Picture from Liu Y et al. (2011) PLoS Genet "A cholinergic-regulated circuit coordinates the maintenance and bi-stable ...."

Figure S3 Expression of ionotropic ACh receptor genes in the male tail. Muscle abbreviations: DSP: dorsal spicule protractor; VSP: ventral spicule protractor; DSR: dorsal spicule retractor; VSR: ventral spicule retractor; ADP: anal depressor; GER: gubernacular erector; GRT: gubernacular retractor; AOB: anterior oblique; POB: posterior oblique; SPH: sphincter. To obtain the expression pattern of unc-29 in the C. elegans male, we initially fused to the YFP gene a 2.2 kb sequence, which includes a 1.3 kb of sequence upstream of the unc-29 start codon and the genomic sequence of the gene up to the second intron (Punc- 29:YFP). This construct is expressed in the anal depressor muscle and the spicule protractor muscles in the male spicule circuit, and it is also expressed in the body wall muscles, some ventral cord neurons and some head neurons. This is consistent with the previous published expression pattern of unc-38, which encodes an a-subunit that forms the L-AChR with unc-29 (Liu et al. 2007). Nonetheless, the unc-29 genomic sequence also contains important information to facilitate transcription of the gene. Therefore, the expression construct that contained the unc-29 genomic sequence(Punc-29:unc-29::YFP) provided a broader expression pattern than Punc-29:YFP. (A-D) Expression of Punc-29:YFP (yellow) and Pgar- 3:CFP (cyan) imaged from the tail of a same L4 larval male. The two images are merged to show that unc-29 is not expressed in the SPC neurons where gar-3 is expressing (C). The Nomarski image of the tail is also shown (D). Scale bar, 10 um. (E, F) Expression of Punc-29:YFP in the male tail. Scale bar, 20 um. We fused the 2.4 kb sequence upstream of the unc-63 start codon, including the first three codons, to the YFP gene. We found that this construct was expressed in the body wall muscles, the sexually dimorphic anal depressor muscle and sphincter muscle, and every male-specific sex muscle, including the spicule protractor and retractor muscles, the diagonal muscles, the gubernacular muscles, and the oblique muscles. However, it was also expressed in the SPC neurons where unc-29 was not expressed (G-J). When we made an unc-38 promoter-genomic DNA fusion construct, we found that, similarly to unc-63, it was also expressed in every male-specific muscle and the SPC neurons (K-N). (G-J) Expression of Punc-63:YFP in the male tail. Scale bar, 10 um. (K-N) Expression of Punc-38:unc- 38::YFP in the male tail. Scale bar, 10 um. (O, P) Expression of Pacr-12:acr-12::YFP in the male tail. Scale bar, 10 um. (Q-S) Expression of Pacr-18:ChR2::YFP in the male tail. Scale bar, 10 um. (T-V) Expression of Pacr-18:ChR2::YFP (yellow) and Ppkd- 2:CFP (cyan) imaged from the tail of a same adult male. The two images are merged to show that acr-18 is expressed in the HOA neuron that is next to the HOB neuron where pkd-2 is expressing (V). Scale bar, 10 um.

Picture from Liu Y et al. (2011) PLoS Genet "A cholinergic-regulated circuit coordinates the maintenance and bi-stable ...."

(E-H) Fluorescence images of the tail region of adult males expressing the Punc-29:unc-29::YFP construct. Scale bar, 20 um. Gubernacular erector (GER), gubernacular retractor (GRT), anterior oblique (AOB), dorsal spicule protractor (DSP), ventral spicule protractor (VSP) and anal depressor (ADP) muscle.

Picture from Liu Y et al. (2011) PLoS Genet "A cholinergic-regulated circuit coordinates the maintenance and bi-stable ...."

Muscle abbreviations: DSP: dorsal spicule protractor; VSP: ventral spicule protractor; DSR: dorsal spicule retractor; VSR: ventral spicule retractor; ADP: anal depressor; GER: gubernacular erector; GRT: gubernacular retractor; AOB: anterior oblique; POB: posterior oblique; SPH: sphincter. (K-N) Expression of Punc-38:unc- 38::YFP in the male tail. Scale bar, 10 um. When we made an unc-38 promoter-genomic DNA fusion construct, we found that, similarly to unc-63, it was also expressed in every male-specific muscle and the SPC neurons (K-N).

Picture from Liu Y et al. (2011) PLoS Genet "A cholinergic-regulated circuit coordinates the maintenance and bi-stable ...."

Muscle abbreviations: DSP: dorsal spicule protractor; VSP: ventral spicule protractor; DSR: dorsal spicule retractor; VSR: ventral spicule retractor; ADP: anal depressor; GER: gubernacular erector; GRT: gubernacular retractor; AOB: anterior oblique; POB: posterior oblique; SPH: sphincter. (G-J) Expression of Punc-63:YFP in the male tail. Scale bar, 10 um. We fused the 2.4 kb sequence upstream of the unc-63 start codon, including the first three codons, to the YFP gene. We found that this construct was expressed in the body wall muscles, the sexually dimorphic anal depressor muscle and sphincter muscle, and every male-specific sex muscle, including the spicule protractor and retractor muscles, the diagonal muscles, the gubernacular muscles, and the oblique muscles. However, it was also expressed in the SPC neurons where unc-29 was not expressed (G-J).

