Phalloidin staining reveals the presence of actin-rich rod-like projections, distinct from OSN microvilli and cilia, in the zebrafish larval and juvenile olfactory epithelium. (A) Maximum intensity projection of an Airyscan confocal image of phalloidin stain in an olfactory pit of a 5 dpf wild-type larva; anterior to the top right, lateral to the bottom right. Arrowhead marks one example olfactory rod. Scale bar = 20 μm. (A′) Enlargement of olfactory rods in panel (A). Scale bar = 5 μm. (B) Dorsal view low power image of phalloidin stain in the head of an 18 dpf (5 mm) wild-type juvenile zebrafish; anterior to the top. Arrowhead marks the position of two olfactory rods in an olfactory pit. Scale bar = 50 μm. (B′) Enlargement of OE in panel (B). Arrowhead marks two olfactory rods. Scale bar = 10 μm. (C–C″) Airyscan confocal image of Alexa-phalloidin signal (C), acetylated α-tubulin immunohistochemistry signal (C′), and merged signals (C″) in an olfactory pit of a 4 dpf wild-type larva; anterior to the top, lateral to the right. Arrowhead marks one example olfactory rod. Scale bar = 20 μm. (C″′) Enlargement of olfactory rod in panel (C″). Scale bar = 5 μm. (D) Differential interference contrast (DIC) image and phalloidin stain (red) in an olfactory pit of a 5 dpf wild-type larva; anterior to the top, lateral to the right. Arrowhead marks one example olfactory rod. Scale bar = 20 μm. (D′) Enlargement of olfactory rods in panel (D). Surrounding olfactory cilia are visible and unlabelled by Alexa-phalloidin. Scale bar = 5 μm. (E) A map of the positions of olfactory rod cell projection bases in olfactory pits of 4 dpf wild-type larvae (N of olfactory pits = 5), based on 2D maximum intensity projections of confocal images of phalloidin stains; anterior “A” to the top, lateral “L” to the right. One dot represents one olfactory rod. Different coloured dots represent olfactory rods from different larvae. (F) Airyscan confocal image of phalloidin stain in an inner ear crista of a 5 dpf wild-type larva. Hair cell stereocilia are labelled with Alexa-phalloidin, and are arranged in a stepped array. In the stereociliary bundle on the extreme left, four different stereociliary lengths are visible [compare with panel (A′)]. Scale bar = 5 μm.

Olfactory rod cells arise early during zebrafish olfactory pit development. (A) Maximum intensity projections of Airyscan confocal images showing the wild-type development of olfactory pit and olfactory rod cells at various embryonic and larval stages, using Alexa-phalloidin as a marker; anterior “A” to the top, lateral “L” to the right. Grayscale values from the original fluorescence image have been inverted. Arrowhead marks one example olfactory rod. Scale bar = 20 μm. Selected inserts show olfactory rods at higher magnification. (B) The change in number of olfactory rod cells per olfactory pit during embryonic development—36 hpf (N of olfactory pits = 4), 38 hpf (N = 5), 40 hpf (N = 7), 42 hpf (N = 4), 44 hpf (N = 7), 46 hpf (N = 6), 48 hpf (N = 9), 3 dpf (N = 5), 4 dpf (N = 10), and 5 dpf (N = 7). Bars indicate mean ± SEM for each stage. Linear regression analysis; * * * * indicates P < 0.0001. (C) The change in lengths of olfactory rod cell projections during embryonic development—36 hpf (N of olfactory pits = 2, n of olfactory rods = 4), 38 hpf (N = 4, n = 17), 40 hpf (N = 6, n = 11), 42 hpf (N = 3, n = 7), 44 hpf (N = 5, n = 7), 46 hpf (N = 6, n = 20), 48 hpf (N = 9, n = 20), 3 dpf (N = 5, n = 32), 4 dpf (N = 10, n = 82), and 5 dpf (N = 8, n = 71). Bars indicate mean ± S.E.M. for each stage. Linear regression analysis; * indicates P = 0.0251, * * * indicates P = 0.0009.

