Increased expression of pgph-1, T23F2.4, and cyp-14A5 in C. elegans dpy-7 mutants and by high salt

Extracellular matrices (ECM) are ubiquitous features of metazoan tissues that have structural and signaling functions (Hay 1981). The cuticle of C. elegans is a complex and flexible ECM composed of over 100 cross-linked collagen fibers secreted by underlying epidermal cells. In addition to functioning as flexible exoskeletons, nematode cuticles act as barriers, providing the first line of defense against environmental stressors such as toxins, water imbalances, and pathogens (Page and Johnstone 2007). It was previously reported that loss or mutation of some cuticle collagens caused constitutive activation of genes that are normally induced by environmental stressors such as high osmolarity and pathogens (Lamitina et al. 2006, Pujol et al. 2008, Choe 2013, Zugasti et al. 2016). We recently demonstrated that this effect is restricted to six collagens required for formation of circumferential bundles termed annular furrows and to activation of three conserved stress responses (Dodd et al. 2018). These results are consistent with an ECM damage sensor that is associated with annular furrows and signals to specific downstream environmental response pathways. DPY-7 is one of the annular furrow collagens (Cox et al. 1980, McMahon et al. 2003, Thein et al. 2003). We recently used RNAseq analysis to identify candidate genes that may be activated by dpy-7 mutation; these genes may contribute to stress responses when the cuticle is disrupted (Dodd et al. 2018). In the current study, we used quantitative RT-PCR to measure expression of stress response-related genes in dpy-7(e88) worms; mRNA levels of each potential stress response gene were normalized to rpl-2, which encodes a ribosomal protein. We selected genes based on predicted functions in stress responses and significant activation in dpy-7 worms in prior RNAseq analyses (Dodd et al. 2018). Twelve biological replicates were measured for each strain, and each RNA replicate sample was isolated from either 10 N2 control or 20 dpy-7 first day adult worms grown on agar seeded with OP50 bacteria. As shown in Figure 1A, pgph-1, T23F2.4, and cyp-14A5 were significantly upregulated in dpy-7 mutants compared to N2 in the absence of environmental stress. We also measured these mRNAs in worms exposed to high salt to determine if they are induced by a relevant environmental condition (Lamitina et al. 2006, Choe 2013, Dodd et al. 2018). As shown in Figure 1B, pgph-1, T23F2.4, and cyp-14A5 were induced by 24 h exposu