Abstract
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.
Highlights
Insulin-like signaling contributes to homeostasis in multi-cellular animals by mediating physiological responses to environmental conditions through systemic signaling
Insulin-like signaling regulates an acute form of developmental arrest that occurs in response to complete starvation (L1 arrest) [4,5]
The objectives of this study were to determine the feasibility of using the nCounter platform for insulin-like mRNA expression analysis in C. elegans and to provide an overview of insulin-like gene expression
Summary
Insulin-like signaling contributes to homeostasis in multi-cellular animals by mediating physiological responses to environmental conditions through systemic signaling. Insulin signaling regulates carbohydrate metabolism, and insulin-like growth factor signaling controls growth. Insulinlike signaling regulates growth and metabolism as well as other aspects of developmental physiology [1]. In the nematode C. elegans, insulin-like signaling regulates formation of a stress resistant, non-feeding developmental alternative to the third larval stage known as the dauer larva [2]. Insulin-like signaling regulates an acute form of developmental arrest that occurs in response to complete starvation (L1 arrest) [4,5]. Insulin-like signaling regulates adult lifespan in C. elegans [6,7,8,9], as well as the fly Drosophila melanogaster and mammals [10,11,12]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
