Loss of dpy-2 and dpy-9 has stage-specific effects on DBL-1 pathway signaling

Loss of some cuticle collagens negatively affects DBL-1 pathway signaling in a stage-dependent manner (Lakdawala et al. 2019; Madaan et al. 2019). We previously observed that in one-day old adult animals, loss of dpy-2 or dpy-9 had no effect on GFP::DBL-1 expressed from the dbl-1 promoter (Beifuss and Gumienny 2012; Lakdawala et al. 2019). We also observed that expression of spp-9p::gfp, a reporter that is negatively regulated by the DBL-1 pathway, was not affected in one-day old adult animals (Roberts et al. 2010; Lakdawala et al. 2019). Post-embryonic expression of dpy-2 and dpy-9 is highest in L2 and L3, but low in L4 and even lower in young adults (Gerstein et al. 2010). Because cuticle secreted in one stage creates the cuticle in the next stage, this is consistent with the observation that loss of dpy-2 and dpy-9 has no effect on DBL-1 signaling in the adult (Hall and Altun 2008; Lakdawala et al. 2019). However, the DPY-2 and DPY-9 expression patterns led us to ask if DBL-1 signaling is affected at L4 by loss of dpy-2 or dpy-9. To our surprise, we found that dpy-2(e8) or dpy-9(e12) resulted in significant increases of GFP::DBL-1 fluorescence within DBL-1-secreting cells in L4 animals compared to control populations (Figure 1, Table 1). We also tested DBL-1 pathway reporter activity in these dpy-2 and dpy-9 mutants. Consistent with the increased GFP::DBL-1 fluorescence at L4, we observed significantly decreased fluorescence from the spp-9p::gfp reporter at L4 (Figure 1, Table 1). These results are consistent with DPY-2 and DPY-9 affecting DBL-1 signaling at the L4 stage but not at the adult stage. This suggests that these two collagens have a stage-specific effect on DBL-1 signaling, but this effect is normally inhibitory, as loss of dpy-2 or dpy-9 increased GFP::DBL-1 fluorescence and decreased spp-9p::GFP fluorescence.