Hybrid neurons in a microRNA mutant are putative evolutionary intermediates in insect CO2 sensory systems

Abstract

Carbon dioxide (CO2) elicits different olfactory behaviors across species. In Drosophila, neurons that detect CO2 are located in the antenna, form connections in a ventral glomerulus in the antennal lobe, and mediate avoidance. By contrast, in the mosquito these neurons are in the maxillary palps (MPs), connect to medial sites, and promote attraction. We found in Drosophila that loss of a microRNA, miR-279, leads to formation of CO2 neurons in the MPs. miR-279 acts through down-regulation of the transcription factor Nerfin-1. The ectopic neurons are hybrid cells. They express CO2 receptors and form connections characteristic of CO2 neurons, while exhibiting wiring and receptor characteristics of MP olfactory receptor neurons (ORNs). We propose that this hybrid ORN reveals a cellular intermediate in the evolution of species-specific behaviors elicited by CO2.

Fig. 1

Ectopic CO₂ neurons are formed in the MPs of S0962-07 mutants. (A) Gr21a expression in wild-type and mutant olfactory appendages. (B) Quantification of Gr21a-positive cells in the MP and antenna. (C) Gr63a (magenta, RNA antisense probe) and Gr21a (green, Gr21a-GAL4) are coexpressed in the MP. (D) Electropalpograms comparing the response to ethyl acetate, air, and CO₂ in control and mutant flies. Contrary to lack of response from the control palps, 5 of 12 mutant MPs responded to CO₂ (9). MPs recorded: n = 12 (control), n = 12 (mutant), P = 0.016. (E) Single confocal sections of MPs labeled with antibody to Elav (magenta) at 60 to 80 hours APF or with MPS-GAL4 and UAS-mCD8GFP (green) at 80 hours APF. Two neurons (Elav) or single ORNs (MPS-GAL4) are labeled in wild-type MP sensilla (arrows). Two additional neurons are observed in a subset of mutant sensilla (arrowheads and inset). (F) Mutant neurons in the MP target the V and medial glomeruli. Mutant flies without MP (upper right) and without antenna (lower right). Magenta, anti-NC82. In (A) and (E), dashed lines outline MPs.

Fig. 2

miR-279 is expressed in precursor cells in the developing MP. Expression of miR-279 was visualized with miR-279-GAL4 and UAS-CD8GFP (green). The arrowhead and arrow in both the schematic and the image panels point to a big cell and a cluster of small cells, respectively. The open arrow in the schematic points to a nerve fiber from one of the cell clusters. Nuclear counterstain TOTO-3 is used in the bottom panels. Dashed lines outline the developing MPs.

Fig. 3

Nerfin-1 is a target of miR-279. (A to C) Expression pattern of Nerfin-1 (magenta) and miR-279 (green, see Fig. 2) in developing MPs at early (A) and later [(B) and (C)] stages. (D) Nerfin-1 (magenta) is expressed in ectopic CO₂ neurons (green) in the mutant MPs (arrows). (E) Quantification of Nerfin-1–positive nuclei in wild-type and mutant MPs at 60 to 80 hours APF. MPs scored: wild type, n = 7; mutant n = 9; **P < 0.001. (F) miR-279 inhibits nerfin-1 expression in cultured Drosophila S2 cell lines (**P < 0.001). (G) nerfin-1 is a dominant suppressor of miR-279 (P < 0.001).

Fig. 4

Ectopic neurons exhibit mixed sensory identity. (A) MPs of mutant flies labeled with RNA antisense probes (magenta) and with Gr21a-GAL4 and UAS-mCD8GFP (green). Or59c and Or42a transcript (magenta) overlaps partly with Gr21a-expressing cells (green, solid arrowhead). Cells only positive for Or59c or Or42a are labeled only in magenta (open arrowhead). (B and C) MPs of mutant flies contain more Or42a- and Or59c-expressing cells. (B) Labeling of MP ORNs with GAL4 reporter constructs (Or42a-GAL4 and MPS-GAL4) or Or59c or Or42a RNA probe. (C) Quantification of the data from (B). Total increase in the number of cells in mutants using MPS-GAL4 driver corresponds to the number of ectopic CO₂ neurons (see Fig. 1B).