Olfactory rod cells are present in the olfactory epithelia of ift88^–/– zebrafish mutants, which lack cilia. (A,B) Maximum intensity projections of Airyscan confocal images of phalloidin stains of a 5 dpf wild-type (A) and ift88^–/– mutant (B) larva; dorsal views, anterior to the top. Grayscale values from the original fluorescence image have been inverted. Abbreviations: nm, cranial neuromast; op, olfactory pit. Several olfactory rods (arrowheads mark examples) are visible in each olfactory pit. Scale bar = 50 μm. (C) SEM of the head of a 4 dpf wild-type larva. Scale bar = 100 μm. (D,E) SEM of 4 dpf larval wild-type olfactory pits [enlarged from panel (C)]. Scale bars = 10 μm. Insert in panel (D) shows enlarged view of boxed area in panel (D). Arrowhead marks the tip of an olfactory rod cell apical projection surrounded by olfactory cilia. (E′) Enlarged view of boxed area in panel (E). Arrowhead marks one olfactory rod. Scale bar = 5 μm. (E″) Enlargement of olfactory rod in panel (E′) (arrowhead). Scale bar = 1 μm. (F) Frontal view SEM of the head of a 4 dpf ift88^–/– mutant larva. Scale bar = 100 μm. (G) A map of the positions of olfactory rod cell projection emergence through the OE in ift88^–/– mutant larvae (N of olfactory pits = 3), based on SEM images at 4 dpf; anterior “A” to the top, lateral “L” to the right. One dot represents one olfactory rod. Different coloured dots represent olfactory rods from different larvae. (Compare with Figure 1E). (H) SEM of 4 dpf larval ift88^–/– mutant olfactory pit [enlarged from panel (F)]. Scale bar = 10 μm. (H′) Enlarged view of boxed area in panel (H). Arrowhead marks one example olfactory rod cell projection present despite the loss of cilia. Scale bar = 5 μm. (I) Enlarged SEM of olfactory rods (arrowhead marks example) in 4 dpf larval ift88^–/– mutant olfactory pit (from a different individual). Scale bar = 1 μm.

Olfactory rods are labelled in the olfactory epithelia of live zebrafish larvae by the Tg(actb2:Lifeact-RFP) transgene. (A) Maximum intensity projection of dorsal view image of the olfactory pits of a live 6 dpf Tg(actb2:Lifeact-RFP) transgenic larva; anterior to the top. Arrowhead marks one example olfactory rod positive for the Lifeact-RFP transgene. Scale bar = 50 μm. (B) Enlargement of olfactory rods in panel (A) [arrowhead in panel (A)] oscillating during raster-scanned image capture. (Raster scanning was performed from top to bottom in the image, as it has been rotated 90° clockwise) (see Supplementary Movie 2). Scale bar = 5 μm. (C) Maximum intensity projection image of a live 4 dpf Tg(actb2:Lifeact-RFP);Tg(elavl3:H2B-GCaMP6s) double-transgenic larval olfactory pit; anterior to the top, lateral to the right. Arrowhead marks one example olfactory rod positive for the Lifeact-RFP transgene (magenta). Neuronal nuclei are labelled in green. Larvae were fully mounted in agarose, so olfactory rods were not moving. Scale bar = 20 μm (see Supplementary Movie 1). (D) A map of the positions of olfactory rod cell projection bases in olfactory pits of 4 dpf Tg(actb2:Lifeact-RFP);Tg(elavl3:H2B-GCaMP6s) double-transgenic larvae (N of olfactory pits = 2), based on 2D maximum intensity projections of confocal images; anterior “A” to the top, lateral “L” to the right. One dot represents one olfactory rod. Different coloured dots represent olfactory rods from different larvae, with purple corresponding to panel (C). (Compare with Figure 1E). (E) A quantitative comparison of the lengths of olfactory rod cell projections in fixed larvae, using Alexa-phalloidin as a marker (N = 10, n of olfactory rods = 82) versus live larvae, using Lifeact-RFP as a marker (N = 2, n = 43). Violin plot; bars indicate the median and lower and upper quartiles for each group. Mann-Whitney U test; ns, not significant (P = 0.232).

Olfactory rod cells are labelled by the cytoplasmic neuronal markers Tg(Xla.Tubb:jGCaMP7f) and Tg(elavl3:GCaMP6f). (A) Olfactory pit of a 4 dpf Tg(Xla.Tubb:jGCaMP7f) larva; anterior to the top, lateral to the right. Arrowhead marks one example olfactory rod, albeit faintly labelled. Scale bar = 20 μm. (A′) Enlargement of olfactory rod marked by arrowhead in panel (A) (grayscale values inverted). Scale bar = 10 μm. (B) Olfactory pit of a 5 dpf Tg(elavl3:GCaMP6f) larva; anterior to the top, lateral to the right. Arrowhead marks one example olfactory rod, albeit faintly labelled. Scale bar = 20 μm. (B′) Enlargement of olfactory rod marked by arrowhead in panel (B) (grayscale values inverted). Scale bar = 10 μm. (C–C″) Lifeact-RFP signal (C), GCaMP6f signal (C′), and merged signals (C″) in an olfactory pit of a 5 dpf Tg(elavl3:GCaMP6f);Tg(actb2:Lifeact-RFP) double-transgenic larva; anterior to the top, lateral to the right. The trace shows levels of red and green fluorescence along the dotted line, which passes through three olfactory rods positive for both Lifeact-RFP and GCaMP6f. The olfactory rod highlighted with the arrowhead shows similar levels of fluorescence in both the red and green channels. Scale bar = 20 μm.

