OMP class OSNs project to a limited number of identifiable protoglomeruli but express a wide variety of ORs. a A confocal optical section through the olfactory bulb of a double transgenic 3 dpf embryo showing that OMP class ciliated (green) and TRPC2 class microvillous (red) sensory neurons target non-overlapping protoglomeruli. b OMP class neurons innervate the CZ, DZ, LG3, and to a lesser extent, MG protoglomeruli. c RNA-seq data from 2 dpf FAC sorted and pooled OMP:RFP expressing OSNs. Approximately ½ of the OR gene repertoire is expressed at an average level of over 1 cpm in the pool as a whole (dashed line). d Expression of or111–11 as visualized by in situ hybridization of its mRNA in a 3 dpf embryo. e The average numbers of OSNs expressing selected ORs at 3 dpf. Scale bars: 20 μm

Dual in situ hybridization with probes to multiple ORs are consistent with OSNs having chosen a single odorant receptor to express by 3 dpf. a Examples of pairs of probes or cocktails of probes used to access OR expression in 3 dpf embryos. The first row of panels demonstrates that probes against individual or111 subfamily members or111–7 and or111–11 are highly specific to the target subfamily member, and further suggest, that individual or111 subfamily members are very rarely, if ever, co-expressed in the same OSN. The second row of panels demonstrates that or111 subfamily members are very rarely co-expressed in OSNs expressing or103, or106, or or128 subfamily members. An exceptional case where or111 and or103 subfamily members were coexpressed is marked with an arrowhead. b Quantification of the numbers of probed OR expressing OSNs per olfactory pit. Cells with overlapping expression are indicated (red). Scale bar: 25 μm

Mapping axon projections by sparse labeling of OMP class OSNs. a A single OSN in a 3 dpf embryo was co-labeled by transient transfection with a combination of OMP:Gal4 and UAS:Citrine plasmids and hybridization with a cocktail of or111 subfamily in situ probes (arrow head). The CZ protoglomerulus is visible as a cell free zone in the olfactory bulb (arrow). b Single OMP:Gal4;UAS:Citrine labeled OSNs project to the CZ, DZ, LG3, and MG protoglomeruli (blue bars). Those that are co-labeled with a cocktail of or111 subfamily probes project exclusively to the CZ protoglomeruls (red bars). The numbers of cells observed for each condition are noted within the bars. SEM is indicated. Scale bar: 20 μm

Mapping OSN axon projections with recombineered BACs. a IRES:Gal4 was inserted just after the coding sequences of selected ORs and a heart-specific promoter driving fluorescent GFP was inserted at a second location distant from the co-labeled OR (see text and Methods for details). b Live confocal image from a double transgenic embryo in which or111–7 driven Gal4 activates UAS:Gap43-Citirine. A small number of OSNs are labeled with Citrine allowing the visualization of their cell bodies in the olfactory pits, axonal projections, and sites of termination in the olfactory bulb. c OSN projections in embryos from tgBACor111–7:IRES:Gal4x tgUAS:Citrine or tgBACor130–1:IRES:Gal4 x tgUAS:Citrine lines (left two columns). The same axonal projection patterns are observed in UAS:Citrine embryos transiently transfected with the BACs used to make the transgenic lines (right two columns). 3 dpf fixed embryos were co-stained with anti-GFP (green) and Propidium Iodide (Grey). The CZ (yellow), DZ (red), LG3 (blue), and MG (magenta) protoglomeruli are indicated on each single optical section. or111–7 expressing OSNs project to the CZ protoglomerulus, while or130–1 expressing OSNs project to the DZ protoglomerulus. Scale bar: 20 μm

a OSNs expressing Gal4 from each of three selected TAAR loci all project to multiple protoglomeruli. b OSNs labeled by expression from the taar13b locus projected to all four OMP class protoglomeruli, while OSNs expressing from the taar13c or taar20t loci projected to both OMP and TRPC class protoglomeruli. Scale bar: 20 μm