GoKawiilGrowth of C. elegans strains
C. elegans strains were grown on nematode growth medium (NGM) plates seeded with Escherichia coli OP50 at 20 °C. unc-122p::dsRed or gfp were used as co-injection markers (at 40 ng µl−1) to generate transgenic strains unless noted otherwise. vha-6p::NLS::gfp or vha-6p::mCherry was used as a co-injection marker at 10 ng µl−1 with 65 ng µl−1 of 1 kb DNA ladder (New England BioLabs) to a final concentration of 100 ng µl−1 to generate PY12814, PY12815, PY12816, PY12821, PY12822 and PY12823 (Supplementary Table 1). Strains were generated using standard genetic crosses. The PY12829 strain was obtained by deleting glr-9 via gene editing to generate glr-9(oy224) in the PY12818 strain. The genotypes of all strains were verified by sequencing or phenotypic examination. All experiments were performed using one-day-old adult hermaphrodites grown under well-fed conditions for at least three generations. A list of all strains used in this work is provided in Supplementary Table 1.
Molecular biology
1.1 kb of sequences upstream of and including the glr-9 START codon and 1.9 kb of sequences upstream of and including the srg-42 START codon were amplified from wild-type worm lysate and used to drive I3-specific or I3-selective expression. Expression in I1 was driven using 1.1 kb of sequences upstream of the lgc-8 START codon. To generate lgc-8p::glr-7::mStayGold::SL2::mScarlet-I3 and lgc-8p::glr-9::mStayGold::SL2::mScarlet-I3, mStayGold::SL2::mScarlet-I3 was amplified by PCR from gBlock fragments and used to generate PSAB1400 and PSAB1401 by Gibson assembly (Supplementary Table 2). glr-9 cDNA or genomic DNA was amplified from wild-type worm lysate and tagged with gfp for localization and functional rescue experiments. Since glr-7 has a and b isoforms, the genomic region of glr-7 from the START codon of the glr-7b isoform (X: 2,419,477) to the STOP codon of both glr-7a and glr-7b isoforms (X:2,414,064) was amplified from wild-type worm lysate. These sequences were tagged with fluorescent reporter sequences to examine localization of glr-7. Full-length cDNAs encoding GLR-9, GLR-7a and GLR-7b amplified from worm lysates were cloned into the pME18s vector for expression in HEK293T cells. All plasmids were verified by Sanger sequencing. A list of plasmids used in this work is provided in Supplementary Table 2.
CRISPR–Cas9-mediated genome editing
Deletion alleles of glr-9, osm-9 and fipr-26 were generated using an injection mix containing 0.5 μl of Cas9 (IDT), 1.5 μl of 100 μM trans-activating CRISPR RNA (tracrRNA), 1 μl of 50 μM 5′ CRISPR RNA (crRNA), 1 μl of 50 μM 3′-crRNA, 1 μl of 17 μM dpy-10 crRNA as a co-CRISPR injection marker, and nuclease-free water, for a total volume of 10 μl. No DNA donor was used. Worms were maintained at 25 °C after injection. Three days following injection, dumpy or roller F 1 progeny were segregated onto individual plates. After the singled F 1 worms laid eggs, parents were lysed and then genotyped using PCR.
To generate the flp-6::loxP insertion strain (PY12288, Supplementary Table 1), the injection mix included 0.5 μl of Cas9 (IDT), 2.8 μl of 34 μM crRNA, 0.2 μl of 100 μM tracrRNA, 2 μl of 26 μM donor DNA, 50 ng µl−1 of column-purified unc-122p::gfp, and nuclease-free water for a total volume of 20 μl. Following injection, worms were kept at 25 °C. F 1 progeny carrying the unc-122p::gfp co-injection marker were singled onto individual plates and genotyped. The C-terminal loxP site was inserted first, followed by insertion of the N-terminal loxP site. All tracrRNA, crRNAs, and donor DNAs were obtained from IDT.
To generate nuclear SL2::mStayGold insertions (PY12818, PY12819, PY12820, Supplementary Table 1), the injection mix included 0.5 μl of Cas9 (IDT), 2.8 μl of 34 μM crRNA, 0.5 μl of 100 μM tracrRNA, and pRF4 rol-6(gf) plasmid as an injection marker. Repair templates were obtained by PCR from each plasmid, gel purified and cleaned up using AMPureX beads, and 500 ng was added to the injection mix. Injected worms were singled and 96 F 1 progeny were singled from plates that contained rollers, allowed to starve out the plate, and then screened by PCR for the expected change. The sequences of the crRNAs and donor DNA are listed in Supplementary Table 3.
Fluorescent reporter imaging
One-day-old adult worms were immobilized with 10 mM (−)-tetramisole hydrochloride (Sigma-Aldrich L9756) and mounted on 10% agarose pads on microscope slides. Images in Fig. 1b, left, and Fig. 1d, left, were acquired on an inverted Zeiss Axiovert with a Yokogawa CSU-X1spinning disk confocal unit and a Photometrics Quantum 512SC camera. Slidebook 6.0 (Intelligent Imaging Innovations, 3i) software was used to acquire images with z-step sizes of 0.2 or 0.27 μm using a 63× oil immersion objective. High resolution images in Fig. 1b, right, Fig. 1d, right, and Fig. 1e were acquired at 0.21–0.23 μm z-intervals using a 100× oil immersion objective on an inverted LSM880 Airyscan confocal microscope using ZEN software (Zeiss). Histograms were adjusted for visualization after Airyscan processing to optimize resolution and contrast. Images in Figs. 1f, 5d and Extended Data Figs. 4c and 5a,b were acquired at 0.2 μm z-intervals using a 63× oil immersion objective on an inverted two-camera spinning disk confocal microscope (Leica DMI6000B with a CSU-W1 spinning disk head and two Andor Neo sCMOS cameras) and Andor IQ3.5 Software. All images were acquired in the Brandeis Light Microscopy Facility (RRID: SCR_025892) and processed using FIJI/Image J.
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