Multilayer regulation underlies the functional precision and evolutionary potential of the olfactory system

Sensory neurons must be reproducibly specified to permit accurate neural representation of external signals but also able to change during evolution. We studied this paradox in the Drosophila olfactory system by establishing a single-cell transcriptomic atlas of all developing antennal sensory lineages, including latent neural populations that normally undergo programmed cell death (PCD). This atlas reveals that transcriptional control is robust, but imperfect, in defining selective sensory receptor expression. A second layer of precision is afforded by the intersection of expression of functionally-interacting receptor subunits. A third layer is defined by stereotyped PCD patterning, which masks promiscuous receptor expression in neurons fated to die and removes “empty” neurons lacking receptors. Like receptor choice, PCD is under lineage-specific transcriptional control; promiscuity in this regulation leads to previously-unappreciated heterogeneity in neuronal numbers. Thus functional precision in the mature olfactory system belies developmental noise that might facilitate the evolution of sensory pathways Overall design: 10-15 virgin peb-Gal4 females were placed in vials with 5-10 UAS-unc84:GFP or UAS-p35,UAS-un84:GFP males for 5 days, after which adults were removed. White pupae (corresponding to 0 h after puparium formation (APF)) were carefully transferred to fresh vials and aged for 18, 24, 30, 36, 42, 48, 56, 64, 72 or 80 additional hours. Developing antennae from aged pupae were dissected in ice cold Schneider's medium and immediately transferred to 1.8 ml Eppendorf tubes containing 100 µl Schneider's medium, flash-frozen in liquid nitrogen and stored at -80°C. The numbers of antennae dissected in control (peb-Gal4/+,UAS-unc84:GFP/+) and PCD-blocked (peb-Gal4/+,UAS-p35/+,UAS-unc84:GFP/+) genotypes are as follows (time-point in h APF (n antennae control / n antennae PCD-blocked): 18 (49 / 57), 24 (72 / 52), 30 (67 / 56), 36 (52 / 51), 42 (45 / 48), 48 (45 / 55), 56 (59 / 49), 64 (48 / 60), 72 (54 / 62) and 80 (47 / 53). Samples were thawed on dry ice and nuclear suspensions prepared as described (Li et al., 2022; McLaughlin et al., 2021). Suspensions from “early” (18/24/30 h APF), “mid” (36/42/48 h APF) and “late” (56/64/72/80 h APF) developmental time points were pooled together, with the exception of time point 80 h APF in the control genotype, which was pooled with mid time points. This latter pooling reflected the initial experimental design, but 80 h APF control cells could be effectively re-classified in the late time point for all subsequent analyses. After addition of Hoechst 33342 (Thermo Fisher Scientific, 62249), samples were loaded into a FACSAria flow cytometer. For each pooled sample, 2 ´ 20,000 GFP-positive nuclei were sorted (20,000 for early nuclei in PCD-blocked condition), which were immediately loaded onto the Chromium Next GEM Chip (10x Genomics)