Article Figures & Data
Figures
- Figure 1.
GFP reporter transgenes for 26 neurotransmitter GPCRs. A, Small-molecule neurotransmitter GPCRs in C. elegans. B, Schematics of GFP reporters used in this study. C-terminal::GFP constructs are illustrated by the reporter for the orphan receptor ADOR-1. The construct provides 21 kb of promoter region DNA 5′ to the coding exons and 9 kb of 3′ DNA. GFP coding sequences were inserted into the last ADOR-1 coding exon so that the transgene expresses the receptor with GFP fused to its C-terminus. SL2::GFP constructs are illustrated by the dopamine receptor DOP-1 reporter. This reporter is similar to that for ADOR-1, except that an SL2 trans-splicing signal followed by GFP coding sequences were inserted immediately downstream of the stop codon so that this reporter coexpresses the receptor and GFP as separate proteins. Analogous schematics of all 26 receptor::GFP reporter transgenes are found in Extended Data Figure 1-1 and described in Extended Data Figure 1-2.
- Figure 2.
Anatomy of the egg-laying system visualized with mCherry markers. A, Schematic of the C. elegans egg-laying system. Neuron and muscle cells (colored and labeled with cell names) are overlaid on the uterus (gray) containing eggs (white). VC4 and VC5 neurons are at the ventral midline. All other cells depicted are on the left side of the animal, and their equivalents on the right side of the bilaterally symmetric anatomy are not shown. B, Schematic of the egg-laying neurons. C, Confocal image of the egg-laying neurons labeled by the ida-1::mCherry marker. Dashed lines outline the cell bodies. D, Schematic of the egg-laying muscles. E, Confocal image of the egg-laying muscles labeled by the unc-103e::mCherry marker. um1 (outlined) and um2 (not indicated) are rarely and faintly labeled by unc-103e::mCherry; but even so, labeling of vm1/vm2 cells provides landmarks to help identify um1 and um2. F, Schematic of cells other than neurons and muscles of the egg-laying system. Red dashed or solid lines indicate cell junctions labeled by the ajm-1::mCherry marker. In addition to cells of the egg-laying system, the ajm-1::mCherry marker also labels junctions of seam and intestinal cells. G, Confocal image of the egg-laying system labeled by the ajm-1::mCherry marker. The following conventions are used in this and other figures in this work. Scale bars, 10 µm. Anterior is left and ventral down. du, Dorsal uterine cell; sp, spermatheca; um, uterine muscle cell; ut, uterine toroid cell; utse, uterine seam cell; uv, uterine ventral cell; vm, vulval muscle cell; vt, vulval toroid cells. All C. elegans strains used in this work are described in Extended Data Figure 2-1.
- Figure 3.
Scoring expression of a GFP reporter for the octopamine receptor OCTR-1 in cells of the egg-laying system. A, B, Two-dimensional renderings of a confocal image of the egg-laying system in an octr-1::gfp; ajm-1::mCherry young adult showing GFP (A) or GFP and mCherry fluorescence (B). The ut2 uterine toroid cells are outlined by the ajm-1::mCherry marker, allowing them to be identified as GFP-positive despite not being uniformly filled out by OCTR-1::GFP. Solid line with arrowheads labels the ventral nerve cord (vnc), a bundle of axons that run past the vulva but that are not known to affect egg laying. In these and other figure panels, white asterisks indicate position of the vulval opening, cells labeled with both GFP and mCherry are named with yellow text, cells labeled with GFP only are named with green text, the number of + symbols indicates the relative intensity of GFP labeling, and identified cells are outlined by faint dotted lines. B, White box represents the region shown at higher magnification in C-F. C, D, An octr-1::gfp; ida-1::mCherry animal with GFP fluorescence displayed at high gain to make ++ GFP labeling visible in the HSN and VC4/VC5 neurons. These neurons are double-labeled by ida-1::mCherry in D. E, F, An octr-1::gfp; unc-103e::mCherry animal with GFP fluorescence displayed at high gain and at a different focal plane than in C, D to visualize + GFP labeling in vm1 and vm2 egg-laying muscles. Movie 1 is a video that illustrates in more detail how octr-1::gfp strains were analyzed. Extended Data Figure 3-1 illustrates how animal-to-animal variations in GFP expression were analyzed.
