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. 2011 Jun 21;108(25):10320-5.
doi: 10.1073/pnas.1016531108. Epub 2011 Jun 6.

Loss of Bardet-Biedl syndrome protein-8 (BBS8) perturbs olfactory function, protein localization, and axon targeting

Abigail L D Tadenev  1 , Heather M Kulaga, Helen L May-Simera, Matthew W Kelley, Nicholas Katsanis, Randall R Reed
Affiliations

Loss of Bardet-Biedl syndrome protein-8 (BBS8) perturbs olfactory function, protein localization, and axon targeting

Abigail L D Tadenev et al. Proc Natl Acad Sci U S A. .

Abstract

Bardet-Biedl syndrome (BBS) is a pleiotropic, heterogeneous human disease whose etiology lies primarily in dysfunctional basal bodies and/or cilia. Both BBS patients and several BBS mouse models exhibit impaired olfactory function. To explore the nature of olfactory defects in BBS, a genetic ablation of the mouse Bbs8 gene that incorporates a fluorescent reporter protein was created. The endogenous BBS8 protein and reporter are particularly abundant in olfactory sensory neurons (OSNs), and specific BBS8 antibodies reveal staining in the dendritic knob in a shell-like structure that surrounds the basal bodies. Bbs8-null mice have reduced olfactory responses to a number of odorants, and immunohistochemical analyses reveal a near-complete loss of cilia from OSNs and mislocalization of proteins normally enriched in cilia. To visualize altered protein localization in OSNs, we generated a SLP3(eGFP) knock-in mouse and imaged the apical epithelium, including dendritic knobs and proximal cilia, in ex vivo tissue preparations. Additionally, protein reagents that reflect the characteristic neuronal activity of each OSN revealed altered activity in Bbs8-null cells. In addition to previously known defects at the ciliary border, we also observed aberrant targeting of OSN axons to the olfactory bulb; axons expressing the same receptor display reduced fasciculation and project to multiple targets in the olfactory bulb. We suggest that loss of BBS8 leads to a dramatic and variable reduction in cilia, the essential signaling platform for olfaction, which alters the uniformity of responses in populations of OSNs expressing the same receptor, thereby contributing to the observed axon-targeting defects.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Genetic ablation of the Bbs8 gene. (A) In situ hybridization reveals Bbs8 mRNA enrichment in OE neurons (Left) and absence in the OE of Bbs8−/− mice (Right). (B) Immunofluorescence localizes BBS8 protein (green) predominantly to dendritic knobs marked by γ-tubulin (red), a basal body protein. The BBS8 protein signal is absent in Bbs8−/− tissue but γ-tubulin staining persists (Right). (C) High-magnification image of OE cryosections at the cilia layer reveals distinct localization within the dendritic knob, as seen in a cross-section (Upper, a higher magnification of B) and in an en face section (Lower). The basal body-associated γ-tubulin staining resides in the core of each knob, and the BBS8 immunofluorescence forms a surrounding shell. (D) Low-power image of an OB cryosection from a Bbs8+/− mouse reveals intense intrinsic fluorescence from the tau:YFP reporter. Signal is seen specifically in OSN axons projecting from the OE through the olfactory nerve layer and terminating in the olfactory glomeruli. [Scale bar: 10 μm (B and C); 500 μm (D).]
Fig. 2.
Fig. 2.
Bbs8−/− mice exhibit reduced responses to odorants. Peak voltage responses in EOG recordings from Bbs8+/− or Bbs8−/− OE exposed to odorants at the indicated molar concentrations. Each bar represents the average responses for Bbs8+/− (n = 12 electrodes/4 mice) or Bbs8−/− mice (n = 13 electrodes/4 mice) ± SD. (Inset) Representative responses to a pulse of 10−4 M amyl acetate.
Fig. 3.
Fig. 3.
