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Supporting Information Supporting Information Yoon et al. 10.1073/pnas.1506058112 SI Materials and Methods Fisher Scientific); Cy3-conjugated donkey anti-rat IgG (Jackson RNA in Situ Hybridization. Mice were perfused with 4% (wt/vol) ImmunoResearch). paraformaldehyde (fix) and the interior of the snout removed and Sections were next incubated for 10 min at 37 °C in PBS soaked in fix overnight. The tissue was decalcified by incubation containing 1 mM EGS (Sigma) and 0.1% Tween20 and then for 3 d in 0.25 M EDTA/2% (wt/vol) paraformaldehyde and then washed three times for 5 min in PBS. They were next incubated embedded in OCT (Sakura). Serial coronal cryostat sections in 0.1 M HCl for 7 min to fragment DNA, washed for 5 min in (14 μm) were obtained and stored at −80 °C. PBS, and then treated with RNase A (100 mg/mL in 2× SSC) for Digoxigenin-labeled cRNA probes were prepared using cloned 30 min at 37 °C. After two 5-min washes in 2× SSC, sections were coding regions of individual Taar genes or the Nqo1 gene as equilibrated in 50% (vol/vol) formamide/2× SSC, pH 7.0. Probe templates. In situ hybridization to OE sections was performed as mix [15 μL containing 50–100 ng BAC DNA probe and 10 μg described (1), except that up to 30 μg/mL proteinase K was used mouse Cot-1 DNA in 50% formamide/2× SSC/10% (wt/vol) to permeabilize tissue sections before hybridization. If needed, dextran sulfate] was applied to each slide, and the slides were proteinase K activity was then eliminated by washing in PBS for coverslipped, sealed with rubber cement, and then incubated at 5 min at 85 °C. 80–82 °C on a slide warmer to denature the probe and cellular DNA. Slides were next incubated overnight at 37 °C in a humid Radioactive in Situ Hybridization. Radioactive in situ hybridization chamber. Sections were then washed three times in 50% was performed as described (2) with minor modifications. Mice formamide/2× SSC at 42 °C for 5 min, twice in 2× SSC at 42 °C were perfused as above and serial coronal sections (12 μm) ob- for 5 min, and then in 4× SSC at room temperature for 5 min. tained from the olfactory bulbs. Sections were treated as for Sections were then incubated with Alexa-488–labeled streptavidin nonradioactive in situ hybridization above, except that 5 μg/mL (Thermo Fisher Scientific) or with FITC-labeled anti-DIG (Sigma), proteinase K was used for permeabilization, and hybridization and nuclei then counterstained with DAPI, and the slides mounted was to 33P-labeled cRNA probes. Slides were coated with NTB-2 in 90% (vol/vol) glycerol/2.3% (wt/vol) DABCO (Sigma). Kodak emulsion, exposed for 3–4 wk at room temperature, and then developed. BAC Probes. BAC probes were prepared using a digoxigenin or biotin nick translation kit (Roche), according to the manufacturer’s Immunfluorescence DNA in Situ Hybridization (Immuno-FISH). instructions. BAC plasmids were obtained from the BACPAC Immuno-FISH was performed as described (3–5) with minor Resources Center (BPRC). BAC plasmids used for this study were modifications. Sections were postfixed for 5 min in 4% (wt/vol) as follows: RP23-43A7 (ch10: 23,642,668-23,851,574), RP23-318I18 paraformaldehyde fix in PBS, washed for 5 min in PBS, and then, (ch9: 37,485,034-37,665,416), RP23-88A24 (ch14: 54,862,839- for antigen retrieval, incubated at 80–85 °C for 23 min in 10 mM 55,005,904), RP23-6M15 (ch15: 97,961,306-98,195,573), RP23- Tris/1 mM EDTA. 155M18 (ch16: 59,174,022-59,376,005), RP24-368I6 (ch1: Sections were permeabilized with 0.5% Triton X-100 in PBS, 174,841,505-174,981,890); all chromosome mapping information washed three times in PBS, blocked with 2% (wt/vol) IgG-free was based on the NCBI37/mm9 assembly of the mouse genome. BSA/0.1% Tween20 in PBS, and then incubated with primary antibodies followed by secondary antibodies, with each antibody Immunohistochemistry and Antibodies. Immunohistochemistry was incubation followed by three 5-min washes in 0.1% Tween20 in conducted using the same initial procedures specified above for PBS. Primary antibodies: Goat anti-Lamin B1 (Santa Cruz, immuno-FISH. Primary antibodies used were against: H3K9me3 sc-6217); Rat anti-mCherry (KeraFAST, EST202). Secondary (Abcam, ab8898), H4K20me3 (Abcam, ab9053), lamin B (Santa antibodies: Alexa-647-conjugated donkey anti-goat IgG (Thermo Cruz, sc-6217), LAP2 (BD, 611000), or lamin A (Abcam, ab133256). 1. Liberles SD, Buck LB (2006) A second class of chemosensory receptors in the olfactory 4. Croft JA, et al. (1999) Differences in the localization and morphology of chromosomes epithelium. Nature 442(7103):645–650. in the human nucleus. J Cell Biol 145(6):1119–1131. 