C. elegans insulin-like peptides.

Insulin-like peptides are a group of hormones crucial for regulating metabolism, growth, and development in animals. Invertebrates, such as C. elegans, have been instrumental in understanding the molecular mechanisms of insulin-like peptides. Here, we review the 40 insulin-like peptide genes encoded in the C. elegans genome. Despite the large number, there is only one C. elegans insulin-like peptide receptor, called DAF-2. The insulin and insulin-like growth factor signaling (IIS) pathway is evolutionarily conserved from worms to humans. Thus C. elegans provides an excellent model to understand how these insulin-like peptides function. C. elegans is unique in that it possesses insulin-like peptides that have antagonistic properties, unlike all human insulin-like peptides, which are agonists. This review provides an overview of the current literature on C. elegans insulin-like peptide structures, processing, tissue localization, and regulation. We will also provide examples of insulin-like peptide signaling in C. elegans during growth, development, germline development, learning/memory, and longevity.

A Compilation of the Diverse miRNA Functions in Caenorhabditis elegans and Drosophila melanogaster Development.

MicroRNAs are critical regulators of post-transcriptional gene expression in a wide range of taxa, including invertebrates, mammals, and plants. Since their discovery in the nematode, Caenorhabditis elegans, miRNA research has exploded, and they are being identified in almost every facet of development. Invertebrate model organisms, particularly C. elegans, and Drosophila melanogaster, are ideal systems for studying miRNA function, and the roles of many miRNAs are known in these animals. In this review, we compiled the functions of many of the miRNAs that are involved in the development of these invertebrate model species. We examine how gene regulation by miRNAs shapes both embryonic and larval development and show that, although many different aspects of development are regulated, several trends are apparent in the nature of their regulation.

C. elegans vab-6 encodes a KIF3A kinesin and functions cell non-autonomously to regulate epidermal morphogenesis.

Cells undergo strict regulation to develop their shape in a process called morphogenesis. Caenorhabditis elegans with mutations in the variable abnormal (vab) class of genes have been shown to display epidermal and neuronal morphological defects. While several vab genes have been well-characterized, the function of the vab-6 gene remains unknown. Here, we show that vab-6 is synonymous with a subunit of the kinesin-II heterotrimeric motor complex called klp-20/Kif3a, a motor well-understood to be involved in developing sensory cilia in the nervous system. We show that certain klp-20 alleles cause animals to develop a bumpy body phenotype that is variable but most severe in mutants containing single amino-acid substitutions in the catalytic head-domain sites of the protein. Surprisingly, animals carrying a klp-20 null allele do not show the bumpy epidermal phenotype suggesting genetic redundancy and only when mutant versions of the KLP-20 protein are present, the epidermal phenotype is observed. The bumpy epidermal phenotype was not observed in other kinesin-2 mutants, suggesting that KLP-20 is functioning independently from its role in intraflagellar transport (IFT) during ciliogenesis. Interestingly, despite having such a prominent epidermal phenotype, KLP-20 is not expressed in the epidermis, strongly suggesting a cell non-autonomous role in which it regulates epidermal morphogenesis.

Characterizing Variants of Unknown Significance of the PTEN tumour suppressorHomolog DAF-18.

Insulin and insulin-like growth factor signaling (IIS) is an anabolic pathway conserved among humans and Caenorhabditis elegans . In humans, the tumour suppressor protein Phosphatase and Tensin Homolog (PTEN) inhibits IIS, preventing excessive growth. PTEN variants are associated with disease, but how they affect PTEN function is not well understood. Here, we characterized variants of unknown significance (VUSs) implicated in autism spectrum disorder by studying homologous mutations in the C. elegans protein DAF-18 to infer how they play a role in human disease.We found that variants D66E and L115V are likely benign, H168Q is intermediate while variants H138R and T176I are likely pathogenic.

An overview of the insulin signaling pathway in model organisms Drosophila melanogaster and Caenorhabditis elegans.

The insulin/insulin-like growth factor signaling pathway is an evolutionary conserved pathway across metazoans and is required for development, metabolism and behavior. This pathway is associated with various human metabolic disorders and cancers. Thus, model organisms including Drosophila melanogaster and Caenorhabditis elegans provide excellent opportunities to examine the structure and function of this pathway and its influence on cellular metabolism and proliferation. In this review, we will provide an overview of human insulin and the human insulin signaling pathway and explore the recent discoveries in model organisms Drosophila melanogaster and Caenorhabditis elegans. Our review will provide information regarding the various insulin-like peptides in model organisms as well as the conserved functions of insulin signaling pathways. Further investigation of the insulin signaling pathway in model organisms could provide a promising opportunity to develop novel therapies for various metabolic disorders and insulin-mediated cancers.