- Figure 4.
Examples of neurotransmitter GPCR::GFP expression in neuronal, muscle, and other cells of the egg-laying system. A-H, Examples of GPCR::GFP expression in neurons of the egg-laying system. Top panel of each image pair shows reporter GFP reporter fluorescence for the receptor named in green, with all GFP-labeled cells of the egg-laying system indicated by white cell names and arrowheads. Bottom, GFP plus the ida-1::mCherry fluorescence used to confirm identity of egg-laying neurons, with only the double-labeled neurons indicated by yellow names and white arrowheads. I-P, Examples of GPCR::GFP expression in muscle cells of the egg-laying system. Images are labeled as in A-H, but bottom panels in each pair show the unc-103e::mCherry fluorescence used to confirm identity of muscle cells. mCherry labeling of um1 is not visible in J. Q-V, Examples of GPCR::GFP expression in cells of the egg-laying system other than neurons and muscles. Images are labeled as in A-H, but bottom panels in each pair show ajm-1::mCherry fluorescence. sp, ut, utse, uv3, and vt cells are identified by their outlines visualized with ajm-1::mCherry. Extended Data Figure 4-1 presents examples of GPCR::GFP expression for all 26 neurotransmitter GPCRs.
- Figure 5.
Summary of neurotransmitter GPCR::GFP expression in the C. elegans egg-laying system. A, Summary of neurotransmitter GPCR::GFP expression by cells in and near the egg-laying system. Dark green represents GFP expression that was on average strong (scored as +++, among brightest of any cell in the animal) or easily detectable (++). Light green represents weak GFP expression (+, just above background in at least 2 different animals). White represents cells in which GFP was not reproducibly observed. v, Variable (denotes cells in which GFP detected in more than two but less than half of animals observed). bwm, Body wall muscles; sp-ut, spermathecal-uterine valve. B, Number of strongly and weakly expressed receptors in each cell type of the egg-laying system. Receptors with variable expression are included. Totals are shown broken down by the Gα protein thought to be activated by the receptors in each cell type when that information is known. C, Neurons and muscles of the egg-laying system that express neurotransmitter GPCRs, indicating which cells have the corresponding neurotransmitter synaptically released onto them (synaptic target cells) or not (extrasynaptic target cells), based on the connectome (Albertson and Thomson, 1976; White et al., 1986; Xu et al., 2013) and neurotransmitter maps (Serrano-Saiz et al., 2013; Pereira et al., 2015; Gendrel et al., 2016). Cell names in dark/light green represent strong/weak receptor expression. Italics indicate that receptor expression is variable. Extended Data Figure 5-1 presents a more detailed summary of our GPCR::GFP expression results, including comparison with previously published work and details of variability in GFP expression.
- Figure 6.
