Functional integration of "undead" neurons in the olfactory system

Abstract

Programmed cell death (PCD) is widespread during neurodevelopment, eliminating the surpluses of neuronal production. Using the Drosophila olfactory system, we examined the potential of cells fated to die to contribute to circuit evolution. Inhibition of PCD is sufficient to generate new cells that express neural markers and exhibit odor-evoked activity. These "undead" neurons express a subset of olfactory receptors that is enriched for relatively recent receptor duplicates and includes some normally found in different chemosensory organs and life stages. Moreover, undead neuron axons integrate into the olfactory circuitry in the brain, forming novel receptor/glomerular couplings. Comparison of homologous olfactory lineages across drosophilids reveals natural examples of fate change from death to a functional neuron. Last, we provide evidence that PCD contributes to evolutionary differences in carbon dioxide-sensing circuit formation in Drosophila and mosquitoes. These results reveal the remarkable potential of alterations in PCD patterning to evolve new neural pathways.

Fig. 1. Inhibition of developmental PCD results in increased neuron numbers in the antenna.

(A) Schematic of the D. melanogaster third antennal segment highlighting different sensory structures. (B) Schematic of the lineage of an antennal disc SOP cell that gives rise to a sensillum containing two neurons (illustrated on the right). The expression of a subset of molecular markers is shown: Elav is expressed in only three of four neural precursors; one of these (Naa) and the Elav-negative cell (Nbb) are eliminated by PCD. The lineage is based on data from (13, 14, 19). (C) Simplified summary of the PCD pathway in D. melanogaster, highlighting the elements relevant for this study. Several intermediate steps between the proapoptotic proteins (Rpr, Grim, Hid, and Skl) and the executioner caspases are not shown. (D) Representative images of anti-Elav immunofluorescence in whole-mount antennae from control [Df(3L)H99/+; the wild-type chromosome here and in other genotypes was derived from a w¹¹¹⁸ parent] and PCD-deficient [Df(3L)H99/Df(3L)XR38] animals. Scale bar, 10 μm. Right: Quantifications of antennal neuron numbers of the indicated genotypes, including an additional control genotype [Df(3L)XR38/+] (n = 14, 14, and 13, respectively). Mixed sexes were analyzed; in all other experiments, female flies were used, except where noted otherwise. ***P = 0.0007216 for the comparison to Df(3L)H99/+ and P = 0.0013224 for the comparison to Df(3L)XR38/+ (Wilcoxon rank sum test, corrected for multiple comparisons using a Bonferroni correction). In this and subsequent panels, individual data points are shown, overlaid with boxes indicating the median and first and third quartiles of the data; whiskers show the limits of the distribution. (E) Representative images of anti-Elav immunofluorescence in whole-mount antennae from control (peb-Gal4/+) and PCD-blocked [peb-Gal4/+;UAS-miR(rpr,hid,grim)/+] animals. Scale bar, 10 μm. Right: Quantifications of neuron numbers of these genotypes. ***P = 2.4 × 10⁻⁷ (t test) (n = 19 and 21; control and PCD-blocked, respectively). (F) Representative images of anti-Elav immunofluorescence in whole-mount antennae from control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals. Scale bar, 10 μm. Right: Quantifications of neuron numbers of these genotypes. *P = 0.024 (t test) (n = 10 and 11; control and PCD-blocked, respectively).

Fig. 2. Undead OSNs are functional.

(A) Representative extracellular electrophysiological traces of basal activity from neurons in an at1 sensillum of control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals. Automatically detected spikes (see Materials and Methods) from the neuron expressing OR67d are shown in blue, and those of the additional, undead neuron(s) in black, as schematized in the cartoons on the left (cells fated to die are shown with dashed outlines). (B) Quantifications of the proportion of sensilla containing one neuron (gray) or two (or more) neurons (red) in control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals. (C) Quantifications of the basal activity of the indicated neurons for the control and PCD-blocked genotypes. (D) Representative electrophysiological traces from at1 sensillum recordings in control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals upon stimulation with a 0.5-s pulse (black horizontal bar) of the pheromone cVA [10⁻² dilution (v/v) in paraffin oil] or a mix of fruit odors [butyl acetate, ethyl butyrate, 2-heptanone, hexanol, isoamyl acetate, pentyl acetate; each odor at 10⁻² dilution (v/v) in paraffin oil]. Automatically detected spikes from the neuron expressing OR67d are shown in blue, and those of the undead neuron(s) in black. (E) Quantifications of odor-evoked responses to fruit odors (see Materials and Methods) in control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals (n = 25 and 34, respectively).

