Noncell- and cell-autonomous G-protein-signaling converges with Ca2+/mitogen-activated protein kinase signaling to regulate str-2 receptor gene expression in Caenorhabditis elegans

Abstract

In the sensory system of C. elegans, the candidate odorant receptor gene str-2 is strongly expressed in one of the two AWC neurons and weakly in both ASI neurons. Asymmetric AWC expression results from suppression of str-2 expression by a Ca2+/MAPK signaling pathway in one of the AWC neurons early in development. Here we show that the same Ca2+/MAPK pathway promotes str-2 expression in the AWC and ASI neurons together with multiple cell-autonomous and noncell-autonomous G-protein-signaling pathways. In first-stage larvae and adult animals, signals mediated by the Galpha subunits ODR-3, GPA-2, GPA-5, and GPA-6 and a Ca2+/MAPK pathway involving the Ca2+ channel subunit UNC-36, the CaMKII UNC-43, and the MAPKK kinase NSY-1 induce strong str-2 expression. Cell-specific rescue experiments suggest that ODR-3 and the Ca2+/MAPK genes function in the AWC neurons, but that GPA-5 and GPA-6 function in the AWA and ADL neurons, respectively. In Dauer larvae, the same network of genes promotes strong str-2 expression in the ASI neurons, but ODR-3 functions in AWB and ASH and GPA-6 in AWB. Our results reveal a complex signaling network, encompassing signals from multiple cells, that controls the level of receptor gene expression at different developmental stages.

Figure 1.—

str-2 expression is reduced in several Gα-mutants. In wild-type animals, str-2∷gfp is expressed at high levels in one AWC neuron (A), whereas more than half of the odr-3; gpa-6 mutants show reduced str-2∷gfp expression (B). Expression is partially restored in gpa-2 gpa-3 gpa-13 odr-3; gpa-5 gpa-6 mutants (C). unc-36 mutants show strong str-2∷gfp expression in both AWC neurons (D; Troemel et al. 1999). This expression is lost in unc-36; odr-3; gpa-5 gpa-6 animals (E), but restored in unc-36; gpa-2 gpa-3 gpa-13 odr-3; gpa-5 gpa-6 mutants (F). Bars, 20 μm. A–F are at ×400 magnification.

Figure 2.—

G-protein and Ca²⁺/MAPK genes together regulate str-2 expression in the AWC neurons. (A–C) In adult animals, ODR-3 and two or three other Gα-subunits (GPA-2, GPA-5, and GPA-6) and UNC-36, UNC-43, or NSY-1 are required for strong str-2 expression. GPA-3 and GPA-13 have inhibiting functions. (D) str-2 expression at different time points after egg laying at 20°. The corresponding developmental stage is indicated: the four larval stages, young adult (ya), and adult (a). Wild-type animals were indistinguishable from unc-36 mutants (not shown) and show strong expression throughout development, whereas unc-36; odr-3; gpa-5 gpa-6 mutants show delayed str-2 expression in L2 larvae, which is abolished in adult animals. odr-1 mutants lose expression following the L1 stage. (E) In adults, str-2 expression is restored when ODR-3 is reintroduced following heat shock (+) of unc-36; odr-3; gpa-5 gpa-6 animals carrying a hsp-16.2∷odr-3 transgene. Each bar represents at least 50 animals. Shown are the percentages of animals with strong str-2 expression in the AWC neurons (detectable at ×100 in A, B, C, and E and ×160 in D). The number of animals for each bar is, from left to right: (A) 98, 63, 63, 55, 74, 66, 52, 74, 51, 61, 61, 50; (B) 95, 97, 85, 99, 84, 75, 77, 114, 53, 105; (C) 32, 88, 51, 40, 86, 86, 123, 114, 60; (E) 55, 64, 81, 98. In D, each data point represents at least 27 animals. Error bars denote standard error of proportion. Shown are significant differences in A compared to unc-36 (*: P < 0.001), unc-36; odr-3 (+: P < 0.001), unc-36; odr-3; gpa-6 (o: P < 0.001), and unc-36; gpa-2 odr-3; gpa-5 gpa-6 (#: P < 0.001, ##: P < 0.05); in B compared to unc-43 (*: P < 0.001), unc-43; odr-3; gpa-5 gpa-6 (+: P < 0.001), nsy-1 (o: P < 0.001), and nsy-1; gpa-2 odr-3; gpa-5 gpa-6 (#: P < 0.001); in C compared to tir-1; odr-3; gpa-5 gpa-6 (**: P < 0.05), sek-1 (++: P < 0.05), and odr-3; gpa-5 sek-1 gpa-6 (oo: P < 0.05); in D compared to wild type (*: P < 0.001); and in E compared to control (*: P < 0.001).

