Table 1.
Tools for assessing mammalian cilia and ciliary function
| Antibody | Transgenic Reporter Mice | |
|---|---|---|
| Ciliated cells, % | γ-Tubulin (Sigma Aldrich) with acetylated α-tubulin (Sigma Aldrich) or ARL13B (Proteintech) or ACIII (since tubulin is not acetylated in neurons; Lifespan Biosciences) |
Cilia-GFP (123) (Mouse Genome Informatics,
MGI:5524281) ARL13B-mCherry (7); centrin2-GFP (59) (MGI:5645691; MGI:3793421) FUCCI2a (mCherry-hCdt1 and mVenus-hGem); ARL13B-cerulean (46) (MGI:6193732) |
| Cilia length | ||
| General shape |
| General structure (antibody) | |
|---|---|
| Axoneme | Acetylated α-tubulin (Sigma Aldrich); IFT-B components: IFT88, IFT81, IFT20, IFT27, IFT52, IFT57 (Proteintech); IFT-A components: IFT122, IFT43 (Proteintech) |
| Ciliary membrane | INPP5E, ARL13B (Proteintech) |
| Transition zone | CEP290, MKS1, NPHP4 (Proteintech) |
| Basal body | γ-Tubulin (Sigma Aldrich), centrin (EMD Millipore) |
| Distal appendage | CEP164 (Sigma Aldrich) |
| Centriole | TTBK2 (Sigma Aldrich), CP110 (Proteintech) |
| Ciliary vesicle | RAB11, RAB8a (Cell Signaling) |
| Signaling | ||||
|---|---|---|---|---|
| Antibody | Reporter Construct | qRT-PCR | Transgenic Reporter Mice | |
| Vertebrate hedgehog | SMO (Santa Cruz) | 8X Gli-eGFP (69) (Addgene no. 84602) |
Gli1 Ptch1 Cell-specific targets (i.e., cyclinD1) |
Gli1-LacZ (4)
(MGI:2449767) Ptch1-LacZ (53) (MGI:1857447) |
| Wnt | β-Catenin (DSHB) | BAT Red (Addgene no. 20674) TOPFlash (Addgene no. 12456) |
Axin2 (or in situ via RNAScope) Find cell-specific targets at http://web.stanford.edu/group/nusselab/cgi-bin/wnt/target_genes |
BATgal (111) (MGI:3697064) |
| Calcium | 5HT6-mCherry-G-GECO1.0 (148) (Addgene no. 47500) |
Arl13b-mCherry-GECO1.2 (31) (MGI:5898420) | ||
Listed are antibodies and reporter constructs along with mouse alleles that are useful to assess general mammalian ciliary structure and function. Table prioritizes commercially available reagents to provide a basic framework for investigators and is not intended to be exhaustive. By staining basal body using γ-tubulin, the location of the cilium can be identified so that cilium itself or ciliary compartment or structure can be assessed by costaining with appropriate ciliary marker and imaging. Transgenic mouse lines expressing fluorescent cilia proteins can be used for in vivo analysis. The FUCCI2; Arl13b-cerulean line enables cilia to be live imaged relative to the stage of the cell cycle. To examine the function of cilia, several signaling pathways linked to cilia can be analyzed. Three examples are included. Vertebrate hedgehog (Hh) signaling is the best characterized signaling pathway linked to the cilium. Components of the vertebrate Hh pathway such as smoothened (SMO) enrich dynamically within cilia in response to pathway stimulation. The pathway can be stimulated with ligand (N-Shh; Fisher) or smoothened agonist, SAG (VWR), and blocked with cyclopamine (Toronto Research Chemicals); the downstream transcription output can be monitored with endogenous transcriptional targets (Gli1, Ptch1 by qRT-PCR and Gli1-lacZ, Ptch1-lacZ in vivo) or reporter constructs, such as 8XGli-eGFP. For Wnt signaling, β-catenin localizes to the nucleus when the pathway is stimulated and induces transcription of Axin2, which can be monitored by qRT-PCR or RNAScope. β-Catenin binds TCF/LEF-binding sites that are used in Wnt reporter constructs such as TOPFlash (for transfection), BAT Red (for transduction), or BATgal (for in vivo). Finally, the calcium flux in cilia can be monitored in vitro or in vivo using genetically encoded calcium indicator for optical imaging (GECO) reporters that are targeted to cilia. Targeting sensitive biosensors to cilia is a current active area in the field. ARL, ADP-ribosylation factor-like; IFT, intraflagellar transport; INPP5E, inositol polyphosphate-5-phosphatase E; DSHB, Developmental Studies Hybridoma Bank.