Structural and protein localization defects in Bbs8−/− OE. OE cryosections from Bbs8+/− or Bbs8−/− animals were immunostained and imaged by confocal microscopy (A: single plane, B and C: flattened Z-stacks). (A) Cilia abundance, visualized by acetylated tubulin, is dramatically reduced in Bbs8−/− mice. (B) The signal transduction proteins ACIII and ORI7 show reduced intensity in the cilia layer and mislocalization within the dendrite in Bbs8−/− OE. (C) SLP3 shows reduced signal at the cilia layer and accumulation within the dendrite and apical cell body. (D) SEM of the olfactory epithelium of 37-wk-old littermates. [Scale bar: 10 μm (A–C); 2 μm (D).]
Fig. 4.
Fig. 4.
Localization of endogenous SLP3 in OSNs. SLP3:eGFP localizes to the dendritic knob and proximal regions of cilia in low-power (Left) and high-power (Right) confocal images of the apical surface of unfixed whole-mount OE at the level of the dendritic knobs. The membrane that forms the surface of the dendritic knob and the proximal portions of cilia appear as a “starburst” pattern when visualizing the intrinsic SLP3:eGFP signal. (Scale bar: 20 μm.)
Fig. 5.
Fig. 5.
SLP3:eGFP expression and localization is altered in Bbs8−/− OSNs. The fluorescent signals from the Bbs8 promoter-driven tau:YFP reporter (red) and SLP3:eGFP (green) were separated by linear unmixing. (A) In en face images of the cilia layer, SLP3:eGFP is expressed in most, if not all, dendritic knobs of Bbs8+/− mice, but is visible in only a few scattered cells of the Bbs8−/− OE. (B) At high magnification, the altered localization of SLP–3eGFP in Bbs8−/− OSNs is apparent. Specifically, in Bbs8+/− OE, SLP3:eGFP is restricted to the dendritic knob and proximal cilia (A and B, Left). In Bbs8−/− OE (A and B, Right), it is distributed along the length of longer ciliary processes, which also display unusual swellings throughout the extent of the cilia. (Scale bar: 20 μm.)
Fig. 6.
Fig. 6.
Bbs8−/− OSNs display increased variability in S100A5 protein expression. Sections of OE from Bbs8/M72TL mice were stained for lacZ and S100A5 to determine the expression level of S100A5 in individual OSNs from a defined OR population. Images show a lacZ-positive Bbs8+/− OSN with relatively low S100A5 levels (Upper panels) and a Bbs8−/− OSN with high S100A5 levels (Lower panels). (Graph) The mean S100A5 pixel fluorescence intensity, normalized to lacZ intensity, was determined within each cell body for 30 M72-expressing OSNs (n = 3 animals) for each genotype. (Scale bar: 5 μm.)
Fig. 7.
Fig. 7.
OSN axon targeting is perturbed in Bbs8−/− mice. The OR reporter line M72TL was crossed into a Bbs8 mutant background and axon targeting was analyzed by whole-mount X-Gal staining of PD15 mice. In dorsal views, axons project from the OE (on the bottom) to their targets in the OB. In Bbs8+/+ mice, M72-tau:lacZ–expressing axons converge to one dorsal glomerulus in each bulb. In the OB of Bbs8-deficient mice, multiple glomeruli are innervated and individual fibers wander on the surface of the bulb.
Fig. 8.
Fig. 8.
Model for olfactory defects seen in Bbs8−/− mice. (Upper) In WT mice, the transduction proteins are restricted to a uniform complement of long cilia, resulting in consistent activity levels among cells expressing the same OR. Activity levels are correlated with abundance of axon guidance and cell adhesion molecules (colored rings). (Lower) Bbs8−/− OSNs display variability in cilia number and morphology. This leads to increased heterogeneity in neuronal activity and corresponding variability in S100A5 levels among OSNs expressing the same OR, depicted as differently shaded OSNs. The greater variation in abundance and diversity of axon guidance and cell adhesion molecules on the OSN axons would affect the fidelity of targeting.
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