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 mouse models exhibit impaired olfactory function. To explore the olfactory defects in BBS, we ablated the mouse Bbs8 gene. BBS8 protein is particularly abundant in olfactory sensory neurons (OSNs) and localizes to the dendritic knob in a shell-like structure surrounding the basal bodies. Bbs8-null mice have reduced olfactory responses and immunohistochemical analyses reveal a dramatic loss of cilia from OSNs and mislocalization of proteins normally enriched in cilia. To better visualize altered protein localization in OSN cilia, we generated a SLP3eGFP knock-in mouse and imaged the apical epithelium ex vivo. Additionally, protein reagents reflecting the characteristic neuronal activity of each OSN revealed altered activity in Bbs8-null cells. We also observed aberrant targeting of OSN axons to 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.

We next developed systems to study protein transport mechanisms within the OSN dendrite and cilia. A photoswitchable fluorescent tag was knocked into the 3' end of the adenylyl cyclase type III (AC3) gene to generate a fusion protein. The fusion protein is enzymatically functional and retains wild-type localization to cilia. Our inability to generate homozygous AC3^psCFP/psCFP mice lead to the finding that the knock-in construct ablated an overlapping gene that is likely essential for cell viability. Crosses with mice heterozygous for an AC3 null allele produced AC3^psCFP/- mice, which will be useful in further imaging and biochemical experiments.

Finally, we used the SLP3eGFP mouse line to study the proteomics of SLP3 function in OSNs. We performed immunoprecipitation with anti-GFP antibodies and tandem mass spectrometry to identify interacting proteins, ultimately identifying a short list of candidates. Thus, the SLP3eGFP mouse is a useful model for elucidating OSN biology.