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

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.

RNAseq Analysis of the Parasitic Nematode Strongyloides stercoralis Reveals Divergent Regulation of Canonical Dauer Pathways

“…These results are consistent with findings in C. elegans , where IIS signaling is thought to be regulated at the level of the ILPs, while intracellular signaling components are always present [41]. However, we did observe developmental regulation of several IIS signaling component transcripts in the post-free living generation, including increases in the transcript abundances for Ss-daf-2 , Ss-age-1 , Ss-pdk-1 , Ss-akt-1 , and Ss-daf-16 (Figure S8).…”

The Tumor Suppressor Rb Critically Regulates Starvation-Induced Stress Response in C. elegans

“…Because both genes were shown to be up-regulated during L1 diapause and in dauer larvae of wild-type animals [20], they may function as the IIS receptor antagonists. Their down-regulation in starved L1 lin-35/Rb(lf) mutants is consistent with Rb’s role in repressing the IIS pathway.…”

Complex expression dynamics and robustness in C. elegans insulin networks

“…6C). In a recent study it was reported that endogenous ins-7 is expressed at higher levels than ins-8 throughout development (Baugh et al 2011). ins-7 and ins-8 are less than 1 kb apart in the genome; therefore, in the ins-7(tm1907), the ins-8 gene resides in closer proximity to the (stronger) ins-7 promoter (data not shown).…”