Neuropeptide and microRNA regulators of juvenile hormone production.

The sesquiterpenoid juvenile hormone(s) (JHs) of insects are the primary regulators of growth, metamorphosis, and reproduction in most insect species. As a consequence, it is essential that JH production be precisely regulated so that it is present only during appropriate periods necessary for the control of these processes. The presence of JH at inappropriate times results in disruption to metamorphosis and development and, in some cases, to disturbances in female reproduction. Neuropeptides regulate the timing and production of JH by the corpora allata. Allatostatin and allatotropin were the names coined for neuropeptides that serve as inhibitors or stimulators of JH biosynthesis, respectively. Three different allatostatin neuropeptide families are capable of inhibiting juvenile hormone but only one family is utilized for that purpose dependent on the insect studied. The function of allatotropin also varies in different insects. These neuropeptides are pleiotropic in function acting on diverse physiological processes in different insects such as muscle contraction, sleep and neuromodulation. Genome projects and expression studies have assigned individual neuropeptide families to their respective receptors. An understanding of the localization of these receptors is providing clues as to how numerous peptide families might be integrated in regulating physiological functions. In recent years microRNAs have been identified that down-regulate enzymes and transcription factors that are involved in the biosynthesis and action of juvenile hormone.

C. elegans PTEN and AMPK block neuroblast divisions by inhibiting a BMP-insulin-PP2A-MAPK pathway.

Caenorhabditis elegans that hatch in the absence of food stop their postembryonic development in a process called L1 arrest. Intriguingly, we find that the postembryonic Q neuroblasts divide and migrate during L1 arrest in mutants that have lost the energy sensor AMP-activated protein kinase (AMPK) or the insulin/IGF-1 signaling (IIS) negative regulator DAF-18/PTEN. We report that DBL-1/BMP works upstream of IIS to promote agonistic insulin-like peptides during L1 arrest. However, the abnormal Q cell divisions that occur during L1 arrest use a novel branch of the IIS pathway that is independent of the terminal transcription factor DAF-16/FOXO. Using genetic epistasis and drug interactions we show that AMPK functions downstream of, or in parallel with DAF-18/PTEN and IIS to inhibit PP2A function. Further, we show that PP2A regulates the abnormal Q cell divisions by activating the MPK-1/ERK signaling pathway via LIN-45/RAF, independently of LET-60/RAS. PP2A acts as a tumor suppressor in many oncogenic signaling cascades. Our work demonstrates a new role for PP2A that is needed to induce neuroblast divisions during starvation and is regulated by both insulin and AMPK.

A functional study of all 40 Caenorhabditis elegans insulin-like peptides.

The human genome encodes 10 insulin-like genes, whereas the Caenorhabditis elegans genome remarkably encodes 40 insulin-like genes. Knockout strategies to determine the roles of all the insulin/insulin-like peptide ligands (INS) in C. elegans has been challenging due to functional redundancy. Here, we individually overexpressed each of the 40 ins genes pan-neuronally, and monitored multiple phenotypes including: L1 arrest life span, neuroblast divisions under L1 arrest, dauer formation, and fat accumulation, as readouts to characterize the functions of each INS in vivo Of the 40 INS peptides, we found functions for 35 INS peptides and functionally categorized each as agonists, antagonists, or of pleiotropic function. In particular, we found that 9 of 16 agonistic INS peptides shortened L1 arrest life span and promoted neuroblast divisions during L1 arrest. Our study revealed that a subset of ß-class INS peptides that contain a distinct F peptide sequence are agonists. Our work is the first to categorize the structures of INS peptides and relate these structures to the functions of all 40 INS peptides in vivo Our findings will promote the study of insulin function on development, metabolism, and aging-related diseases.

A Caenorhabditis elegans Nutritional-status Based Copper Aversion Assay.