2. Ressler KJ, Sullivan SL, Buck LB (1994) Information coding in the olfactory system: ev- 5. Clowney EJ, et al. (2012) Nuclear aggregation of olfactory receptor genes governs their idence for a stereotyped and highly organized epitope map in the olfactory bulb. Cell monogenic expression. Cell 151(4):724–737. 79(7):1245–1255. 3. Ragoczy T, Bender MA, Telling A, Byron R, Groudine M (2006) The locus control region is required for association of the murine beta-globin locus with engaged transcription factories during erythroid maturation. Genes Dev 20(11):1447–1457. Yoon et al. www.pnas.org/cgi/content/short/1506058112 1of5 Fig. S1. Coexpression of mutant and functional Taar alleles in individual OSNs. OE sections from Taar5lacZ/+ (A–C)orTaar6lacZ/+ mice (D–F) were costained with a Taar9 riboprobe and with β-gal antibodies, which detect OSNs expressing the mutant Taar allele. Some β-gal+ OSNs were labeled for Taar9, indicating that cells expressing the mutant Taar5 or Taar6 allele can coexpress a functional Taar allele. (Scale bars, 30 μm.) Yoon et al. www.pnas.org/cgi/content/short/1506058112 2of5 Fig. S2. Expression of Taar mutant alleles in Taar5 and Taar6 knockout mice. The OE distribution of OSNs expressing wild-type versus mutant Taar5 and Taar6 alleles. (A–C) OE sections from wild-type (WT) mice were hybridized with a Taar5 (A), Taar6 (B), or Nqo1 (C) riboprobe. Taar5+ neurons are seen only in the lacZ/+ dorsal, Nqo1-labeled domain whereas Taar6+ neurons are seen in both the dorsal Nqo1+ and ventral Nqo1- domains. (D–G) OE sections from Taar5 (D and lacZ/+ E)orTaar6 (F and G) mice were immunostained for β-gal (D and F) or hybridized to an Nqo1 riboprobe (E and G). β-gal+ OSNs expressing the mutant Taar5 allele are located in only the dorsal Nqo1+ domain whereas those expressing the Taar6 mutant allele are located in both the Nqo1+ dorsal domain and Nqo1- ventral domain. White dots indicate the locations of OSNs labeled with the Taar riboprobe (A and B) or immunostained for β-gal (D and F). (Scale bars, 300 μm.) Yoon et al. www.pnas.org/cgi/content/short/1506058112 3of5 Fig. S3. The presence of a neomycin selection cassette alters the expression of a Taar5 mutant allele. The DNA construct used to generate Taar5 knockout mice contained a neomycin selection cassette. Following removal of the selection cassette, OSNs expressing the mutant Taar5 allele (β-gal+ OSNs) were seen predominantly in the dorsal OE domain, similar to OSNs expressing the wild-type Taar5 allele (see Fig. S2). However, before removal of the selection cassette, β-gal+ OSNs were seen in both the dorsal and ventral OE domains (A). This is apparent from costaining the same OE section with a Taar4 riboprobe to label Taar4-expressing OSNs, which are found predominantly in the dorsal domain (B). White dots indicate β-gal OSNs in OE regions that lack Taar4+ OSNs. (Scale bar, 300 μm.) Table S1. Many OSNs expressing a mutant Taar allele coexpress one other Taar gene Taar5lacZ/+ Taar6lacZ/+ Animal 1 Animal 2 Animal 3 Animal 4 Animal 1 Animal 2 Animal 3 Animal 4 Sum of percentages of β-gal+ OSNs labeled by 67.6 57.1 65.0 63.4 52.4 60.5 51.3 68.3 different Taar probes (Taar2-9)* (%) Percentage of β-gal+ OSNs labeled by a mix 56.7 60.3 57.9 55.1 65.2 48.7 47.7 55.9 containing all Taar probes* (%) + + *20 sections for Taar5lacZ/ , 7 sections for Taar6lacZ/ . Table S2. OE domains expressing wild-type versus mutant Taar5 and Taar6 alleles Taar5lacZ/+ Taar6lacZ/+ Taar5+ β-gal+ Taar6+ β-gal+ No. of labeled cells 297 1,071 654 1,521 No. of labeled cells in dorsal Nqo1+ OE domain 296 1,057 151 987 % of labeled cells in dorsal domain 99.7 98.7 23.1 64.9 No. of animals (male/female) 2/1 2/1 No. of sections scored per animal 20 7 The number of OSNs labeled for Taar5, Taar6,orβ-gal in the entire OE or the dorsal Nqo1+ domain. Yoon et al. www.pnas.org/cgi/content/short/1506058112 4of5 Movie S1. Taar localization and chromocenter distribution in OSNS and other OE cells. This movie shows a 4.75-μm-deep z-stack through the OE in 0.25-μm steps. Apical sustentacular cells are located at the top and basal cells at the bottom, with OSNs occupying a large central region. DNA FISH using a Taar BAC probe (green) shows that, in OSNs, Taar alleles mostly overlap with lamin B (red), which marks the nuclear lamina, whereas this localization is seldom seen in sustentacular cells and is less often seen in basal cells.
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