Neurotransmitter GPCRs knockouts rarely reveal egg-laying defects. A, Micrographs of animals with egg-laying phenotypes. WT adults (top) typically accumulate 15-20 unlaid eggs, whereas hyperactive egg-laying mutants, such as like goa-1 (second from top), accumulate fewer unlaid eggs and egg-laying defective mutants, such as egl-10 (third from top), accumulate more unlaid eggs. Eggs from WT animals have developed to the ∼100 cell stage by the time they are laid (top right), whereas eggs from hyperactive egg-laying mutants are laid earlier, often with fewer than eight cells (bottom right). B, Number of unlaid eggs for neurotransmitter GPCR single knockout strains. egl-10(md176) and goa-1(n1134) served as controls (these control data are replotted in Fig. 7C and Extended Data Fig. 6-1B). n ≥ 30 for each strain. *Statistical significance (p < 0.05) compared with the WT using one-way ANOVA with Bonferroni correction for multiple comparisons. Significant p values were as follows: goa-1 (p < 0.0001); egl-10 (p < 0.0001); ser-5 (p = 0.0002); dop-4 (p = 0.0004); tyra-2 (p = 0.001); gar-3 (p = 0.0021); gbb-1 (p = 0.0253); gbb-2 (p < 0.0001); mgl-1 (p < 0.0001); ador-1 (p < 0.0001); F35H10.10 (p < 0.0001); dop-5 (p = 0.0001); and dop-6 (p < 0.0001). Error bars indicate 95% CIs. Here and in other graphs in this work, different colors are used to plot data for receptors for different neurotransmitters, or for the orphan GPCR class. C, Percent of early-stage eggs laid by neurotransmitter GPCR gene single knockout strains. C, D, goa-1(n1134) is used as a control hyperactive egg-laying mutant (these control data are replotted in Fig. 6D and Extended Data Fig. 7-1A). n = 100 eggs per strain. *Statistical significance (p < 0.05) compared with the WT using the Fisher's exact test. Significant p values were as follows: goa-1 (p < 0.00001); ser-5 (p = 0.0349); and F35H10.10 (p = 0.0349). The Wilson-Brown method was used to determine the 95% CI for binomial data. D, Percent of early-stage eggs laid by strains carrying combinations of KOs in genes encoding Gαo-coupled neurotransmitter GPCRs. goa-1(n1134) is used as a control hyperactive egg-laying mutant. Red “X” identifies the receptors knocked out in a given strain. Statistical analysis was as in C. The five significant p values (for measurements from left to right that are denoted with asterisks) were p < 0.00001, p = 0.0101, p = 0.0055, p < 0.00001, and p = 0.0184. Extended Data Figure 6-1 presents further analysis of neurotransmitter GPCR knockout strains.
- Figure 7.
Neurotransmitter GPCR overexpressors reveal strong egg-laying defects. A, Strategy to reveal and validate egg-laying defects caused by increased signaling through overexpressed neurotransmitter GPCRs. B, Overexpression of the serotonin GPCR SER-1 (ser-1(oe)) causes hyperactive egg-laying that is suppressed by knocking out serotonin biosynthesis with a null mutant in the tryptophan hydroxylase enzyme (TPH-1). n = 100 eggs per strain. Error bars indicate 95% CIs determined by the Wilson-Brown method. *Statistical significance (p < 0.05) compared with the WT (Fisher's exact test). p values for the two asterisked comparisons were as follows: p < 0.00001 (left comparison) and p < 0.00001 (right comparison). C, Number of unlaid eggs for neurotransmitter GPCR gene overexpressor strains. egl-10(md176) and goa-1(n1134) served as controls. n ≥ 30 for each strain. Error bars indicate 95% CIs. *p < 0.05, compared with the WT (one-way ANOVA with Bonferroni correction for multiple comparisons). p values for the comparisons to the WT were as follows: goa-1 (p < 0.0001); egl-10 (p < 0.0001); ser-1(oe) (p = 0.0011); ser-5(oe) (p = 0.0025); ser-7(oe) (p = 0.0189); dop-2(oe) (p < 0.0001); dop-3(oe) (p < 0.0001); dop-4(oe) (p = 0.0019); octr-1(oe) (p = 0.0223); ser-6(oe) (p < 0.0001); ser-2(oe) (p = 0.009); tyra-2(oe) (p < 0.0001); gar-1(oe) (p < 0.0001); gar-2(oe) (p < 0.0001); gbb-1(oe) (p < 0.0001); gbb-2(oe) (p < 0.0001); mgl-1(oe) (p < 0.0001); mgl-3(oe) (p < 0.0001); ador-1(oe) (p = 0.0006); dop-5(oe) (p < 0.0001); and dop-6(oe) (p = 0.0389). D, Overexpression of