Identification of a novel gene family involved in osmotic stress response in Caenorhabditis elegans

Abstract

Organisms exposed to the damaging effects of high osmolarity accumulate solutes to increase cytoplasmic osmolarity. Yeast accumulates glycerol in response to osmotic stress, activated primarily by MAP kinase Hog1 signaling. A pathway regulated by protein kinase C (PKC1) also responds to changes in osmolarity and cell wall integrity. C. elegans accumulates glycerol when exposed to high osmolarity, but the molecular pathways responsible for this are not well understood. We report the identification of two genes, osm-7 and osm-11, which are related members of a novel gene family. Mutations in either gene lead to high internal levels of glycerol and cause an osmotic resistance phenotype (Osr). These mutants also have an altered defecation rhythm (Dec). Mutations in cuticle collagen genes dpy-2, dpy-7, and dpy-10 cause a similar Osr Dec phenotype. osm-7 is expressed in the hypodermis and may be secreted. We hypothesize that osm-7 and osm-11 interact with the cuticle, and disruption of the cuticle causes activation of signaling pathways that increase glycerol production. The phenotypes of osm-7 are not suppressed by mutations in MAP kinase or PKC pathways, suggesting that C. elegans uses signaling pathways different from yeast to mount a response to osmotic stress.

Figure 1.—

Correlation between Dec and Osr phenotypes. N2 worms have altered cycle periods (P < 0.0001) on high-salt plates vs. normal NGM agar plates (50 mm NaCl). The defecation cycle period of all Osr mutants is longer than that of wild type (P < 0.0001). dpy-5 and dpy-13 have normal cycle periods. Average cycle periods are the unweighted average of all measured cycle periods. Error bars show the standard deviation.

Figure 2.—

Molecular identification of osm-7 and osm-11. (A) Genomic region of osm-7, with deletions and cosmids used for mapping, not to scale. (B) Genomic structure of osm-7 and osm-11, showing locations of signal sequences (s.s., red), OSR domains (blue), and sequenced mutations. (C) CLUSTAL alignment of the osm-7 OSR domain with its closest relatives from C. elegans and C. briggsae. Conserved regions of the OSR domain are boxed in blue.

Figure 3.—

Expression of osm-7∷GFP in the hypodermis. Nomarski (A and C) and fluorescence (B and D) images of two adult animals are shown. (A and B) The head region of one animal, with the anterior and posterior bulbs of the pharynx marked by closed arrows. Hypodermal cells expressing GFP are visible along the edges of the animal, surrounding the pharynx (B). (C and D) The midsection of another animal, with GFP-expressing hypodermal cells. Both images are at the top focal plane of the animal, so that the hypodermis is in focus but the intestine and other internal structures are not visible. The seam cells, which are not readily apparent in C, become visible as non-GFP cells along the center of the animal (open arrows) in D.