Fig. 3. Undead neurons express a subset of olfactory receptor genes.

(A) Gene expression differences measured by RNA-seq (see Materials and Methods) between control and PCD-blocked antennae. The volcano plot shows the differential expression (on the x axis) of D. melanogaster Or, Ir, and Gr gene transcripts (each gene represented by a dot), as well as the four proapoptotic genes (grim, rpr, hid, and skl; red labels), plotted against the statistical significance (on the y axis). The mean expression level of individual genes across all samples is shown by the shading of the dot, as indicated by the gray scale on the right [units: log₂(counts per million)]. Only chemosensory genes showing a >1.5-fold increase in PCD-blocked antennae are labeled: Blue labels indicate genes whose expression in the antenna has previously been demonstrated by RNA in situ hybridization; magenta and green labels indicate receptors normally only expressed in the adult maxillary palps and larval dorsal organ, respectively; and black labels indicate receptors that are expressed in gustatory organs. The horizontal dashed line indicates a false discovery rate threshold of 5%. Data for all Or, Ir, Gr, and proapoptotic genes are provided in table S2. (B) Representative images of RNA FISH for the indicated Or genes in whole-mount antennae of control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals. Scale bar, 10 μm. Quantifications of neuron numbers are shown at the bottom. *** indicates Or19a P = 6.526 × 10⁻⁵ (t test) [n = 17 and 10 (control and PCD-blocked, respectively)], Or43a P = 5.888 × 10⁻⁷ (t test) (n = 10 and 10), Or47a P = 3.088 × 10⁻¹⁰ (t test) (n = 13 and 13), Or65b P = 2.2 × 10⁻¹⁶ (t test) (n = 17 and 11) (see fig. S2A for additional examples). The pink dashed lines encircle cells in PCD-blocked antennae that express the visualized Ors outside their usual spatial domain (see also figs. S2B and S3A). Comparison of the variation in OSN number between control and PCD-blocked antennae indicated only one neuron type—of those in this panel and in figs. S2A and S4—displayed greater variance in PCD-blocked antennae (Or19a P = 0.01; F test for equality of variance, with Bonferroni correction for multiple comparisons). (C) Representative images of RNA FISH for the indicated Or genes in whole-mount antennae of control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals. Scale bar, 10 μm. Quantifications of neuron numbers are shown at the bottom. *** indicates Or33a P = 1.812 × 10⁻⁷ (t test) (n = 23 and 23), ns indicates Or85e P = 0.053 (Wilcoxon rank sum test) (n = 12 and 22). We never detected any Or85e mRNA-positive neurons in control antennae but frequently detected one (or more) labeled cells in PCD-blocked antennae. (D) Schematic summarizing the normal olfactory organ/sensillum expression pattern of the subset of Or genes with higher expression in PCD-blocked antennae that display coexpression in wild-type neurons (highlighted in red; receptor genes showing no changes in transcript levels are in gray). The neuronal precursor identity of these OSNs is shown below. We represent Or69aA and Or69aB as distinct receptors here because, although they share 3′ exons, they are transcribed from different promoters and encode receptors with different odor specificities (15, 56). (These isoforms were not, however, distinguished in the RNA-seq and RNA FISH analyses). (E) Representative images of combined RNA FISH for Or65a (green) and Or65b (magenta) in whole-mount antennae of control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals (n = 4 and 5, respectively), showing coexpression of these receptors in both endogenous and undead neurons. Scale bar, 10 μm. (F) Representative images of RNA FISH for Or85f and anti-GFP in whole-mount antennae of control (peb-Gal4/+;Or49a-GFP/+) and PCD-blocked (peb-Gal4/+;Or49a-GFP/UAS-p35) animals. Scale bar, 10 μm. Quantifications of coexpression are shown to the right. *** indicates Or49a-GFP⁺/Or85f mRNA⁻ population P = 5.48 × 10⁻¹² (t test) (n = 16 and 15; control and PCD-blocked, respectively). The pink dashed line encircles cells in the PCD-blocked antenna that express Or49a-GFP outside the usual spatial domain (see also fig. S2B). We used an Or49a-GFP reporter due to our inability to reliably detect Or49a transcripts in situ; the higher number of Or49a-GFP⁺/Or85f mRNA⁻ negative cells is not an artifact of the detection method, as an Or85f-GFP reporter revealed a similarly limited increase in neuron number (fig. S3B). (G) Representative images of combined RNA FISH for Or47a (green) and Or33b (magenta) in whole-mount antennae of control (peb-Gal4/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+) animals. In control animals, Or33b is coexpressed with Or47a in the larval dorsal organ and is never detected in the antenna. In PCD-blocked antennae, Or33b- and Or47a-expressing undead neurons are almost completely nonoverlapping: 4% of Or33b-positive undead OSNs weakly coexpress Or47a (n = 79 cells from 10 antennae). Scale bar, 10 μm.

