Transcriptional profiling of Caenorhabditis elegans nematodes treated with L4440 (Empty Vector - EV) or grd-1 RNAi from L1 hatch, and collected at L3 of development.

Hedgehog morphogen GRD-1 regulates C. elegans growth and metabolism downstream of TOR complex 2. Both Hedgehog (Hh) signaling and target of rapamycin complex 2 (TORC2) are evolutionarily conserved pathways that regulate growth and metabolism. In C. elegans, loss of Hh morphogens often leads to developmental arrest, while loss of essential TORC2 component RICTOR (rict-1) causes delayed development, reduced brood, small size, increased fat, and shortened lifespan. Here we report that knockdown of Hh-related morphogen grd-1 causes developmental acceleration rather than arrest by speeding up molting transitions. Further, we show that RNAi to grd-1 not only suppresses the slow growth of rict-1 mutants, but also normalizes multiple abnormalities downstream of TORC2 including restoration of lifespan to near normal and partial rescue of low brood, small body size, and increased fat. Mechanistically, grd-1 slows growth downstream of TORC2 at least in part by increasing transcription of paraquat mediator 1 (PQM-1) target genes. Given the important role of TORC2 in governance of development, these data implicate grd-1 and pqm-1 as critical executors of organismal growth slowing in response to unfavorable growth conditions downstream of the nutrient sensor TORC2 in C. elegans. Overall design: Three independent passages of wildtype N2 animals were bleach synchronized and grown from larval stage 1 (L1) of development to larval stage 3 (L3) of development on three independently grown RNAi cultures of HT115 E. coli bacteria expressing L4440 (Empty Vector - EV Control) or grd-1 dsRNA to induce RNAi knockdown. Animals were collected via manual inspection at the L3 stage, and snap frozen for lysis, RNA extraction and downstream mRNA sequencing analysis. This experiment contains 3 control and 3 grd-1 knockdown replicates, for a total of 6 samples.