Figure 3.—

Cell-specific expression of ODR-3, GPA-5, GPA-6, and UNC-36 restores str-2 expression in AWC. (A) ODR-3 restores str-2 expression when expressed in the AWC and ADF neurons. (B) GPA-5 restores str-2 expression when expressed in the AWA neurons. (C) GPA-6 restores str-2 expression when expressed in the ADL neurons. (D) UNC-36 expression in the AWC neurons strongly increases str-2 expression. Shown are the percentages of unc-36; odr-3; gpa-5 gpa-6 adult animals in which str-2∷gfp could be detected in the AWC neurons at ×100 magnification. The number of animals for each bar, from left to right, is: (A) 244, 106, 110, 92, 107, 133, 140; (B) 244, 121, 73, 120, 112; (C) 244, 100, 108, 156, 118, 164, 119, 117; and (D) 244, 103. Significant differences (*: P < 0.001, **: P < 0.05) compared to unc-36; odr-3; gpa-5 gpa-6 animals are shown. Additional results are presented in Tables S1–S4 at http://www.genetics.org/supplemental/.

Figure 4.—

ODR-3 and GPA-5 under control of cell-specific promoters are correctly expressed in the AWC, ADF, and AWA cells. Shown are antibody stainings of cilia in unc-36; odr-3; gpa-5 gpa-6 mutants that express ODR-3 or GPA-5 in a cell-specific manner. The contours of the head of each animal are indicated by a dotted line. Left is anterior. Bars, 5 μm. (A) Anti-ODR-3 staining of an unc-36; odr-3; gpa-5 gpa-6 animal expressing odr-3 under the gpa-13 promoter. ODR-3 is predominantly expressed in the cilia and cell bodies of the AWC cells, of which the wing-like cilia can be clearly distinguished. Occasionally, weak staining of the cilia of the ASH and/or ADF neurons was observed. (B) Anti-ODR-3 staining of an unc-36; odr-3; gpa-5 gpa-6 animal expressing odr-3 under the srh-142 promoter. ODR-3 is exclusively expressed in the dual cilia of the ADF neurons. (C) Anti-GPA-5 staining of an unc-36; odr-3; gpa-5 gpa-6 animal expressing gpa-5 under the odr-10 promoter. GPA-5 staining was observed exclusively in the cell bodies, axons, and clearly recognizable, branched cilia of the AWA neurons.

Figure 5.—

Cell-specific regulation of str-2 expression in the ASI neurons of Dauer larvae. (A) str-2 expression is reduced in the ASI neurons of unc-36; odr-3; gpa-5 gpa-6 Dauer larvae. (B) ODR-3 partially restores str-2 expression when expressed in AWB and ASH. (C) GPA-6 partially restores str-2 expression when expressed in AWB. (D) GPA-5 partially restores str-2 expression when expressed in AWA. (E) UNC-36 partially restores str-2 expression when expressed in AWC. Shown are the percentages of unc-36; odr-3; gpa-5 gpa-6 Dauer larvae with detectable str-2∷gfp in the ASI neurons at ×100 magnification. Only those cell-specific rescue experiments that gave considerable restoration of expression are shown (see Table S5 at http://www.genetics.org/supplemental/). Expression of odr-3 in AWA or ADF and gpa-5 and gpa-6 in ADL did not significantly restore str-2 expression (results not shown). The number of animals for each bar is, from left to right: (A) 76, 25, 107; (B) 76, 65, 38, 50, 48; (C) 76, 66, 41, 37, 56; (D) 76, 42, 67; and (E) 76, 40. Significant differences (*: P < 0.001, **: P < 0.05) compared to unc-36; odr-3; gpa-5 gpa-6 animals are shown.

Figure 6.—

Models for regulation of str-2 expression. (A) In the AWC neurons in adults, G-protein-mediated signals from the AWA, ADL, and ADF neurons converge with cell-autonomous G-protein signals and the Ca²⁺/MAPK pathway to regulate str-2 expression. The cells in which GPA-3 and GPA-13 function have not been identified, but the AWC neurons are likely candidates. (B) In Dauer larvae, signals from the AWA, AWB, ASH, and possibly AWC and/or ADF neurons regulate str-2 expression in the ASI neurons. (C) A model of the genetic pathway that regulates str-2 expression. The G-protein/Ca²⁺/MAPK network probably signals partially via daf-3. odr-1 is epistatic to the G-protein/Ca²⁺/MAPK network, indicating a parallel or downstream function. The position of osm-9 has been extrapolated from its presumed cellular function.