Contribution of the receptor guanylyl cyclase GC-D to chemosensory function in the olfactory epithelium

Leinders-Zufall et 10.1073/pnas.0704965104.XXYYYYY103.

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SI Figure 6
SI Figure 7
SI Figure 8
SI Figure 9
SI Materials and Methods

Fig. 6. Gene targeting of Gucy2d. (A) Exon-intron structure of the targeted region of Gucy2d (Upper) and the targeting vector (Lower). Gray, exons; black, homology arms; red, IRES-Mapt-lacZ reporter cassette; green, neo selection cassette. Key restriction endonuclease sites, loxP recombination sites, and exon numbers are indicated. (B) Southern blot analysis of genomic DNA from Gucy2d +/+, +/-, and -/- mice, confirming proper gene targeting. (C) RT-PCR of PDE2, GC-D, and CNGA3 cDNA from MOE of Gucy2d +/- and -/- mice and from MOE of Cnga3 -/- mice (5). Amplicon sizes are: PDE2, 441 bp; GC-D, 1.6 kb; CNGA3, 284 bp. (D) b-Gal immunohistochemistry in MOE of Gucy2d +/- and - /- mice, taken from equivalent regions on the endoturbinates. b-Gal-immunopositive cells were much less dense in other areas of the epithelium (e.g., the septum; see Figs. 4A and 5A), consistent with previous in situ hybridization results (9). (Scale bars: 10 mm.) (Magnification is slightly different for upper and lower panels.)

SI Figure 7

Fig. 7. GC-D is not required for effective suckling. Mean mass of Gucy2d +/+ (open circles), +/- (gray filled circles), and -/- (black filled circles) pups on each postnatal day until weaning. n = 33 pups (10 Gucy2d +/+, 17 Gucy2d +/-, and six Gucy2d -/- pups from five litters). Error bars are SD. All pups were observed to have milk in their stomachs within 12 h of birth. Gucy2d -/- pups born of sires and dams that were both Gucy2d -/- similarly suckled effectively, indicating that priming by +/- or +/+ littermates was not required for -/- pups to identify and attach to a nipple (12) (data not shown).

Fig. 8. X-Gal histochemical labeling of the olfactory bulb of a Gucy2d -/- mouse. A single bulb and attached olfactory epithelium were dissected from the cranium and forebrain and stained with X-Gal. (A-D) The same bulb is shown in lateral (A), medial (B), ventral (C), and dorsal (D) views. The white arrows point rostrally. Black arrowheads indicate two necklace glomeruli of different size (C), and the black arrow indicates GC-D axons passing toward more dorsal regions of the medial side (C).

Fig. 9. GC-D neurons can be divided into three functional subclasses (A-C), each exhibiting a somewhat different peptide recognition profile. The bar histograms summarize DF/F peak Ca²⁺ responses to stimulation with uroguanylin (1 mM), guanylin (1 mM), and dilute urine (1:100) in guanylin-sensitive/uroguanylin-insensitive (7/26, 27%) (A), uroguanylin-sensitive/guanylin-insensitive (7/26, 27%) (B), and uroguanylin- and guanylin-sensitive (12/26, 46%) (C) knobs. Ligands were focally applied to the ciliated surface of the sensory epithelium by a multibarreled stimulation pipette. The 26 b-gal-positive knobs (from seven Gucy2d +/- mice) were challenged sequentially and in random order. Data are expressed as means ±SD. The number of experiments is indicated above each bar.