the glutamate GPCR MGL-1 causes defective egg laying that is suppressed by knocking out glutamate signaling with a mutation in the vesicular glutamate transporter EAT-4. D-G, n ≥ 30 for each strain. Error bars indicate 95% CIs. *p < 0.05 (one-way ANOVA with a Tukey's test to determine statistical significance for multiple comparisons). p values for the two asterisked comparisons in D were as follows: p < 0.0001 (left comparison) and p < 0.0001 (right comparison). E, Overexpression of the glutamate GPCR MGL-3 causes defective egg laying that is suppressed by knocking out glutamate signaling with a mutation in the vesicular glutamate transporter EAT-4. p values for the two asterisked comparisons were as follows: p < 0.0001 (left comparison) and p < 0.0001 (right comparison). F, Overexpression of the tyramine GPCR SER-2 causes defective egg-laying that is suppressed by knocking out the tyramine biosynthesis enzyme tyrosine decarboxylase (TDC-1). p values for the two asterisked comparisons were as follows: p < 0.0001 (left comparison) and p < 0.0001 (right comparison). G, Overexpression of the octopamine GPCR SER-6 causes defective egg laying that is suppressed by knocking out the octopamine biosynthesis enzyme tyramine β-hydroxylase (TBH-1). p values for the two asterisked comparisons were as follows: p < 0.0001 (left comparison) and p = 0.0257 (right comparison). Extended Data Figure 7-1 presents additional validation tests of neurotransmitter GPCR overexpression phenotypes.
Movies
- Movie 1.
Video of methods used to score expression of a GFP reporter for the octopamine receptor OCTR-1 in cells of the egg-laying system. The video illustrates how images of the egg-laying system were analyzed in three dimensions to identify GFP-expressing cells and score GFP expression levels.
Figure 1-1
GFP reporters for 26 neurotransmitter GPCRs. Schematic of GFP reporters used in this study. For all reporters, the lengths of the 5' promoter and 3' downstream regions are indicated in kilobases (kb). Coding exons are indicated by gray boxes, predicted transmembrane domain coding regions by yellow boxes, GFP coding regions by a green box, and the SL2 trans-splicing signal region by an orange box. Flanking genomic DNA is indicated in red. Introns 1kb or less are drawn to scale with a black line. Introns greater than 1 kb are not drawn to scale and indicated with slashed black lines. C-terminal::GFP constructs were used for 19 neurotransmitter receptors and express a receptor with GFP fused to its C-terminus. SL2::GFP constructs were used for four neurotransmitter receptors: an SL2 trans-splicing signal followed by GFP coding sequences were inserted immediately downstream of the neurotransmitter GPCR stop codon so that such reporters coexpresses a neurotransmitter GPCR and GFP as separate proteins. Three transgenes were constructed in other manners. The ser-4::gfp transgene (Tsalik et al., 2003; Gürel et al., 2012) has GFP coding sequences fused to the third coding exon of ser-4. Repetitive sequences in the ser-4 genomic DNA 3' of this position prevented us from including the downstream regions of ser-4 in the transgenes. The gbb-2::gfp reporter had GFP coding sequences inserted between two arginine codons in exon 14 of the gbb-2 gene. The details of splicing downstream of exon 14 remain uncertain, preventing us from inserting GFP coding sequences more 3' to this exon. The mgl-2::gfp reporter is a transcriptional fusion with a promoter fragment extending 7.9 kb 5' of the mgl-2 start codon inserted upstream of GFP coding sequences in the plasmid pPD955_75 (Addgene). Download Figure 1-1, TIF file.
Figure 1-2
Construction and properties of GPCR::GFP transgenes. This extended data file is a table showing the technical details of constructing each GPCR::GFP transgene and its transformation into C. elegans. Download Figure 1-2, XLSX file.
Figure 2-1
C. elegans mutations, transgenes, and strains used in this work. This extended data table provides technical details sufficient to allow others to select and use these resources for future work. Download Figure 2-1, XLSX file.