Olfactory rod cells in the zebrafish olfactory epithelium are not hair-cell-like. (A–A″) Maximum intensity projection of Airyscan confocal image of Alexa-phalloidin signal (A), Tg(pou4f3:GFP) signal (A′), and merged signals (A″) in a cranial neuromast of a 5 dpf larva. Scale bar = 10 μm. (B–B″) Airyscan confocal image of Alexa-phalloidin signal (B), Tg(pou4f3:GFP) signal (B′), and merged signals (B″) in an olfactory pit of a 5 dpf larva; anterior to the top, lateral to the right. Arrowhead marks one olfactory rod. Scale bar = 20 μm. (C,F) Widefield imaging of 3 dpf Tg(pou4f3:GFP) larvae showing the damaging effects of 500 μM neomycin treatment for 60 min on lateral line neuromast hair cells. Fluorescence is lost or greatly reduced in both trunk (arrowhead) and cranial neuromasts, whereas fluorescence in hair cells of the inner ear maculae and cristae (arrow) is unaffected. Scale bars = 500 μm. (D,G) Maximum intensity projections of Airyscan confocal images showing the damaging effects of 500 μM neomycin treatment for 60 min on hair cells in a cranial neuromast of a 3 dpf larva, using Tg(pou4f3:GFP) (green) and Alexa-phalloidin (magenta) as markers. Scale bars = 10 μm. (E,H) Maximum intensity projections of Airyscan confocal images showing no effect of 500 μM neomycin treatment for 60 min on olfactory rods, using Alexa-phalloidin as a marker; anterior to the top, lateral to the right. Arrowheads mark olfactory rods. Scale bars = 20 μm. (I) The number of olfactory rod cell projections per olfactory pit of 3 dpf Tg(pou4f3:GFP) larvae after 500 μM neomycin treatment for 60 min (N of olfactory pits = 4), compared with an untreated group (N = 4). Bars indicate mean ± SEM. Welch’s unpaired two-tailed t-test; ns, not significant (P = 0.8018).

Olfactory rod cells are apically located in the zebrafish olfactory epithelium, with a rounded cell body and no detectable axon. (A–B″) Airyscan confocal image of Alexa-phalloidin signal (A,B), Tg(sox10:Lifeact-mRFPruby) signal (A′,B′), and merged signals (A″,B″) in olfactory pits of 4–5 dpf larvae; anterior to the top, lateral to the right. Arrowhead marks one olfactory rod negative for Lifeact-mRFPruby. Arrow marks one olfactory rod positive for Lifeact-mRFPruby. Scale bars = 20 μm. (C) Number of olfactory rod cells positively marked by Alexa-phalloidin (n of olfactory rods = 59), compared with the number of those also marked by Tg(sox10:Lifeact-mRFPruby) (n = 38), in olfactory pits of 4–5 dpf larvae (N of olfactory pits = 5). Connecting lines indicate olfactory rods from the same olfactory pit. Paired two-tailed t-test; * indicates P = 0.0146. (D) Enlargement of two microvillous OSNs, expressing Lifeact-mRFPruby, in the OE of a 4 dpf larva; Alexa-phalloidin signal (green), Tg(sox10:Lifeact-mRFPruby) signal (magenta). Arrowhead marks the microvillous apical projections. The gamma value for the magenta channel in the bottom half of the panel has been set to 0.5 to show the axon from one of the cells (arrow). Scale bar = 5 μm. (E) Enlargement of olfactory rod cells (of which both the apical actin projections and cell bodies are labelled by the Tg(sox10:Lifeact-mRFPruby) transgene) in the OE of a 4 dpf larva; Alexa-phalloidin signal (green), Tg(sox10:Lifeact-mRFPruby) signal (magenta). Arrowhead marks an olfactory rod cell apical projection, positive for both markers. The gamma value for the bottom half of the panel has been set to 0.5 as in panel (D); no axon is visible. Scale bar = 5 μm. See also Supplementary Movie 3.

Olfactory rod cells are present in the olfactory epithelia of sox10^–/– zebrafish mutants. (A) Maximum intensity projection of Airyscan confocal image of phalloidin stain in a 5 dpf larval wild-type olfactory pit; anterior to the top, lateral to the right. Arrowhead marks one example olfactory rod. Scale bar = 20 μm. (B) Airyscan confocal image of phalloidin stain in a 5 dpf larval sox10^–/– mutant olfactory pit; anterior to the top, lateral to the right. Arrowhead marks one example olfactory rod. Scale bar = 20 μm.