Article Figures & Data
Figures
- Figure 1.
Discovery of Goofy (#123) by signal sequence trap screening. A, Strategy of the signal sequence trap screening for identification of novel transmembrane and secreted molecules expressed in the OE. B, A summary table for individual 49 cDNA clones obtained by the signal sequence trap screening. C, mRNA expression patterns of six novel genes in P3 OE coronal sections assessed by in situ hybridization analysis. The clone #123 (Goofy) is abundantly and specifically expressed in the OE. NG, Nasal gland. Scale bar: C, 500 μm.
- Figure 2.
Structure of Goofy protein. A, Hydrophobicity plot of mouse Goofy protein. Two highly hydrophobic regions are present: one at the N terminus and the other around amino acid number 400, which potentially serve as a signal peptide (SP) and a transmembrane segment (TM), respectively. B, Amino acid sequence of mouse Goofy protein. Red underline, Signal peptide; blue underline, transmembrane segment; green double underline, canonical SH3-binding motif; black triangles, potential Asn-linked glycosylation sites. Ser, Thr, and Pro residues are highlighted in blue, green, and red letters, respectively. C, A schematic diagram depicting the domain structure of Goofy protein. Goofy consists of an N-terminal signal peptide (SP, red), a Ser/Thr/Pro-rich extracellular region (yellow) with three potential glycosylation sites (black triangles), a transmembrane segment (TM, blue), and a Pro-rich cytoplasmic region (green).
- Figure 3.
Expression of Goofy mRNA. A, Tissue distribution of Goofy mRNA in adult mouse assessed by Northern blot analysis. Top, Autoradiogram of a blot probed with 32P-labeled Goofy cDNA. Bottom, Same blot stained with methylene blue. Goofy mRNA is specifically expressed in the olfactory epithelium. B–K, Ontogenic expression of Goofy and OMP mRNAs assessed by in situ hybridization analysis. Coronal sections containing the OE and VNO from E13 (B, G), E15 (C, H), P0 (D, I), and P14 (E, F, J, K) mice were hybridized with 35S-labeled Goofy (B–F) and OMP (G–K) cRNA probes. In both OE and VNO, the expression of Goofy mRNA precedes that of OMP mRNA. Scale bar: B–K, 500 μm.
- Figure 4.
Promoter analysis of Goofy gene. A, Organization of mouse Goofy gene (top). Exons are indicated by boxes with numbers: light blue boxes indicate the open reading frame, and white boxes represent 5′-nontranslated and 3′-nontranslated regions. Red asterisks denote canonical Olf-1-binding sites. Transgene construct (bottom). The Goofy gene's 5′-flanking region (3 kb) was fused to rabbit β-globin intron (gray bar), membrane-targeted Venus cDNA [gapVenus (gV)], and polyadenylation (pA) signals. B, C, Whole-mount fluorescence views of Goofy-gV transgenic mouse at E15.5. Intense gapVenus fluorescence is specifically observed in the olfactory axons projecting from OE to OB even from the outside of embryo. A merged view of fluorescence and visible light is shown in C. D–G, Whole-mount fluorescence views of OE, VNO, OB (D, E), and brain (F, G) from adult Goofy-gV transgenic mouse. Specific and intense fluorescence of gapVenus is detected in the olfactory and vomeronasal axons in all the transgenic mice examined (n = 5). Merged views of fluorescence and visible light are shown in E and G. Cx, Cerebral neocortex; Cb, cerebellum. Scale bars: B, C, 1 mm; D–G, 2 mm.
- Figure 5.
Localization of Goofy protein. A–F, Double immunofluorescence labeling of a coronal section of P3 mouse head with anti-Goofy (A, C, D, F) and anti-OMP (B, C, E, F) antibodies. D–F, Higher magnification of OE. C, F, Merged images showing Goofy (red), OMP (green), and DAPI (blue). G–R, Double fluorescence labeling of adult OE sections with various organelle markers (green) and anti-Goofy antibody (M–R, magenta). The organelle markers include anti-BiP (G, M, endoplasmic reticulum;), anti-GM130 (H, N, cis-Golgi), WGA (I, O, trans-Golgi), anti-EEA1 (J, P, early endosome), anti-Lamp-1 (K, Q, lysosome), and anti-Mcl-1 (L, R, mitochondria). Goofy is predominantly localized to cis-Golgi and trans-Golgi apparatus (N, O). Reproducibility of the result was confirmed for individual markers in at least three experiments. Scale bars: A–C, 500 μm; D–F, 100 μm; G–R, 20 μm.
- Figure 6.
