Olfactory epithelium (OE) in 2.5-dpf zebrafish. Multi-ciliated motile cilia are present at the border, ciliated OSNs in the centre; maximum intensity projection. a Basal bodies stained with anti-cep290 (red), a’ cilia stained with anti-acetylated tubulin (green), a” merged image. Inset in a” closeup of sensory (left) and motile (right) cilia showing basal body staining of anti-Cep290. b EM image of dendritic knob of ciliated zebrafish OSNs containing multiple basal bodies (arrowheads). c Tg(omp:mCherry,trpc2:Venus) zebrafish with ciliated Omp-positive OSNs (red) and c’ microvillus TrpC2-positive OSNs (green), c” merged image; single confocal section. d Quantification of C, N = 5 fish. e Tg(omp:mCherry,elavl3:GCaMP5) zebrafish show overlap between ciliated Omp-positive OSNs (red) and e’ GCaMP5-positive OSNs (green) OSNs, e” merged single confocal section. f Quantification of E, N = 6 fish. Bars represent mean and SEM. Scale bar is 10 µm, except b bar is 1 µm

Cilia defects have functional effects in OSNs in Ift-deficient zebrafish at 2.5 dpf. a Anti-acetylated tubulin (green) stains all cilia axonemes in wildtype embryos as well as neuronal processes projecting away from the olfactory placode. a’ Anti-Ga/olf (red) is a marker for OSN cilia. a” Merged image of a and a’. b ift88 mutant homozygotes show loss of acetylated tubulin positive axonemes and b’ loss of anti-Ga/olf staining. b” Merged image of b and b’. c Control morpholino injected olfactory placode stained with anti-acetylated tubulin and c’ anti-Ga/olf. c” Merged image of c and c’. d ift172 morphant olfactory placodes show loss of anti-acetylated tubulin staining and d’ loss of anti-Ga/olf staining. d” Merged image of d and d’. Remaining anti-acetylated tubulin staining is restricted to neuronal processes [67] (b, d). For quantification see Table 1. e Significant change in percentage of odour responding OSNs per embryo after addition of bile acids and food odour in Tg(elavl3:GCaMP5) ift88 mutant OSNs compared to sibling OSNs (N = 637 OSNs, 10 fish for sibling and N = 720 OSNs, 9 fish for mutant). f Significantly reduced response amplitude to bile acids and food odour in Tg(elavl3:GCaMP5) ift88 mutant responding OSNs compared to sibling responding OSNs. g Whole mount in situ hybridization using an omp probe demonstrates clear omp expression in the OE in both wild type siblings g and g’ ift88 mutants and siblings at 2 dpf. (For other time points see Additional file 1: Fig. S6A). h Ciliated OSNs stained with anti-GFP (green) and anti-Ga/olf (red) present in both h sibling and h’ ift88 mutant, based on cell shape (arrows) in Tg(elavl3:GCaMP5) fish. i No difference in number of GCaMP5 positive OSNs per fish (P =  0.10). Bars represent mean and SEM (*P < 0.05, **P < 0.01, ***P < 0.001 Mann–Whitney U test for e and f, student’s t test for i. Scale bar is 10 μm, except panel g bar is 100 μm

Effect of ift172-deficiency on cilia-dependent olfactory sensory neuron signalling in 2.5-dpf-old zebrafish. a Significant change in percentage of responding cells per embryo to nucleotides in Tg(elavl3:GCaMP5) ift172 MO OSNs compared to control MO OSNs (N = 517 OSNs, 5 fish for control MO and N = 495 OSNs, 6 fish for ift172 MO). b Significantly reduced response amplitude to bile acids and food odour in Tg(elavl3:GCaMP5) ift172 MO responding OSNs compared to control MO responding OSNs. c No difference in Omp-positive OSNs between control and ift172 MO Tg(omp:gal4,UAS:YPF) fish. d Quantification of C (N = 3 control MO, N = 4 ift172 MO, P = 0.33). e No difference in number of GCaMP5-positive OSNs per embryo (P = 0.16). Bars represent mean and SEM (*P < 0.05, **P < 0.01, ***P < 0.001 Mann–Whitney U test for a, b, Student’s t test for d, e). Scale bar is 10 µm

Severe reduction of IFT88 protein (anti-IFT88 antibody in red) expression is present in the cilia of the OE (marked by anti-acetylated tubulin staining) of the ift88 mutant (B) compared to the ift88 wildtype sibling (A). (C) Quantification of the signal in the red (IFT88) channel demonstrated a 99% decrease in intensity of the red staining. (N=3 fish per condition, P=5.5E-7, Student's t-test). Bars represent mean and SEM. Scale bar is 10 µm.

(A) Phenotype of ift172 MO fish, arrow points at kidney cyst. (B) Larger ift172 PCR product using ift172 cDNA as a template in ift172 morphant. (C) Sequence analysis reveals that injection of ift172 MO leads to retention of intron 1 in cDNA of morphants, leading to a predicted 16 amino acids peptide instead of the 1745 amino acids of the original protein.

Olfactory sensory cilia deficit in oval/ift88 -/- mutants. (A) Olfactory sensory cilia in the center of a wild type olfactory placode stained with anti-Ift88 (red) and anti-acetylated tubulin (green; cilia) and imaged in confocal Z-stacks. Ift88 immunoreactivity was strongest in basal bodies (bb's). Scale bar in (A) equals 1 µm. (B) Wild type olfactory placode stained with anti-acetylated tubulin (green; cilia and neuronal cell body processes) and anti-cep290 (red; basal bodies). (C) oval mutant olfactory placode stained with anti-G _/olf (red) and anti-acetylated tubulin (green) shows cilia loss with some short, residual G _{/olf-positive} axonemes (arrowheads). All panels are set at equivalent scale and represent a 3µm thick maximum intensity projection of the center of the olfactory placode.

(A) Whole mount in situ hybridization using the omp probe showing omp expression in olfactory epithelia both in ift88 mutants and wildtype siblings at 2, 3, 4, and 5 dpf. (B) Tg(omp:mCherry) fish demonstrate presence of Omp-positive OSNs in ift88 wildtype siblings (B) and ift88 mutants (B') at 5 dpf. (C) Anti-GFP (green) and anti-G _/olf (red) staining shows presence of ciliated OSNs in the ift88 wildtype sibling, in the ift88 mutant omp-positive cells are detected based on their characteristic flask like shape (arrows) in Tg(elavl3:GCaMP5) fish at 6 dpf. Scale bar is 50 µm in B, and 10 µm in C.

Ciliated OSNs stained with anti-GFP (green) and anti-G _/olf (red) present in both sibling and ift88 mutant. Lower panels: the anti-G _/olf (red) signal only, demonstrating presence of anti-G _/olf (red) staining in the cell bodies of both sibling and ift88 mutant. Scale bar is 10 µm.

Anti-Cep290 antibody validation. In a 10 somite stage zebrafish embryo, the Cep290CT antibody (red) stain punctate structures at the base of Kuppfer's vesicle cilia (anti-acetylated tubulin in green). Staining is lost when the antibodies are preincubated with antigen, demonstrating antibody specificity. Inset: magnified view of a single basal body region. Scale bar is 5 µm.

GCaMP6f expression in the olfactory placode in 2.5 dpf Tg(omp:GCaMP6) larvae. GCaMP6f in Tg(omp:GCaMP6) larvae was selectively expressed in flask shaped OSNs, representing a subset of ciliated OSNs adjacent to non-expressing (asterisk), presumably microvillus OSNs (see figure 1).