Fig. 4. Undead OSNs form novel receptor/glomerular couplings in the brain.

(A) Representative images of anti-GFP immunofluorescence in whole-mount antennae of control [peb-Gal4/+;;grim^{MI03811(EGFP)}/+] and PCD-blocked [peb-Gal4/+; UAS-p35/+;grim^{MI03811(EGFP)}/+] animals. Blind scoring by two independent observers of antennae as control (n = 10) or PCD-blocked (n = 9) was 100% accurate. Scale bar, 10 μm. (B) Representative images of combined anti-GFP and nc82 immunofluorescence in whole-mount brains of control (peb-Gal4/+;;grim^{MI03811(EGFP)}/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+;grim^{MI03811(EGFP)}/+) animals with antennae intact (left) or excised (right). Blind categorization of brains as control (n = 9) or PCD-blocked (n = 12) was 85% accurate (two independent observers). Scale bar, 10 μm. (C) Representative images of nc82 immunofluorescence in whole-mount brains of control (peb-Gal4/+;;grim^{MI03811(EGFP)}/+) and PCD-blocked (peb-Gal4/+;UAS-p35/+;grim^{MI03811(EGFP)}/+) animals. Equivalent glomeruli found in antennal lobes of control and PCD-blocked animals are labeled for reference with dashed blue lines; potential novel glomerular structures (or displaced original glomeruli) in PCD-blocked animals are shown with dashed pink lines. Further planes of the same brains are shown in fig. S5A. Blind categorization of brains as control (n = 11) or PCD-blocked (n = 13)—visualizing only the nc82 channel of confocal stacks of animals containing a diversity of fluorescent reporters in the genetic background; see genotypes below—was 85% accurate (two independent observers). Scale bar, 10 μm. (D) Representative images of combined anti-GFP and nc82 immunofluorescence in whole-mount brains of control (peb-Gal4/+;Or49a-GFP/Or49a-GFP, peb-Gal4/+;;Or19a-GFP/+, peb-Gal4/+;Or43a-GFP/+) and PCD-blocked (peb-Gal4/+;Or49a-GFP/Or49a-GFP,UAS-p35, peb-Gal4/+;UAS-p35/+;Or19a-GFP/+, peb-Gal4/+;Or43a-GFP/UAS-p35) animals. Blind categorization of brains as control or PCD-blocked—visualizing only the GFP channel of confocal stacks and based exclusively on additional glomerular innervation patterns—was, for each reporter, respectively, Or49a-GFP: 85% (n = 10 and 10; control and PCD-blocked, respectively); Or19a-GFP: 89.5% (n = 20 and 18); and Or43a-GFP: 93.9% (n = 19 and 15). Scale bar, 10 μm. Additional representative images are provided in fig. S5B. (E) Representative images of combined anti-GFP, anti–red fluorescent protein (Tomato), and nc82 immunofluorescence in whole-mount brains of control (peb-Gal4/+;Or49a-GFP/Or49a-GFP;GH146-QF,QUAS-Tomato/+) and PCD-blocked (peb-Gal4/+;Or49a-GFP/Or49a-GFP,UAS-p35;GH146-QF,QUAS-Tomato/+) animals. The dashed line encircles the novel Or49a-GFP–labeled glomerulus (i.e., not the normal DL4 glomerulus, which is not visible in this anterior plane of the antennal lobe). Scale bar, 10 μm. (F) Representative images of PN soma (bounded by the dashed lines) labeled by GH146>Tomato in whole-mount brains of control (peb-Gal4/+;Or49a-GFP/Or49a-GFP;GH146-QF,QUAS-Tomato/+) and PCD-blocked (peb-Gal4/+;Or49a-GFP/Or49a-GFP,UAS-p35;GH146-QF,QUAS-Tomato/+) animals. Scale bar, 10 μm. Quantifications of labeled PN numbers are shown to the right. ns indicates P = 0.819 (t test) (n = 6 and 5, control and PCD-blocked, respectively).