Picture from Liu Y et al. (2011) PLoS Genet "A cholinergic-regulated circuit coordinates the maintenance and bi-stable ...."

(A-D) Fluorescence images of the tail region of adult males expressing the Pacr-16:YFP construct. Scale bar, 20 um. Gubernacular erector (GER), gubernacular retractor (GRT), anterior oblique (AOB), dorsal spicule protractor (DSP), ventral spicule protractor (VSP) and anal depressor (ADP) muscle.

Picture from Liu Y et al. (2011) PLoS Genet "A cholinergic-regulated circuit coordinates the maintenance and bi-stable ...."

Figure 2. Male tail expression of acr-16 and unc-29. Gubernacular erector (GER), gubernacular retractor (GRT), anterior oblique (AOB), dorsal spicule protractor (DSP), ventral spicule protractor (VSP) and anal depressor (ADP) muscle. (A-D) Fluorescence images of the tail region of adult males expressing the Pacr-16:YFP construct. Scale bar, 20 um. (E-H) Fluorescence images of the tail region of adult males expressing the Punc-29:unc-29::YFP construct. Scale bar, 20 um.

Picture from Liu Y et al. (2011) PLoS Genet "A cholinergic-regulated circuit coordinates the maintenance and bi-stable ...."

Muscle abbreviations: DSP: dorsal spicule protractor; VSP: ventral spicule protractor; ADP: anal depressor. To obtain the expression pattern of unc-29 in the C. elegans male, we initially fused to the YFP gene a 2.2 kb sequence, which includes a 1.3 kb of sequence upstream of the unc-29 start codon and the genomic sequence of the gene up to the second intron (Punc- 29:YFP). This construct is expressed in the anal depressor muscle and the spicule protractor muscles in the male spicule circuit, and it is also expressed in the body wall muscles, some ventral cord neurons and some head neurons. This is consistent with the previous published expression pattern of unc-38, which encodes an a-subunit that forms the L-AChR with unc-29 (Liu et al. 2007). Nonetheless, the unc-29 genomic sequence also contains important information to facilitate transcription of the gene. Therefore, the expression construct that contained the unc-29 genomic sequence(Punc-29:unc-29::YFP) provided a broader expression pattern than Punc-29:YFP. (A-D) Expression of Punc-29:YFP (yellow) and Pgar- 3:CFP (cyan) imaged from the tail of a same L4 larval male. The two images are merged to show that unc-29 is not expressed in the SPC neurons where gar-3 is expressing (C). The Nomarski image of the tail is also shown (D). Scale bar, 10 um. (E, F) Expression of Punc-29:YFP in the male tail. Scale bar, 20 um.

Picture from Liu Y et al. (2007) J Neurosci "G alpha(q)-coupled muscarinic acetylcholine receptors enhance nicotinic ...."

Figure 6. Native and heterologous expression of the gar-3::YFP translational fusion protein. A, B, DIC (A) and fluorescence (B) images of the left lateral tail region of a mid L4 male expressing both Pgar-3A::gar-3::YFP (pYL8) and Pgar-3B::gar-3::YFP (pYL9). Nuclei of the neurons are labeled with arrows. In B, positions of these neurons are indicated with circles, and localizations of GAR-3::YFP on the cell membranes are denoted by arrows. C, D, DIC (C) and fluorescence (D) images of left lateral tail region of an adult male expressing Punc-103 E::gar-3::YFP (pYL10). E, F, Merged DIC and fluorescence (E) and fluorescence (F) images of left lateral tail region of a mid L4 male expressing the Punc-103 F ::gar-3::YFP (pYL11). In E, nuclei of neurons are labeled with arrows. GAR-3::YFP localization on cell membranes can be seen as white puncta. In F, neuronal positions are indicated with circles, and localizations of GAR-3::YFP on the cell membranes are denoted by arrows. Scale bar, 10 μm.

Picture from Liu Y et al. (2007) J Neurosci "G alpha(q)-coupled muscarinic acetylcholine receptors enhance nicotinic ...."

Figure 3. Male tail expression of unc-38 and gar-3 promoters. Fluorescence images of the right lateral tail region of an adult male expressing the unc-38 promoter::YFP construct (A), early L4 male expressing the unc-38 promoter::YFP construct (B), and adult (C) and late L4 (D) males expressing the Pgar-3B::YFP construct pYL1. Scale bar, 10 um.

Picture from Liu Y et al. (2007) J Neurosci "G alpha(q)-coupled muscarinic acetylcholine receptors enhance nicotinic ...."

Male tail expression of unc-38 promoter. Fluorescence images of the right lateral tail region of an adult male expressing the unc-38 promoter::YFP construct (A), early L4 male expressing the unc-38 promoter::YFP construct (B). Scale bar, 10 um.