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doi: 10.1242/10.1242/dev.00497

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The C. elegans G-protein-coupled receptor SRA-13 inhibits RAS/MAPK signalling during olfaction and vulval development

Zoologisches Institut, Universitaet Zurich, Winterthurerstrasse 190, CH 8057, Zurich, Switzerland

View larger version (46K): [in a new window]   Fig. 1. Identification of sra-13. (A) Physical map of the dpy-10 unc-4 interval on chromosome II. The YAC and cosmid clones tested are shown. The three long-PCR fragments derived from the cosmid F49E12 that were tested are shown at the bottom. The F49E12.4, F49E12.5 (sra-13) and F49E12.6 PCR products spanned positions 27,000 to 30,140; 29,500 to 33,085; and 34,600 to 38,300 in the F49E12 cosmid sequence, respectively. `-' indicates no suppression and `+' indicates suppression of the Muv phenotype. (B) Sequence alignment of SRA-13 with SRA-11 (gi2500866), SRA-4 (gi1176649) and SRA-6 (gi1176651). Conserved residues are indicated by the grey shading. The regions underlined in bold are the predicted transmembrane domains. The broken line indicates the extent of deletion in the sra-13 open reading frame in the zh13 allele. (C) Dendogram of the SRA family of GPCRs calculated with the neighbour-joining method using the CLUSTALX software (Thompson et al., 1997).

View larger version (31K): [in a new window]   Fig. 3. sra-13 inhibits the RAS/MAPK pathway during olfaction. The chemotaxis indices were calculated as described in the Materials and Methods. An index of 0 indicates no attraction and 1 indicates maximal attraction. The error bars indicate the standard deviation of the mean observed in at least three independent trials. `with HS' in I,K indicates that young adult animals were heat shocked twice at 33°C for 30 minutes with a 3 hour interval and chemotaxis was assayed after a 4 hour recovery period. Alleles used: sra-13(zh13), let-60(n2021), lin-45(sy96), mek-2(n2678), let-60(n1046gf), gpa-2(pk16), gpa-3(pk35), gpa-5(pk376), gpa-7(pk610).

View larger version (40K): [in a new window]   Fig. 2. SRA-13 expression pattern. (A) Expression of the sra-13::gfp translational reporter in the two pairs of AWA and AWC chemosensory neurones in the head of an adult animal (arrows). A 3D reconstruction of confocal sections through the head region is shown. (B) Expression of the sra-13::gfp transcriptional reporter in the body wall muscles and hypodermis of an L4 animal and (C) in the vulval muscles that control egg-laying in the adult hermaphrodite. (D) Nomarski image of the animal shown in C. Scale bars: 10 µm in A,D; 50 µm in B.

View larger version (52K): [in a new window]   Fig. 4. DP-MPK-1 staining in AWC after stimulation with isoamlyalcohol. (A-C) Wild type; (D-F) let-60(rf); (G-I) sra-13(0); let-60(rf) animal. DP-MPK-1 staining in A,D,G is shown in red, GFP staining in B,E,H is in green. (C,F,I) Merged images. Anterior is towards the left and dorsal is upwards in all panels. The arrows indicate the AWC neurones. The frequency at which DP-MPK-1 staining was observed in each strain is given on the right. (n) refers to the number of animals scored. Scale bar: 10 µm.