SI Materials and Methods

Gene Targeting. A genomic clone containing exons 2-5 of the mouse Gucy2d gene (1) (MGI:106030) was isolated from a 129/Sv phage library (Invitrogen, Carlsbad, CA) and fully sequenced. Note that the gene names for receptor guanylyl cyclases are inconsistent between mouse and human. The orthologue of the mouse Gucy2d gene is a pseudogene in humans (ref. 2 and L. Newman and S.D.M, unpublished data); Gucy2d is not an orthologue of the human GUCY2D gene, the latter of which encodes a retinal receptor guanylyl cyclase involved in phototransduction known as GC-E/RETGC-1. The targeting vector was constructed in pBlueScript KS- (pBS) after modification of the vector as follows: an EcoRI-AscI-PacI-AscI-EcoRI polylinker was inserted into the EcoRI site of pBS, to give pBS-APA; an EcoRV-BglII-NsiI-HindIII polylinker was inserted into the EcoRV and HindIII sites of pBS-APA to give pBS-APA-BN. For the upstream arm, a 2.3-kb PstI fragment containing a portion of the first intron and 8 bp of exon 2 was inserted into the NsiI site in pBS-APA-BN to give pBS-APA-BN(up). For the downstream arm, a 3.7-kb XbaI-ApaI fragment containing the terminal 120 bp of exon 3 as well as exons 4 and 5 and their intervening introns was inserted into pBK-CMV and then excised with XbaI and SmaI. This XbaI-SmaI fragment was inserted into pBS-APA-BN(up) digested with XbaI and SmaI. Finally, the ETLp(A)+/LDTNL reporter/selection cassette, containing a Mapt-lacZ reporter following an internal ribosomal entry site (IRES), as well as the neomycin resistance (neo) gene and a partially deleted thymidine kinase (tk) gene flanked by loxp recombination sites (3), was inserted into the targeting vector after AscI digestion. The completed targeting vector was linearized with SalI and transfected by electroporation into mouse 129/Sv ES cells, and neo-positive clones were isolated. After injection into C57BL/6J blastocysts, chimeric animals were identified, and the selection cassette was removed by cre-dependent recombination after mating of heterozygous progeny to cre recombinase-expressing lines (4); breeding colonies were established. All animal procedures were approved by the Institutional Animal Care and Use Committees of the University of Maryland School of Medicine, the Johns Hopkins University School of Medicine, or the Ludwig-Maximilians-Universität München.

Growth and Survival Studies. For assays of gain in body mass, individual pups born of matings of Gucy2d +/- mice were measured on a calibrated balance each morning for the first 21 days after birth. Pups were genotyped after completion of the measurement period. Means for each genotype on each day were obtained.

RT-PCR. Main olfactory epithelium (MOE) tissue was dissected from Gucy2d +/-, -/-, and Cnga3 -/- mice (two mice each), pooled, and total RNA was extracted with Trizol reagent (Invitrogen) or RNeasy Mini (Qiagen, Valencia, CA). Two micrograms of total RNA was reverse-transcribed with oligo(dT) primers, and cDNA was used as template for PCR amplification with TaqPro Complete (Denville Scientific, Metuchen, NJ).

X-Gal Histochemistry. Adult Gucy2d +/- and -/- mice were intracardially perfused with cold PBS and then 4% paraformaldehyde in PBS (pH 7.4). Tissue was harvested and incubated in buffer A (0.1 M NaPO₄, pH 7.4/2 mM MgSO₄/5 mM EGTA) for 35 min followed by twice for 5 min each time in buffer B (0.1 M NaPO₄, pH 7.4/2 mM MgSO₄/0.01% sodium deoxycholate/0.02% Nonidet P-40), all at room temperature. Tissues were then incubated in buffer B containing 5 mM K₃Fe(CN)₆, 5 mM K₄Fe(CN)₆, and 1 mg/ml X-Gal at 37°C until a strong blue color developed.

Immunohistochemistry. Adult Gucy2d +/- and -/- mice and Cnga3 -/- mice (5) were intracardially perfused with cold PBS and then either Bouin's fixative (Sigma, St. Louis, MO) or 4% paraformaldehyde in PBS (pH 7.4). Tissue was harvested and postfixed for 2 h (4°C) and immersed in 30% (wt/vol) sucrose/1´ PBS overnight at 4°C. After imbedding in Tissue-Tek O.C.T compound, 20-mm frozen sections were collected on Superfrost Plus glass slides (Fisher Scientific, Pittsburgh, PA). Sections were blocked with 2% heat-inactivated donkey serum/0.2% Triton X-100 in 0.1 M PBS (pH 7.4), then incubated overnight at room temperature with either rabbit anti-tyrosine hydroxylase (1:500; Chemicon, Temecula, CA) or goat anti-phosphodiesterase 2 (PDE2) (1:100; Santa Cruz Biotechnology, Santa Cruz, CA) and mouse anti-b-galactosidase (1:2,000; Promega, Madison, WI) with 2% donkey serum/0.2% Triton X-100 in PBS. Immunoreactivity was visualized with Cy2-labeled donkey anti-rabbit, Cy2-labeled donkey anti-goat, and/or Cy3-labeled donkey anti-mouse secondary antibodies followed by confocal microscopy.