Fig. 5. Examples of naturally occurring extra neurons in at1 sensilla.

(A) Phylogeny of 26 drosophilid species, representing most of the Drosophila genus subgroups, based on the protein sequences of housekeeping loci (see Materials and Methods). Species names are colored to reflect the presence of one or two neurons in at1 sensilla. Numbers next to the tree nodes indicate the support values. The scale bar for branch length represents the number of substitutions per site. (B) Representative electrophysiological traces from recordings of at1 sensilla of the indicated drosophilid species (n = 5 per species) upon stimulation with a 0.5-s pulse (black horizontal bar) of solvent (dichloromethane) or cVA [10⁻² dilution (v/v)]. A single cVA-responsive neuron (known or assumed to express OR67d orthologs) is detected (shown in blue), as schematized in the cartoon on the left. (C) Representative electrophysiological traces from recordings of at1 sensilla of the indicated drosophilid species (n = 5 per species) upon stimulation with a 0.5-s pulse (black horizontal bar) of solvent (dichloromethane) or cVA [10⁻² dilution (v/v)]. Two classes of spike are detected: a cVA-responsive neuron (assumed to express OR67d orthologs) (shown in blue) and a second neuron with a larger spike amplitude, which does not respond to cVA (shown in black). The inferred sensilla organization is shown in the cartoon on the left.

Fig. 6. PCD can explain an evolutionary difference in CO₂-sensing neuron formation in D. melanogaster and mosquitoes.

(A) Left: Scanning electron micrographs of D. melanogaster and Anopheles gambiae heads indicating the two olfactory organs: antennae (blue) and maxillary palps (magenta) [adapted from (3), with permission]. Right: Schematic illustrating that, in D. melanogaster, CO₂-sensing neurons (green) are located in antennal ab1 sensilla and maxillary palp sensilla house only two Or neurons. By contrast, in A. gambiae (and other mosquitoes), CO₂-sensing neurons are located in the maxillary palp, housed together with two Or neurons. (B) Representative images of combined anti-ORCO (the OR coreceptor, which labels all OR neurons) and anti-GFP immunofluorescence in whole-mount maxillary palps of control (peb-Gal4/+;Gr21a-GFP/+) and PCD-blocked (peb-Gal4/+;Gr21a-GFP/UAS-p35) animals. Scale bar, 10 μm. Quantifications of Gr21a-GFP–positive neuron numbers are shown at the bottom; *** indicates P = 6.7 × 10⁻⁹ (Wilcoxon rank sum test) [n = 22 and n = 21 (control and PCD-blocked, respectively)]. Schematic of inferred maxillary palp sensilla organization in each genotype is illustrated on the right. (C) Representative images of combined RNA FISH for the indicated Or genes (magenta) and anti-GFP immunofluorescence (green) in whole-mount maxillary palps of control (peb-Gal4/+;Gr21a-GFP/+) and PCD-blocked (peb-Gal4/+;Gr21a-GFP/UAS-p35) animals. Schematic of the inferred maxillary palp sensilla organization in each genotype is illustrated on the right. GFP-positive neurons pair with Or85d OSNs in pb3 sensilla (encircled by dashed lines) but not Or85e OSNs in pb2 sensilla (arrowheads). (D) Representative images of combined anti-GFP and nc82 immunofluorescence in whole-mount brains of control (peb-Gal4/+;Gr21a-GFP/+) and PCD-blocked (peb-Gal4/+;Gr21a-GFP/UAS-p35) animals with antennae intact (left) or excised (right). Blind categorization of brains from animals lacking antennae as control (n = 19) or PCD-blocked (n = 17) (visualizing only the GFP channel of confocal stacks and based exclusively on the presence of GFP-labeled axons) was 94.4% accurate. Scale bar, 10 μm. Additional representative images are provided in fig. S7.