Temporal changes in PTTH/Egf signaling and ERK target gene expressions during chilling-induced diapause termination in Bombyx mori eggs

Activation of extracellular signal-regulated kinase (ERK) signaling is related to chilling-induced diapause termination in Bombyx mori eggs. However, the changes in expressions of upstream signaling and downstream target genes are not very clear. In the present study, I investigated temporal changes in gene expressions of the prothoracicotropic hormone (PTTH) and epidermal growth factor (Egf) signaling pathways, which are the major stimulators of ERK signaling in insect systems, during a long chilling period. My results showed that when chilling was conducted from 2 days post-oviposition, expressions of PTTH/Egf signaling pathway genes were maintained at relatively higher levels, compared to those kept at 25 °C. In eggs exposed to 5 °C from 15 days post-oviposition, expression of the PTTH gene had dramatically increased by day 45 after chilling. Egf signaling pathway genes, including spitz (spi), vein (vn), rhomboid (rho), and Egf receptor (Egfr), showed gene-specific increasing patterns during the chilling period. I further showed that expression of the pointed (an ERK target) gene was maintained at a relatively higher when chilling was conducted from 2 days post-oviposition. Expression of the pointed gene had dramatically increased by day 45 in eggs chilled beginning after 15 days post-oviposition. Additionally, dechorionation increased pointed gene expression. LY294002 (a specific phosphatidylinositol 3-kinase (PI3K) inhibitor) and U0126 (an ERK inhibitor) inhibited pointed gene expression in dechorionated eggs, indicating that both PI3K and ERK are involved in increased pointed gene expression. When eggs that had been kept at 5 °C for 30 days were transferred to 25 °C, expression levels of PTTH and pointed genes decreased after transfer, clearly indicating that continuous chilling for 45 days is necessary to sustain the higher expressions of these genes. To the best of my knowledge, this study presents the first comprehensive analysis of the transcriptional regulation of both upstream and downstream genes in the ERK signaling pathway during a chilling period, offering valuable insights into the mechanisms underlying chilling-induced diapause termination.