Figure 3-1
Animal-to-animal variations in GFP expression from a chromosomally-integrated octr-1::gfp transgene. A-F, Strong expression of octr-1::gfp in uv1 cells is consistent from animal to animal. Confocal images of octr-1::gfp; ida-1::mCherry in the GFP channel (A,C,E) and GFP plus mCherry channel (B,D,F) for three different young adult animals. In (A,C,E) cells other than uv1 that express GFP are indicated by white text. GFP intensity in the cells of interest is indicated by +++ (among the strongest cells labeled), ++ (easily detectable), + (slightly above background). Labeling in G-R is analogous to that in A-F. G-L, Expression of octr-1::gfp in the HSN neurons varies from easily detectable to undetectable in different animals. The same confocal images as in A-F are shown but in these panels the labels for HSN include the GFP levels scored for this cell. GFP is easily detectable in two animals shown (G-I) but not detectable above background in a third (K,L). M-R, Expression of octr-1::gfp in the VC neurons varies from moderate to undetectable in different animals. Labels for VC neurons indicate the GFP levels scored for these cells. Q-R, the same images shown in E,F,K,L but with new labels, while M-O are images not shown in previous panels. VC4/VC5 neuron GFP varies between animals, from easily detectable (M,N) to slightly above background (O,P) to undetectable (Q,R). In all panels, asterisks indicate position of the vulva; HSN: hermaphrodite-specific neuron; uv: uterine ventral cell; vm: vulval muscle; vnc: ventral nerve cord; scale bars: 10 microns. Download Figure 3-1, TIF file.
Figure 4-1
Examples of GPCR::GFP expression in the egg-laying system for all 26 neurotransmitter GPCRs. A larger subset of images than shown in Figure 4, selected from the 809 confocal images analyzed in this work. Actual analysis of GFP expression was carried out in three dimensions as shown in Movie 1, and these additional two-dimensional images are shown for illustrative purposes. All images are presented in pairs, with the upper panel showing GFP fluorescence expressed from a reporter for the neurotransmitter GPCR named in green and the lower panel adding mCherry fluorescence for the marker transgene named in red. Upper panels use arrowheads and white cell names to indicate every cell type detectably expressing the GPCR::GFP reporter in our full data set. Lower panels use arrowheads and yellow cell names to indicate GFP-expressing cells that are also labeled by the mCherry marker. Cell names in parentheses indicate cells where GFP expression is not visible in the image as displayed, but can be detected in two or more such images from our full data set. White asterisks indicate the position of the vulva and all scale bars are 10 µm. Cell types are denoted by the following abbreviation: bwm: body wall muscles; du: dorsal uterine cell; HSN: hermaphrodite-specific neuron; sp: spermatheca; sp-ut: spermathecal-uterine valve; um: uterine muscle; ut: uterine toroid cell; utse: uterine seam cell; uv: uterine ventral cell; vm: vulval muscle; vt: vulval toroid cell. Download Figure 4-1, TIF file.
