Isolation of single OSNs and identification of mature cells expressing a single predominant OR.

(A) Method of isolation of single OSNs. Transgenic zebrafish embryos expressing OMP:RFP were raised to 48hpf. Olfactory epithelia were dissected and dissociated to obtain single-cell suspensions. OMP:RFP expressing neurons were enriched using FACS and loaded onto a Fluidigm C1 microfluidics chip to isolate single cells in singe wells. cDNA synthesis and amplification were carried out directly on the chip. Samples with high yields of cDNA and smooth distributions of RNA lengths were processed though Nextera library prep and Illumina sequencing. (B) A heatmap of normalized marker gene values for 47 single OSN transcriptomes. 5 cells were enriched in markers of progenitor and/or early mature stages (shaded in black on top bar). 42 cells showed strong expression of mature OSN marker genes and low expression of progenitor/earlier mature genes. High OR expression levels (second bar:cyan) were detected in 29 of these mature cells (shaded in magenta on top bar). Three color keys are shown at the bottom: the first pertains to cell age inferred in the topmost bar, the second pertains to log10 values of normalized (predominant) OR expression, and the third relates to log10 values of normalized developmental gene expression.

Confirmation of differential gene expression using dual fluorescent in situ hybridization.

(A) Dual-FISH of nrp1a with an or111 sub-family probe cocktail. Two cells have been highlighted to show that nrp1a signal (red) is localized in cells with or111 subfamily labelling (green). (B) Summary of dual-FISH expression for four candidate guidance receptors. The percentage of OSNs expressing the indicated OR subfamily that also expressed the indicated candidate guidance transcript are noted. The total number of OR subfamily expressing cells counted are shown in parentheses. Pink shading indicates transcripts that showed higher overlap with the or111 sub-family probe-mix and green shading indicates transcripts that showed higher overlap with or133 sub-family probe mix. nrp1a shows greater overlap with or111 subfamily expression than with or133 subfamily expression. nrp1b, pcdh11, and robo2 show greater overlap with or133 subfamily expression than with or111 subfamily expression.

Nrp1b and robo2, but not nrp1a, are required for axonal targeting to the DZ.

Tg(BACor111-7:IRES:GAL4; UAS:citrine) (‘or111-7’) expressing OSNs project axons to the CZ in wild-type olfactory bulbs (A, D, G) while Tg(BACor130-1:IRES:GAL4; UAS:citrine) (‘or130-1’) expressing OSNs project axons to the DZ in wild type olfactory bulbs at 72 hpf (J, M, P). In nrp1a-/- larvae, or111-7 axons misproject to the DZ (B, C). Mistargeting of or111-7 axons is not observed in nrp1b-/- (E,F) or robo2-/- larvae. (H, I). However, in nrp1b-/- larvae, or130-1 axons misproject to the CZ (N, O). Similarly, robo2-/- larvae show misprojections of or130-1 axons to the MG (Q, R). Mistargeting of or130-1 axons is not observed in nrp1a-/- larvae (K,L). Fisher’s exact test (two-tailed) * = p<0.05, ** = p< = 0.005. CZ: central zone, DZ: dorsal zone, MG: medial glomerulus, LG2: lateral glomerulus 2, LG3: lateral glomerulus 3. OE: olfactory epithelium, OB: olfactory bulb.