Local Field Potential Recording. Adult (3-6 months old) male or female Gucy2d +/- and -/- mice, Cnga2-null mice (6), Cnga3 -/- mice (5), or wild-type C57BL/6 mice (Charles River Laboratories, Wilmington, MA) were anesthetized, decapitated, and hemisected, and the nasal septum was removed to expose the endoturbinate system of the olfactory epithelium. The submerged electroolfactogram technique was used (3, 7) to record local field potentials. The epithelium was superfused continuously at room temperature with oxygenated (95% O₂/5% CO₂) external solution containing 120 mM NaCl, 25 mM NaHCO₃, 5 mM KCl, 5 mM N,N-2,2-bis(hydroxymethyl)-2,2',2"-nitrilotriethanol (BES), 1 mM MgSO₄, 1 mM CaCl₂, and 10 mM glucose. Localized micropulse stimuli were focally ejected by using multibarrel stimulation pipettes (8). The interstimulus interval was 4 min. Stimulus concentration refers to the concentration in the stimulation pipette. Guanylin (mouse/rat; Bachem, King of Prussia, PA), uroguanylin (with N-terminal tyrosine; mouse/rat; Bachem), h-uroguanylin (human; Sigma and Bachem), enterotoxin STp (Escherichia coli STp; Bachem), urodilatin (human, Sigma), 2-heptanone (Sigma), or 2,5-dimethylpyrazine (Aldrich, St. Louis, MO) were diluted to the final concentrations in extracellular bath solution. SQ22536 (Calbiochem, San Diego, CA) and L-cis-diltiazem (Biomol, Plymouth Meeting, PA) were applied by bath flow. The spatial distribution of guanylin- or uroguanylin-evoked potentials fell within a broad central zone of the neuroepithelium, consistent with previous GC-D in situ hybridizations (9). Epithelial hot spots were often found at the dorsal rim of each endoturbinate. We did not observe any obvious differences between males and females in peptide-evoked potentials. If not stated otherwise, data are recorded from at least three different mice and expressed as means ±SEM.

Live-Cell Imaging of b-Gal and Single-Knob Electrophysiology. Intact olfactory epithelium obtained from the nasal septum of adult mice was mounted in an en face view (10) and superfused continuously at room temperature with oxygenated external solution. We developed a protocol for live-cell imaging of b-gal in overexpressing dendritic knobs of olfactory neurons by using the fluorogenic b-gal substrate fluorescein digalactoside (FDG; Invitrogen). FDG was dissolved in 10 mM DMSO, added to oxygenated extracellular solution, and briefly sonicated to give a final concentration of 10 mM FDG/0.1% DMSO. The epithelium was incubated in this solution for 10 min at 37°C (O₂/CO₂ incubator) and washed twice with dye-free oxygenated solution. The tissue was kept in the incubator for another 20 min to allow for sufficient hydrolysis of FDG to fluorescein. Fluorescent knobs were then imaged by using a combination of infrared differential interference contrast and fluorescence videomicroscopy. Action potential-driven capacitive currents (8, 10, 11) were recorded extracellularly from identified knobs by using patch pipettes filled with Hepes-buffered extracellular solution (145 mM NaCl/5 mM KCl/1 mM CaCl₂/1 mM MgCl/10 mM Hepes, pH 7.3; 300 mOsm; seal resistance 100-200 MW) connected to a computerized EPC-9 patch clamp amplifier (HEKA Electronics, Mahone Bay, NS, Canada). Data were written to disk by using Pulse software (sample rate: 100 ms/point; HEKA Electronics). Spikes were analyzed off-line by using IGOR Pro software (WaveMetrics, Lake Oswego, OR).

Simultaneous Imaging of b-Gal and Ca²⁺ by Using Confocal Microscopy. The epithelial preparation was loaded with the Ca²⁺ indicator fluo-4/AM (Invitrogen) (8, 11). Changes in intracellular Ca²⁺ concentration were imaged in single olfactory knobs using a Radiance 2100 confocal laser system (Carl Zeiss Microimaging, Thornwood, NY) (8, 11). Simultaneous visualization of b-gal was achieved by coloading the cells with the fluorogenic substrate resorufin galactoside (200 mM; Invitrogen). The fluorescence emission spectrum of resorufin, in contrast to that of fluorescein, does not interfere with that of fluo-4. In control experiments, we confirmed that there is virtually no spillover of fluo-4 fluorescence onto the resorufin channel and vice versa. Images were acquired at a rate of 0.97 Hz by using LaserSharp software (Carl Zeiss Microimaging) and analyzed by using ImageJ 1.32j (National Institutes of Health, Bethesda, MD) and IGOR Pro software. The PDE inhibitors erythro-9-2-hydroxy-3-nonyl]-adenine (EHNA) and isobutyl-1-methylxanthine, the adenylyl cyclase activator forskolin, and the cyclic nucleotide-gated channel agonist 8-bromo-cGMP were obtained from Sigma. Urine samples were freshly collected from adult male or female C57BL/6 control mice and diluted 1:100. We observed no difference in the effect of male or female urine on electrophysiological or Ca²⁺ responses of GC-D cells in Gucy2d +/- mice. Stimuli were delivered in random order and with identical interstimulus intervals to rule out order effects or desensitization.

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