To ensure survival, organisms must be capable of avoiding unfavorable habitats while ensuring a consistent food source. Caenorhabditis elegans alter their locomotory patterns upon detection of diverse environmental stimuli and can modulate their suite of behavioral responses in response to starvation conditions. Nematodes typically exhibit a decreased aversive response when removed from a food source for over 30 min. Observation of behavioral changes in response to a changing nutritional status can provide insight into the mechanisms that regulate the transition from a well-fed to starved state. We have developed an assay that measures a nematode's ability to cross an aversive barrier (i.e. copper) then reach a food source over a prolonged period of time. This protocol builds upon previous work by integrating multiple variables in a manner that allows for continued data collection as the organisms shift towards an increasingly starved condition. Moreover, this assay permits an increased sample size so that larger populations of nematodes can be simultaneously evaluated. Organisms defective for the ability to detect or respond to copper immediately cross the chemical barrier, while wild type nematodes are initially repelled. As wild type worms are increasingly starved, they begin to cross the barrier and reach the food source. We designed this assay to evaluate a mutant that is incapable of responding to diverse environmental cues, including food sensation or detection of aversive chemicals. When evaluated via this protocol, the defective organisms immediately crossed the barrier, but were also incapable of detecting a food source. Hence, these mutants repeatedly cross the chemical barrier despite temporarily reaching a food source. This assay can straightforwardly test populations of worms to evaluate potential pathway defects related to aversion and starvation.

CDC-42 Orients Cell Migration during Epithelial Intercalation in the Caenorhabditis elegans Epidermis.

Cell intercalation is a highly directed cell rearrangement that is essential for animal morphogenesis. As such, intercalation requires orchestration of cell polarity across the plane of the tissue. CDC-42 is a Rho family GTPase with key functions in cell polarity, yet its role during epithelial intercalation has not been established because its roles early in embryogenesis have historically made it difficult to study. To circumvent these early requirements, in this paper we use tissue-specific and conditional loss-of-function approaches to identify a role for CDC-42 during intercalation of the Caenorhabditis elegans dorsal embryonic epidermis. CDC-42 activity is enriched in the medial tips of intercalating cells, which extend as cells migrate past one another. Moreover, CDC-42 is involved in both the efficient formation and orientation of cell tips during cell rearrangement. Using conditional loss-of-function we also show that the PAR complex functions in tip formation and orientation. Additionally, we find that the sole C. elegans Eph receptor, VAB-1, functions during this process in an Ephrin-independent manner. Using epistasis analysis, we find that vab-1 lies in the same genetic pathway as cdc-42 and is responsible for polarizing CDC-42 activity to the medial tip. Together, these data establish a previously uncharacterized role for polarized CDC-42, in conjunction with PAR-6, PAR-3 and an Eph receptor, during epithelial intercalation.

NPR-9, a Galanin-Like G-Protein Coupled Receptor, and GLR-1 Regulate Interneuronal Circuitry Underlying Multisensory Integration of Environmental Cues in Caenorhabditis elegans.

C. elegans inhabit environments that require detection of diverse stimuli to modulate locomotion in order to avoid unfavourable conditions. In a mammalian context, a failure to appropriately integrate environmental signals can lead to Parkinson's, Alzheimer's, and epilepsy. Provided that the circuitry underlying mammalian sensory integration can be prohibitively complex, we analyzed nematode behavioral responses in differing environmental contexts to evaluate the regulation of context dependent circuit reconfiguration and sensorimotor control. Our work has added to the complexity of a known parallel circuit, mediated by interneurons AVA and AIB, that integrates sensory cues and is responsible for the initiation of backwards locomotion. Our analysis of the galanin-like G-protein coupled receptor NPR-9 in C. elegans revealed that upregulation of galanin signaling impedes the integration of sensory evoked neuronal signals. Although the expression pattern of npr-9 is limited to AIB, upregulation of the receptor appears to impede AIB and AVA circuits to broadly prevent backwards locomotion, i.e. reversals, suggesting that these two pathways functionally interact. Galanin signaling similarly plays a broadly inhibitory role in mammalian models. Moreover, our identification of a mutant, which rarely initiates backwards movement, allowed us to interrogate locomotory mechanisms underlying chemotaxis. In support of the pirouette model of chemotaxis, organisms that did not exhibit reversal behavior were unable to navigate towards an attractant peak. We also assessed ionotropic glutamate receptor GLR-1 cell-specifically within AIB and determined that GLR-1 fine-tunes AIB activity to modify locomotion following reversal events. Our research highlights that signal integration underlying the initiation and fine-tuning of backwards locomotion is AIB and NPR-9 dependent, and has demonstrated the suitability of C. elegans for analysis of multisensory integration and sensorimotor control.

