. The NCR-1 and -2 (NPC1-related) intracellular cholesterol transporters function redundantly in preventing dauer arrest, presumably by regulating the availability of substrates for steroid hormone synthesis. We have identified hsd-1 as a new component of this cholesterol trafficking/processing pathway, using an ncr-1 enhancer screen. HSD-1 is orthologous to 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4) isomerases (3beta-HSDs), which are key steroidogenic enzymes in vertebrates, and is exclusively expressed in two neuron-like XXX cells that are crucial in preventing dauer arrest, suggesting that it is involved in biosynthesis of dauer-preventing steroid hormones. The hsd-1 null mutant displays defects in inhibiting dauer arrest: it forms dauers in the deletion mutant backgrounds of ncr-1 or daf-28/insulin; as a single mutant, it is hypersensitive to dauer pheromone. We found that hsd-1 defects can be rescued by feeding mutant animals with several steroid intermediates that are either downstream of or in parallel to the 3beta-HSD function in the dafachronic acid biosynthetic pathway, suggesting that HSD-1 functions as a 3beta-HSD. Interestingly, sterols that rescued hsd-1 defects also bypassed the need for the NCR-1 and/or -2 functions, suggesting that HSD-1-mediated steroid hormone production is an important functional output of the NCR transporters. Finally, we found that the HSD-1-mediated signal activates insulin/IGF-I signaling in a cell non-autonomous fashion, suggesting a novel mechanism for how these two endocrine pathways intersect in directing development.

Clustering of genetically defined allele classes in the Caenorhabditis elegans DAF-2 insulin/IGF-1 receptor.

The DAF-2 insulin/IGF-1 receptor regulates development, metabolism, and aging in the nematode Caenorhabditis elegans. However, complex differences among daf-2 alleles complicate analysis of this gene. We have employed epistasis analysis, transcript profile analysis, mutant sequence analysis, and homology modeling of mutant receptors to understand this complexity. We define an allelic series of nonconditional daf-2 mutants, including nonsense and deletion alleles, and a putative null allele, m65. The most severe daf-2 alleles show incomplete suppression by daf-18(0) and daf-16(0) and have a range of effects on early development. Among weaker daf-2 alleles there exist distinct mutant classes that differ in epistatic interactions with mutations in other genes. Mutant sequence analysis (including 11 newly sequenced alleles) reveals that class 1 mutant lesions lie only in certain extracellular regions of the receptor, while class 2 (pleiotropic) and nonconditional missense mutants have lesions only in the ligand-binding pocket of the receptor ectodomain or the tyrosine kinase domain. Effects of equivalent mutations on the human insulin receptor suggest an altered balance of intracellular signaling in class 2 alleles. These studies consolidate and extend our understanding of the complex genetics of daf-2 and its underlying molecular biology.

Evolutionary conservation of regulated longevity assurance mechanisms.

BACKGROUND: To what extent are the determinants of aging in animal species universal? Insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an evolutionarily conserved (public) regulator of longevity; yet it remains unclear whether the genes and biochemical processes through which IIS acts on aging are public or private (that is, lineage specific). To address this, we have applied a novel, multi-level cross-species comparative analysis to compare gene expression changes accompanying increased longevity in mutant nematodes, fruitflies and mice with reduced IIS. RESULTS: Surprisingly, there is little evolutionary conservation at the level of individual, orthologous genes or paralogous genes under IIS regulation. However, a number of gene categories are significantly enriched for genes whose expression changes in long-lived animals of all three species. Down-regulated categories include protein biosynthesis-associated genes. Up-regulated categories include sugar catabolism, energy generation, glutathione-S-transferases (GSTs) and several other categories linked to cellular detoxification (that is, phase 1 and phase 2 metabolism of xenobiotic and endobiotic toxins). Protein biosynthesis and GST activity have recently been linked to aging and longevity assurance, respectively. CONCLUSION: These processes represent candidate, regulated mechanisms of longevity-control that are conserved across animal species. The longevity assurance mechanisms via which IIS acts appear to be lineage-specific at the gene level (private), but conserved at the process level (or semi-public). In the case of GSTs, and cellular detoxification generally, this suggests that the mechanisms of aging against which longevity assurance mechanisms act are, to some extent, lineage specific.

RILM: a web-based resource to aid comparative and functional analysis of the insulin and IGF-1 receptor family.

The metazoan receptors for insulin (INSR), insulin-like growth factor 1 (IGF1R), and other insulin-like molecules are transmembrane tyrosine kinases involved in the regulation of cell size, cell proliferation, development, signaling of nutritional and environmental conditions, and aging. Historically, mutations in the human insulin receptor have been studied because such changes often lead to severe insulin resistance. More recently, amino acid sequence alterations in the insulin receptor-like receptors of Drosophila melanogaster and Caenorhabditis elegans, as well as in the mouse insulin receptor have been the focus of attention. These modifications can have profound effects on growth, body size, metabolism, and aging. To integrate the many findings on insulin/IGF1 receptor structure and function across species we have created "Receptors for Insulin and Insulin-like Molecules" (RILM), a curated computer-based resource that displays residue-by-residue information on sequence homology, three-dimensional structure, structure/function annotation, and documented mutations. The resource includes data obtained from sequence and structure analysis tools, primary database resources, and published reports. The information is integrated via a structure-based multiple sequence alignment of diverse members of the family. RILM was designed to provide easy access to multiple data types that could prove useful in the analysis of the effect of mutations on protein structure and ligand binding within this receptor family. RILM is available at www.biochem.ucl.ac.uk/RILM.