Nature Medicine doi: /nm.2860
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1 Supplemental Figure Legends Supplemental Figure 1: Hypomorphic expression of IFT88 results in olfactory signaling proteins no longer localizing to the ciliary layer. (a) ACIII localizes to the cilia and co-localizes with acetylated α tubulin in WT mice but is absent from the cilia layer of ORPK mice. (b) CNGA2 localizes to the cilia layer where it co-localizes with acetylated α-tubulin, but is also absent from the apical surface of ORPK mice. (c) Gy13 localizes to the cilia in WT mice, and is absent from the apical surface of ORPK mice. (d) Quantification of OE thickness in WT and ORPK mice. The OE of P19-P21 ORPK mice are not significantly thinner than WT mice (n = 3 mice). (e) Representative EOG traces from WT and ORPK mutant littermate mice in response to 3 dilutions of amyl acetate. (f) Quantification of fluorescence intensity of TH staining in WT and ORPK showing the decrease seen in ORPK mice ( n = 19 WT, 17 ORPK glomeruli. p < 0.005, students t-test). Scale Bars, a,b: 5µm, e-g: 10µm. Supplemental Figure 2: Localization of IFT88:GFP recapitulates endogenous IFT88. (a-c) IFT88:GFP localizes to the dendritic knobs (arrowheads) of OSNs co-localizing with acetylated α tubulin. Both IFT88:GFP (d-f) and endogenous IFT88 (g-i) localize to the cilia layer, but are not detected in cell bodies or the axons of OSNs. OSN axon bundles are circled in the lamina propria. (j) En face image of fixed tissue showing IFT88:GFP is detected around the dendritic knob (arrows) and restricted to the proximal region of olfactory cilia. (k) Antibody staining for endogenous IFT88 also shows an enrichment of IFT88 in the proximal regions of olfactory cilia, matching IFT88:GFP. (l) Fixation does not affect IFT88:GFP localization, as IFT88:GFP is still enriched in the proximal regions of olfactory cilia in live tissue. (m-n) En face imaging of live OSNs co-infected with IFT88:GFP and Arl13b:mCherry reveals puncta of IFT88:GFP along Arl13b:mCherry labeled cilia. (o) Schematic of the cilia on a single OSN, highlighting the proximal and distal segments. NC; nasal cavity, OE; olfactory epithelium. Scale bars, 10µm Supplemental Figure 3: Adenoviral mediated expression of IFT88 in ORPK OSNs restores olfactory cilia and function. (a-d) OSNs expressing IFT88:GFP have cilia as seen by acetylated α-tubulin staining around GFP positive knobs. (e-h) Adenoviral delivery of IFT88iresMyrPalm:GFP also restores cilia to infected OSNs. Staining for acetylated α tubulin in the OE of IFT88iresMyrPalm:GFP infected ORPK animals shows cilia only around infected neurons. The acetylated α-tubulin staining overlaps with MyrPalm:GFP found in the cilia. (i-l) In WT mice infected with IFT88iresMyrPalm:GFP, a mosaic expression pattern of S100a5 is present. (m-p) Adenovirus infection does not induce neuronal activity on its own as S100a5 (low) OSNs expressing IFT88iresMyrPalm:GFP are found in the WT OE (q-t) Acetylated α-tubulin and S100a5 labeling is absent in regions of OE from ORPK mice infected with IFT88iresMyrPalm:GFP in which OSNs were not transduced with virus. (u-x) Expression of S100a5 within cells and acetylated α-tubulin in the cilia layer returns to OSNs expressing IFT88iresMyrPalm:GFP. Scale Bars, 10µm Supplemental Figure 4: Localization of ectopic IFT88:GFP in ORPK OSNs is similar to WT OSNs. (a) En face image of WT OSNs expressing IFT88:GFP showing enrichment in the proximal regions of the cilia. (b). En face image of ORPK OSNs showing similar enrichment of IFT88:GFP in proximal regions of the cilia. Scale bars, 10µm Supplemental Figure 5: Expression of IFT88:GFP does not alter EOG responses in WT mice. (a) Quantified responses of WT and WT mice treated with IFT88:GFP to dilutions of Amyl Acetate. (n = 5 mice) (b) Linear regression of correlation between TH staining to GFP fluorescence in ORPK mice treated with IFT88:GFP (closed circles, r = 0.37) and to Cherry fluorescence in ORPK mice treated with Arl13b:mCherry (open circles, r = 0.006). Supplemental Figure 6: Arl13b:mCherry expression does not restore olfactory cilia or olfactory function. (a, b) Acetylated α-tubulin staining in WT littermate and ORPK infected with Arl13b:mCherry. Adenoviral infections of ORPK mice with Arl13b:mCherry fail to show acetylated α-tubulin expression along the apical surface of the OE. (c, d) EOG responses of untreated WT littermates and ORPK mice treated with Arl13b:mCherry, showing that Arl13b:mCherry does not restore EOG response (n = 3 mice, p < 0.05, students t-test). (e, f) Ectopic expression of Arl13b:mCherry fails to return S100a5 staining to ORPK mice (g, h) TH staining is still dramatically reduced in ORPK mice treated with Arl13b:mCherry. Scale Bars; 10µm Supplemental Figure 7: Mature OSNs are preferentially infected by adenovirus. (a-c) Analysis of GFP expression in the OE 24hrs post infection reveals that the majority of infected cells are OMP + mature OSNs (92.3%, n = 3 mice), (d-f) Some GFP expressing neurons are immature OSNs as determined by Gap43 staining (8.7%, n = 3 mice). (g) Quantification of cell counts of co-localization of OMP and Gap43 with Nature Medicine doi: /nm.2860
2 GFP. (h-j) WT mice were infected with both IFT88:GFP and mcherry and analyzed 4 weeks post infection. Expression of both constructs was still detected at this time point allowing us to count hundreds of infected OSNs. (k-p) Timed pregnant mice were injected with BrdU at E14.5 to label dividing cells. Pups were then analyzed at P7 to determine maturation state of BrdU + OSNs. (k-m) By P7 all BrdU + OSNs were also OMP +. (n-p) BrdU + OSNs were negative for Gap43 indicating that by P7 all OSNs that are BrdU + are mature. (q-v) Mice were infected with mcherry adenovirus on days P4 and P5 and analyzed at age P9. (q-s) Analysis of mcherry expression in the OE at P9 reveals all infected OSNs are OMP + mature neurons. (t-v) By age P9, mcherry + OSNs were Gap43 negative. Scale Bars; 10µm Supplemental Figure 8: ORPK cortical neurons lack primary cilia (a-c) Dissociated WT cortical neurons at DIV5 cortical possess primary cilia that are positive for both polyglutamylated α-tubulin and ACIII. (d-f) ORPK neurons do not possess and identifiable primary cilium. Scale bars; 10µm. Supplemental Table 1: Ciliopathy samples with IFT88Met383Lys mutations Phenotype Sample ID IFT88 genotype Ethnicity Additional Ciliopathy mutations MKS-like MKS_542 Met383Lys hom France BBS7: p.y671c het; TTC21B: p.f60y het MKS-like 102 Met383Lys het Pakistani MKS1: c.1448_1451dupcagg hom MKS-like 244 Met383Lys het Pakistani MKS-like 247 Met383Lys het Spanish MKS 324 Met383Lys het Pakistani JATD PB15 Met383Lys het E. European JATD PB34 Met383Lys het N. European Nature Medicine doi: /nm.2860
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