C. elegans Methods to Study PTEN.

C. elegans encodes a PTEN homolog called DAF-18 and human PTEN can functionally replace DAF-18. Thus C. elegans provides a valuable model organism to study PTEN. This chapter provides methods to study DAF-18/PTEN function in C. elegans. We provide methods to genotype daf-18/Pten mutants, visualize and quantify DAF-18/PTEN in C. elegans, as well as to study physiological and developmental processes that will provide molecular insight on DAF-18/PTEN function.

Mechanosensation circuitry in Caenorhabditis elegans: A focus on gentle touch.

Forward or reverse movement in Caenorhabditis elegans is the result of sequential contraction of muscle cells arranged along the body. In larvae, muscle cells are innervated by distinct classes of motorneurons. B motorneurons regulate forward movement and A motorneurons regulate backward movement. Ablation of the D motor neurons results in animals that are uncoordinated in either direction, which suggests that D motorneurons regulate the interaction between the two circuits. C. elegans locomotion is dictated by inputs from interneurons that regulate the activity of motorneurons which coordinate muscle contraction to facilitate forward or backwards movement. As C. elegans moves through the environment, sensory neurons interpret chemical and mechanical information which is relayed to the motor neurons that control locomotory direction. A mechanosensory input known as light nose touch can be simulated in the laboratory by touching the nose of the animal with a human eyebrow hair. The recoil reaction that follows from light nose touch appears to be primarily mediated by glutamate release from the polymodal sensory neuron ASH. Numerous glutamate receptor types are found in different neurons and interneurons which suggest that several pathways may regulate the aversive response. Based on the phenotypes of mutants in which neuropeptide processing is abolished, neuropeptides play a role in circuit regulation. The light touch response is also regulated by transient receptor channel proteins and degenerin/epithelial sodium channels which modulate the activity of sensory neurons involved in the nose touch response.

C. elegans as a model to study PTEN's regulation and function.

PTEN (phosphatase and tensin homolog deleted on chromosome 10) has important roles in tumor suppression, metabolism, and development, yet its regulators, effectors, and functions are not fully understood. DAF-18 is the PTEN ortholog in Caenorhabditis elegans. DAF-18's role is highly conserved to human PTEN, and can be functionally replaced by human PTEN. Thus C. elegans provides a valuable model to study PTEN. This review assesses current and emerging methods to study DAF-18's regulators and functions in C. elegans. We propose genetic modify screens to identify genes that interact with daf-18/PTEN. These genes are potential targets for anticancer drug therapies. We also provide a review on the roles DAF-18/PTEN has during C. elegans development and how studying these physiological roles can provide mechanistic insight on DAF-18/PTEN function.

Structures of an Eph receptor tyrosine kinase and its potential activation mechanism.

Eph receptor tyrosine kinases (RTKs) and their ephrin ligands play a crucial role in both physiological and pathophysiological processes, including tumourigenesis. A previous study of Eph RTKs established a regulatory role for the juxtamembrane segment (JMS) in kinase activation through the phosphorylation of two tyrosines within the JMS. Here, structures of EphA2 representing various activation states are presented. By determining the unphosphorylated inactive and phosphorylated active structures as well as an alternative conformation, conformational changes during kinase activation have been revealed. It is shown that phosphorylation of a tyrosine residue (Tyr772) in the activation loop without direct involvement of the JMS is sufficient to activate the EphA2 kinase. This mechanistic finding is in contrast to the mechanism of other Eph RTKs, such as EphB2, in which phosphorylation of the two JMS tyrosines initiates the dissociation of the JMS and triggers activation-loop phosphorylation for kinase activation. Furthermore, experiments demonstrate that the EphA2 substrate PTEN, a phosphatase that has been implicated in tumour suppression, acts to regulate the phosphorylation states of EphA2, exemplifying a unique reciprocal enzyme-substrate system. Based on these studies, it is therefore suggested that EphA2 may possess an alternate activation mechanism distinct from other Eph RTKs.

A unified nomenclature and amino acid numbering for human PTEN.

The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.