Figure 5-1
Neurotransmitter GPCR expression identified in this work compared to that found in previous publications, shown with details of variability. A, GPCR expression results from our study compared to previous findings. Blue indicates that we observed GPCR::GFP expression in a cell where expression of that receptor had previously been reporter, red indicates that we did not observe GPCR::GFP expression in a cell where such expression was previously reported, and orange indicates that we identified expression in a cell where no such expression had been previously reported. The previous studies that reported expression for 13 of the 26 neurotransmitter GPCRs in the egg-laying system were as follows: ser-1: Cho et al., 2000; Carnell et al., 2005; Carre-Pierrat et al., 2006; ser-4: Gürel et al., 2012; ser-5: Carre-Pierrat et al., 2006; Hapiak et al., 2009; ser-7: Carre-Pierrat et al., 2006; dop-3: Chase et al., 2004; octr-1: Wragg et al., 2007; ser-3: Suo et al, 2006; ser-2: Tsalik et al., 2003; Rex et al., 2004; tyra-3: Wragg et al., 2007; Bendesky et al., 2011; gar-2: Lee et al., 2000; gbb-1: Yemini et al., 2019; mgl-2: Yemini et al., 2019; and ador-1: Plummer, 2011. B, Number of observations and strength of GFP expression in each cell type of the egg-laying system for each of the 26 GPCR::GFP reporters. Dark gray indicates cells in which GFP, when detected, was on average at the brightest levels (+++) for a reporter. Medium gray indicates cells in which GFP, when detected, was on average easily detectable (++). Light gray indicates cells that in which GFP, when detected, was on average only slightly above background (+). White indicates cells in which GFP was not observed in at least two animals. Numbers within a box in the form x/y indicate that GFP was detected x times in y animals examined for that cell type. Cell types are denoted by the following abbreviation: bwm: body wall muscles; du: dorsal uterine cell; HSN: hermaphrodite-specific neuron; sp: spermatheca; sp-ut: spermathecal-uterine valve; um: uterine muscle; ut: uterine toroid cell; uv: uterine ventral cell; vm: vulval muscle; vt: vulval toroid cell. Download Figure 5-1, TIF file.
Figure 6-1
Neurotransmitter GPCRs KOs reveal no significant egg-Laying defects. A, A second allele of dop-4 accumulates unlaid eggs. dop-4(ok1321), the dop-4 null allele used in Figure 6, accumulates a WT number of unlaid eggs, while a strain carrying dop-4(tm1392), an independent deletion allele, accumulates significantly more unlaid eggs that does the wild type. As described in the text, the tm1392 phenotype could not be rescued by a WT dop-4 transgene, suggesting this phenotype is an artifact of the genetic background and not the result of loss of dop-4 function. Statistical significance was tested using one-way ANOVA with a Tukey's test to determine statistical significance for multiple comparisons for the unlaid egg assay. n ≥ 30 for each strain. p ≥0.05 was considered not significant (ns), with p = 0.56 for ok1321 and p < 0.0001 for tm1392. B, Combination KOs of Gαo-coupled neurotransmitter GPCRs expressed in the egg-laying system reveal no strong egg-laying defects in the accumulation of unlaid eggs in the uterus. In the table below graph, the red “X” signifies the combination of neurotransmitter Gαo-coupled GPCR genes knocked out. egl-10(md176) V and goa-1(n1134) I served as controls for strong egg-laying defects. Statistical significance was tested using one-way ANOVA with Bonferroni correction for multiple comparisons for the unlaid egg assay. n ≥ 30 for each strain. p <0.05 was considered significant. The eight significant p values (for measurements from left to right that are denoted with asterisks) were p < 0.0001, p < 0.0001, p = 0.0281, p = 0.0105, p < 0.0001, p = 0.0003, p = 0.0082 and p = 0.0214. Download Figure 6-1, TIF file.
