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

Abstract

Mutation or loss of six 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 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 DIC microscopy, and stress response gene expression in three furrow collagen gene mutants at all post-embryonic 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.

RNAi screening for modulators of an osmo-sensitive gene response to extracellular matrix damage reveals negative feedback and interactions with translation inhibition

“…2 , hars-1 , pgph-3 , or rars-1 dsRNA clones are shown in S1 Fig ; relative gpdh-1p :: dsRed2 fluorescence generally matched relative gpdh-1 mRNA changes. Loss of furrows from the cuticle can be observed with high magnification DIC [ 34 , 44 ]; as shown in S2 Fig , furrows were not restored by any of these dsRNA clones. Furthermore, the associated Dpy (short and wide) body shape phenotype [ 34 ] was not suppressed or enhanced by any of these dsRNA clones ( S3 Fig ).…”

“…Loss of furrows from the cuticle can be observed with high magnification DIC [ 34 , 44 ]; as shown in S2 Fig , furrows were not restored by any of these dsRNA clones. Furthermore, the associated Dpy (short and wide) body shape phenotype [ 34 ] was not suppressed or enhanced by any of these dsRNA clones ( S3 Fig ).…”

“…Strong induction of hmit-1 . 1 by high osmolarity and furrow loss suggests that myo-inositol also has a role [ 31 , 34 , 47 ]. Under conditions that induce osmotic stress responses, RNAi of hmit-1 .…”

“…Furrows were imaged by differential interference contrast (DIC) on the same microscope. Body shape was measured as described previously [ 34 ].…”

“…We discovered that loss of any one of a subset of six dpy genes ( dpy-2 , 3 , 7 , 8 , 9 , or 10 ) activates broad osmotic, innate immune, and detoxification stress responses without activating heat shock or organelle-specific stress responses [ 31 ]. Loss or mutation of any one of these six genes disrupts organization of circumferential bands of collagen termed ‘annular furrows’ and at least one encodes a collagen localized to furrows [ 31 – 34 ]. These results are consistent with furrows being part of, or interacting with, a sensor for cuticle damage that regulates three environmental stress responses related to barrier defenses.…”