MicroRNAs Strengthen Gene Regulatory Networks to Maintain Phenotypic Robustness throughout Critical Periods of Genetic and Environmental Change

Animal development normally completes with a remarkably low rate of failure. Canalizing processes lead development into the proper directions. For example, gene regulatory networks parse signals from noise to reproducibly produce discrete phenotypes. A class of genes called microRNAs (miRNAs) might help facilitate the process. Yet surprisingly little is known about how miRNAs confer robustness to gene regulatory networks or the types of stresses they might be able to buffer. Here I examine how miRNAs prevent phenotypic failure against genetic, evolutionary, and environmental perturbations.;To start, I explored how miRNAs wire into gene regulatory network motifs. I placed microRNA-7 into a positive feedback loop by genetically perturbing individual network components and observing how gene expression changes reverberated through the altered networks. This positive feedback loop locks-in stable cell fates even in the case of weak or transient inductive signals by preventing reversion once critical activity thresholds are reached.;The rate of evolution might change if gene regulatory networks are buffered over evolutionary timescales. This would occur through minimizing the phenotypic expression of selectable genetic variation. Here I implicate miRNAs in the process by lowering microRNA-9a dose and observing significantly altered response rates to artificial selection as measured through quantitative genetics. Artificially selected miR-9a mutant and wild type genomes were deep sequenced to detect the genomic regions that responded to selection. Wild type lines absorbed the accumulated cryptic genetic variation, but this was unmasked in miR-9a mutants to cause phenotypic change.;Paradoxically, animals mutant for conserved miRNAs such as miR-7 or miR-9a often fail to exhibit overt phenotypes. I conducted a bioinformatic meta-analysis of miR-7 target genes but uncovered few targets conserved between flies and humans. Thousands were species specific. I hypothesized that some miRNAs might only be essential during periods of environmental stress. When miRNA mutants were perturbed with temperature stress, the robust patterning of the peripheral nervous system was disrupted. This suggests that miRNAs can confer environmental canalization during development.;I conclude that microRNAs canalize gene regulatory networks against genetic, evolutionary, and environmental perturbations to minimize developmental fail rates. This promotes phenotypic robustness on developmental and evolutionary timescales.