Figure 7-1
Egg-laying defects in neurotransmitter GPCRs overexpressors are caused by increased neurotransmitter signaling. A, Overexpression of two neurotransmitter GPCR genes, ser-1 and gbb-1, results in a strong hyperactive egg-laying phenotype as seen by the high percentage of early-staged eggs laid. Bars are color coded for the neurotransmitter corresponding to teach receptor: dark green, serotonin; light green, dopamine; brown, octopamine; orange, tyramine; red, acetylcholine; blue, GABA; yellow, glutamate; magenta, orphan GPCRs. goa-1(n1134) I was used as a control hyperactive egg-laying mutant. For panels A-C, n = 100 eggs per strain. Statistical significance was determined with the Fisher's exact test for the early-staged egg assay. The Wilson-Brown Method was used to determine the 95% CI for binomial data. p <0.05 (*) was considered significant. In A significant p values were as follows: goa-1 (p < 0.00001); ser-1(oe) (p < 0.00001); ser-7(oe) (p < 0.00001); dop-1(oe) (p = 0.0287); dop-3(oe) (p < 0.00001); gar-2(oe) (p = 0.0008); gbb-1(oe) (p < 0.00001). B, Overexpression of the serotonin GPCR gene ser-1 causes a strong hyperactive egg-laying defect that is reproduced in strains carrying an extrachromosomal (Ex.) ser-1 transgene and three independent chromosomally-integrated ser-1 transgenes. Control is WT. p values for the four asterisked measurements compared to the wild type were (left to right) p < 0.00001, p < 0.00001, p < 0.00001 and p < 0.00001. C, One strain carrying an integrated transgene for the GABA GPCR gbb-1 shows a strong hyperactive egg-laying defect that is not reproduced in a second chromosomal integrant or in a strain carrying an extrachromosomal (Ex.) gbb-1 transgene. Controls are WT or a strain with fluorescent neurons carrying a rab-3p::NLS::TagRFP transgene. p values for the two asterisked measurements compared to the wild type were (left to right) p = 0.0052 and p < 0.00001. D, Overexpression of the octopamine GPCR gene ser-6 causes strong egg-laying defects as seen by the accumulation of unlaid eggs in the uterus in strains carrying four independent chromosomally-integrated ser-6 transgenes and one extrachromosomal (Ex.) ser-6 transgene. The strain carrying the integrated transgene ser-6(oe) #1 was outcrossed to wild type to remove the rab-3p::NLS::TagRFP transgene and re-assayed. Controls are WT or rab-3p::NLS::TagRFP; Brown: ser-6 overexpressors. For this panel and panels E-I, statistical significance was tested using one-way ANOVA with a Tukey's test for multiple comparisons. n ≥ 30 for each strain. p <0.05 (*) was considered significant. p values for the upper asterisked comparison was p < 0.0001 and for the five lower asterisked comparisons (left to right) were p = 0.0002, p < 0.0001, p = 0.0024 p < 0.0001 and p < 0.0001. E, Overexpression of the glutamate GPCR gene mgl-1 causes strong egg-laying defects as seen by the accumulation of unlaid eggs in the uterus in both an extrachromosomal (Ex.) and chromosomally-integrated mgl-1 transgenic strains. Controls are WT or rab-3p::NLS::TagRFP. p values for the two asterisked comparisons were p < 0.0001 (upper comparison) and p < 0.0001 (lower comparison). F, Overexpression of the glutamate GPCR gene mgl-3 causes mild egg-laying defects as seen by the accumulation of unlaid eggs in the uterus in three extrachromosomal and one chromosomally-integrated mgl-3 transgenic strains for MGL-3. Control is WT. p values for the four asterisked comparisons (left to right) were p < 0.0001, p < 0.0001, p < 0.0001 and p < 0.0001. G, Overexpression of the acetylcholine GPCR gene gar-1 reveals mild egg-laying defects as seen by the accumulation of unlaid eggs in the uterus in extrachromosomal (Ex.) and chromosomally-integrated gar-1 transgenic strains. p values for the two asterisked values compared to WT were p < 0.0001 (left) and p < 0.0001 (right). H, Extrachromosomal (Ex.) overexpression of the octopamine GPCR gene ser-3 causes strong egg-laying defects as seen by the accumulation of unlaid eggs in the uterus, but this phenotype is not reproduced in by a strain with a chromosomally-integrated ser-3 transgene. p values for comparison to the WT were p < 0.0001 for ser-3(oe) and p = 0.4744 for the integrated ser-3(oe) strain. I, Overexpression of the tyramine GPCR gene ser-2 causes mild egg-laying defects as seen by the accumulation of unlaid eggs in the uterus in extrachromosomal and chromosomally-integrated ser-2 transgenic strains. Controls are WT and rab-3p::NLS::TagRFP. p values for the two asterisked comparisons were p < 0.0001 (upper comparison) and p < 0.0001 (lower comparison). Download Figure 7-1, TIF file.
