Extracellular matrix regulation of stress response genes during larval development in Caenorhabditis elegans

Abstract

Mutation or loss of 6 extracellular matrix collagen genes disrupts annular furrows in adult C. elegans cuticles, causes a wide "Dumpy" body morphology, and activates osmotic, detoxification, and antimicrobial defense genes. High environmental osmolarity reduces internal turgor pressure, physically distorts the epidermis, and activates the same stress responses. Collagen gene mutations that cause Dumpy without furrow disruption do not activate stress responses. These results are consistent with an extracellular damage sensor associated with furrows in the adult cuticle that regulates environmental stress responses in adjacent cells. Several cuticle characteristics change between molts, but all stages have annular furrows and express furrow collagen genes. We compared body shape, furrow organization imaged with differential interference contrast microscopy, and stress response gene expression in furrow collagen gene mutants at all postembryonic stages. We find that most body shape and furrow disorganization phenotypes start at the L3 stage and increase in severity with each molt afterwards. Stress response genes were induced the strongest in adults, correlating with the greatest Dumpy and furrow phenotypes. Although weaker than in adults, osmolyte transporter gene hmit-1.1 and antimicrobial gene nlp-29 were also induced in some early larvae that had weak or undetectable cuticle phenotypes. Our data are consistent with progressive cuticle phenotypes in which each new cuticle is at least partially directed by organization of the former cuticle. Gene expression and cuticle data support the role of furrow disruption as a signal in L4 larvae and adults, but also suggest a role for other cuticle organization or epidermal cell effects in early larvae.

Keywords: Caenorhabditis elegans; development; extracellular matrix; stress response.

Fig. 1.

Cuticles are more resistant to furrow collagen gene mutations in early larvae than adults. a) Mean width/length ratios at each developmental stage plus SE; N = 10–18 individual worms. b) Percentage of worms with clearly organized furrows visible with high magnification DIC at each developmental stage, N = 10–39 worms. c) Mean density of furrows per 10 µm plus SE, N = 10–23 individual worms; furrows were not organized enough to measure density for dpy worms at L4 or young adult (YA) stages. *P < 0.05, **P < 0.01, and ***P < 0.001 vs N2 worms on 51 mM NaCl.

Fig. 2.

Furrow organization is disrupted in L4 and adult dpy-7 worms. Representative micrographs of cuticles in N2 and dpy-7 worms taken at 120× magnification. Scale bars are 5 µm.

Fig. 3.

Mean relative mRNA levels plus SE for a) gpdh-1, b) hmit-1.1, c) nlp-29, and d) gst-4 in all conditions measured at all developmental stages. Values are normalized to 1.0 for N2 worms grown at 51 mM NaCl. N = 4–12 samples of 6–15 worms per replicate. *P < 0.05, **P < 0.01, and ***P < 0.001 vs N2 at the same stage using Welch’s t-tests for uneven variances and Benjamini–Hochberg corrections on log base 2 relative expression values.

Fig. 4.

Localization of DPY-7::GFP. a) Percentage of worms DPY-7::GFP localized to furrows in N2, dpy-3, and dpy-9 worms. b) Representative fluorescence images at all postembryonic stages. Images of the same worms with DIC are in Supplementary Fig. 2. Scale bars are 10 µm.