C. elegans chemotaxis assay.

Many organisms use chemotaxis to seek out food sources, avoid noxious substances, and find mates. Caenorhabditis elegans has impressive chemotaxis behavior. The premise behind testing the response of the worms to an odorant is to place them in an area and observe the movement evoked in response to an odorant. Even with the many available assays, optimizing worm starting location relative to both the control and test areas, while minimizing the interaction of worms with each other, while maintaining a significant sample size remains a work in progress (1-10). The method described here aims to address these issues by modifying the assay developed by Bargmann et al.(1). A Petri dish is divided into four quadrants, two opposite quadrants marked "Test" and two are designated "Control". Anesthetic is placed in all test and control sites. The worms are placed in the center of the plate with a circle marked around the origin to ensure that non-motile worms will be ignored. Utilizing a four-quadrant system rather than one 2 or two 1 eliminates bias in the movement of the worms, as they are equidistant from test and control samples, regardless of which side of the origin they began. This circumvents the problem of worms being forced to travel through a cluster of other worms to respond to an odorant, which can delay worms or force them to take a more circuitous route, yielding an incorrect interpretation of their intended path. This method also shows practical advantages by having a larger sample size and allowing the researcher to run the assay unattended and score the worms once the allotted time has expired.

Eph receptor signaling in C. elegans.

Eph receptor protein-tyrosine kinases are among the oldest known animal receptors and have greatly expanded in number during vertebrate evolution. Their complex transduction mechanisms are capable of bidirectional and bimodal (multi-response) signaling. Eph receptors are expressed in almost every cell type in the human body, yet their roles in development, physiology, and disease are incompletely understood. Studies in C. elegans have helped identify biological functions of these receptors, as well as transduction mechanisms. Here we review advances in our understanding of Eph receptor signaling made using the C. elegans model system.

A genome-wide functional screen shows MAGI-1 is an L1CAM-dependent stabilizer of apical junctions in C. elegans.

BACKGROUND: In multicellular organisms, cell-cell junctions are involved in many aspects of tissue morphogenesis. α-catenin links the cadherin-catenin complex (CCC) to the actin cytoskeleton, stabilizing cadherin-dependent adhesions. RESULTS: To identify modulators of cadherin-based cell adhesion, we conducted a genome-wide RNAi screen in C. elegans and uncovered MAGI-1, a highly conserved protein scaffold. Loss of magi-1 function in wild-type embryos results in disorganized epithelial migration and occasional morphogenetic failure. MAGI-1 physically interacts with the putative actin regulator AFD-1/afadin; knocking down magi-1 or afd-1 function in a hypomorphic α-catenin background leads to complete morphogenetic failure and actin disorganization in the embryonic epidermis. MAGI-1 and AFD-1 localize to a unique domain in the apical junction and normal accumulation of MAGI-1 at junctions requires SAX-7/L1CAM, which can bind MAGI-1 via its C terminus. Depletion of MAGI-1 leads to loss of spatial segregation and expansion of apical junctional domains and greater mobility of junctional proteins. CONCLUSIONS: Our screen is the first genome-wide approach to identify proteins that function synergistically with the CCC during epidermal morphogenesis in a living embryo. We demonstrate novel physical interactions between MAGI-1, AFD-1/afadin, and SAX-7/L1CAM, which are part of a functional interactome that includes components of the core CCC. Our results further suggest that MAGI-1 helps to partition and maintain a stable, spatially ordered apical junction during morphogenesis.

Select Neuropeptides and their G-Protein Coupled Receptors in Caenorhabditis Elegans and Drosophila Melanogaster.

The G-protein coupled receptor (GPCR) family is comprised of seven transmembrane domain proteins and play important roles in nerve transmission, locomotion, proliferation and development, sensory perception, metabolism, and neuromodulation. GPCR research has been targeted by drug developers as a consequence of the wide variety of critical physiological functions regulated by this protein family. Neuropeptide GPCRs are the least characterized of the GPCR family as genetic systems to characterize their functions have lagged behind GPCR gene discovery. Drosophila melanogaster and Caenorhabditis elegans are genetic model organisms that have proved useful in characterizing neuropeptide GPCRs. The strength of a genetic approach leads to an appreciation of the behavioral plasticity that can result from subtle alterations in GPCRs or regulatory proteins in the pathways that GPCRs control. Many of these invertebrate neuropeptides, GPCRs, and signaling pathway components serve as models for mammalian counterparts as they have conserved sequences and function. This review provides an overview of the methods to match neuropeptides to their cognate receptor and a state of the art account of neuropeptide GPCRs that have been characterized in D. melanogaster and C. elegans and the behaviors that have been uncovered through genetic manipulation.