Generation of Goofy-deficient mice. A, Gene targeting strategy to generate Goofy-deficient mice. A pgk-neo selection marker was inserted into the Goofy gene. DTA, Diphtheria toxin A subunit. B, PCR genotyping of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. C, Immunoblot analysis of Goofy protein. Goofy protein is absent in OE homogenates from mutant mice. D–I, Nissl-stained sections of OE (D, E, G, H) and OB (F, I) from wild-type (D–F) and Goofy-deficient (G–I) mice. No obvious difference in gross anatomy is observed at both low (D, F, G, I) and high (E, H) magnifications. J–Y, Immunofluorescence labeling of OB sections from wild-type (J–Q) and Goofy-deficient (R–Y) mice with antibodies against NCAM (J, R), OCAM (K, S), NQO1 (L, T), neuropilin-1 (M, U), neuropilin-2 (N, V), BIG-2 (O, W), Olfr73 (P, X), and Olfr1507 (Q, Y). No difference is observed in the expression of cell adhesion molecules (J–O, R–W) and the olfactory axon convergence to both lateral and medial target glomeruli (P, Q, X, Y). Scale bars: D, G, J–O, R–W, 500 μm; E, H, P, Q, X, Y, 200 μm.
- Figure 7.
Abnormal localization of ACIII in Goofy-deficient mice. A–J, Immunofluorescence labeling of OE (A–D, F–I) and OB (E, J) sections with antibodies against Goofy (A, F), Golf (B, G), CNGA2 (C, H), and ACIII (D, E, I, J). In wild-type mice, Golf, CNGA2, and ACIII are highly localized to olfactory cilia, while strong ACIII immunoreactivity is observed also in olfactory axons and glomeruli of mutant mice. K, Immunoblot analysis of ACIII protein in olfactory cilia, whole OE, and OB from three individual mice of each genotype. In Goofy-deficient mice, ACIII is significantly decreased in the cilia (60 ± 7%), slightly decreased in the OE (94 ± 2%), and dramatically increased in the OB (496 ± 41%), compared with wild-type mice (n = 3). L–N, Double immunofluorescence labeling of wild-type OE section with anti-Goofy (L, N) and anti-ACIII (M, N) antibodies. N, A merged image showing colocalization of Goofy (magenta) and ACIII (green) at Golgi apparatus in OSNs. Scale bars: A–D, F–I, 200 μm; E, J, 500 μm; L–N, 20 μm.
- Figure 8.
Shortened olfactory cilia in Goofy-deficient mice and dnPKA-expressing transgenic mice. A, B, Whole-mount OE preparations from wild-type (A) and Goofy-deficient (B) mice immunolabeled with anti-Olfr2 (I7, magenta) and anti-Olfr6 (M50, green) antibodies. The length of both Olfr2-expressing and Olfr6-expressing olfactory cilia in Goofy-deficient mice is markedly shorter than that in wild-type mice. C–E, Double immunofluorescence labeling of an OE section from Olfr73-ires-gapEGFP transgenic mouse on Goofy-deficient background with anti-Olfr73 (C, E) and anti-GFP (D, E) antibodies. E, A merged image showing perfect overlapping of Olfr73 (magenta) with GFP (green) in the olfactory cilia. F–K, Immunofluorescence labeling of OE sections from wild-type (F–H) and Goofy-deficient (I–K) mice with antibodies against Olfr2 (I7; F, I), Olfr17 (P2; G, J), and Olfr73 (mOR-EG; H, K). L–N, Cumulative frequency plot for length of olfactory cilia expressing Olfr2 (L), Olfr17 (M), and Olfr73 (N) in wild-type (blue) and Goofy-deficient (red) mice. Thin broken lines represent the results from individual mice (n = 3 mice for each genotype; n = 259–805 OSNs for each mouse), while bold lines represent pooled data (n = 1196–1815 OSNs). Insets, Averages of cilia length. For all the three ORs, the length of olfactory cilia in Goofy-deficient mice is significantly shorter than that in wild-type mice. O, Cumulative frequency plot for length of rat I7-expressing olfactory cilia in I7-ires-ECFP (light blue) and I7-ires-dnPKA-ires-EYFP (green) transgenic mice. Thin broken lines represent the results from individual mice (n = 3 mice for each genotype; n = 292–693 OSNs for each mouse), while bold lines represent pooled data (n = 1538 for I7-ires-ECFP; n = 1229 for I7-ires-dnPKA-ires-EYFP). Insets, Averages of cilia length. P, Q, Immunofluorescence labeling of coronal sections of OE from I7-ires-ECFP (P) and I7-ires-dnPKA-ires-EYFP (Q) transgenic mice with anti-rat I7-specific antibody. **p < 0.01 (2-tailed t test). Scale bars: A, B, 20 μm; C–K, P, Q, 50 μm.
- Figure 9.
Reduced olfactory sensitivity in Goofy-deficient mice. A–C, Electrophysiological properties of OSNs assessed by measurement of odor-evoked EOG responses (A, B). Representative EOG traces to eugenol, 2-heptanone, and 1-heptanol in wild-type (A) and Goofy-deficient (B) mice. C, Relationship between applied eugenol concentrations and EOG responses in wild-type (blue) and Goofy-deficient (red) mice (n = 12 for each genotype). *p < 0.05 (2-tailed t test). D, E, Behavioral responses of wild-type (blue) and Goofy-deficient (red) mice to an aversive odor TMT stimulation (n = 8–10 for each genotype and each TMT concentration). Freezing time (D) and avoidance index (E) were measured for the two genotypes to different amounts of TMT. *p < 0.05 (Mann–Whitney U test).
