Regulation of odor receptor genes in trichoid sensilla of the Drosophila antenna

Abstract

This study concerns the problem of odor receptor gene choice in the fruit fly Drosophila melanogaster. From a family of 60 Odor receptor genes, only one or a small number are selected for expression by each olfactory receptor neuron. Little is known about how an olfactory receptor neuron selects a receptor, or how the nucleotide sequences flanking a receptor gene dictate its expression in a particular neuron. Previous investigation has primarily concerned the maxillary palp, the simpler of the fly's two olfactory organs. Here we focus on genes encoding four antennal receptors that respond to fly odors in an in vivo expression system. To investigate the logic of odor receptor expression, we carry out a genetic analysis of their upstream regulatory sequences. Deletion analysis reveals that relatively short regulatory regions are sufficient to confer expression in the appropriate neurons, with limited if any misexpression. We find evidence for both positive and negative regulation. Multiple repressive functions restrict expression to the antenna, to a region of the antenna, and to neurons. Through deletion and base substitution mutagenesis we identify GCAATTA elements and find evidence that they act in both positive and negative regulation.

Figure 1.—

The olfactory organs and sensilla of D. melanogaster. (A) Electron micrograph of a Drosophila head indicating the two main olfactory organs, the antenna (arrowhead) and maxillary palp (arrow). Scale bar is 100 μm. (B and C) Transmitted-light confocal image showing the anterior face of a Drosophila third antennal segment. In C, the orange line separates the proximomedial region, which is devoid of trichoid sensilla, from the central and distolateral regions of the antenna, which are covered by basiconic, coeloconic, and trichoid sensilla. Orange dots indicate the tips of visible trichoid sensilla. Abbreviations: P, proximal; D, distal; M, medial; L, lateral. Image in A reproduced from Carlson(1996) with permission from Elsevier.

Figure 2.—

Antennal expression patterns of initial Or promoter–GAL4 constructs: Or47b−7.9kb, Or67d−6.1kb, Or65a−4.7kb, Or88a−1.6kb. Cuticular autofluorescence appears as magenta. (A) Confocal images of Drosophila antennae homozygous for both the UAS–GFP and the Or–GAL4 construct. Images in A are Z-projected cuticle-level confocal sections of the antennae showing patterns of GFP-labeled olfactory sensilla in the distolateral region (Or47b, Or65a, and Or88a) and midregion (Or67d) of the antenna. (B) Z-projections of interior sections reveal ORN dendrites (open arrowheads) and axons (solid arrowheads). Abbreviations: P, proximal; M, medial; L, lateral; Di, distal.

Figure 3.—

Expression of Or47b–GAL4 truncation constructs. (A) Scheme of Or47b–GAL4 truncation constructs. Black lines represent Or47b sequences upstream of the predicted translation start site, and green arrows represent vector and GAL4 coding sequences. (B–D) Images are interior sections of antennae (top row) or maxillary palps (bottom row) heterozygous for the indicated construct and the UAS–GFP reporter, except Or47b−7.9kb, which is homozygous for both. (B) Constructs with 419 bp or more of Or47b upstream DNA show similar patterns of expression. (C) Variable expression patterns of truncated Or47b–GAL4 constructs. For each construct, confocal Z-projections from two independent insertion lines are shown. All lines of these three constructs exhibited some degree of maxillary palp GFP expression. (D) No expression was observed in antennae or maxillary palps of flies carrying Or47b−119. (E–G) Antennal lobes showing neuropil (nc82 antibody) in magenta and GFP expression (anti-GFP antibody) in green. (E) Antennal lobe from a 0- to 1-day-old fly homozygous for Or47b−7.9kb–GAL4 and UAS–GFP. (F and G) Antennal lobes from 7- to 8-day-old flies heterozygous for the indicated Or47b−219 insertion and the UAS–GFP reporter. Similar patterns of glomerular targeting were seen in 0- to 1-day-old flies (not shown). Abbreviations: P, proximal; Do, dorsal; Di, distal; M, medial; L, lateral; V, ventral.

Figure 4.—

Or67d–GAL4 truncation constructs. (A) Scheme of Or67d reporter constructs. Black lines indicate regulatory DNA and green arrows indicate the GAL4 coding region and vector sequences. (B) Expression of Or67d–GAL4 truncation constructs. Antennae and maxillary palps were dissected from male flies of various ages for Or67d−136, and at 7–8 days for all other constructs. Flies were heterozygous for both the indicated Or67d–GAL4 construct and the UAS–GFP reporter. Images are Z-projections of cuticle-level confocal sections or of entire antennae (top and middle rows, respectively) and confocal Z-projections of maxillary palps (bottom row). GFP-labeled ORNs lie primarily in a band across the midregion of the antenna (most evident in the top row). All Or67d–GAL4 truncation constructs with at least 236 bp of upstream DNA exhibited nonneuronal GFP expression in the antenna (most evident in middle row). Numbers after the construct names identify the specific transgenic line shown. Abbreviations: P, proximal; Do, dorsal; M, medial; L, lateral; Di, distal; V, ventral.

Figure 5.—

Expression of Or65a–GAL4 truncation constructs. (A) Constructs. (B) Z-projections of interior confocal antennal sections from flies heterozygous for both the indicated Or65a–GAL4 truncation construct and the UAS–GFP reporter. Expression levels are indicated beneath the images. (C) DNA sequence upstream of Or65a. A GCAAATT element is underlined. Positions relative to the predicted Or65a translation start site are indicated. Abbreviations: P, proximal; M, medial; L, lateral; Di, distal.

Figure 6.—

Expression of Or88a–GAL4 constructs. (A) Constructs. (B) Expression patterns of the indicated Or88a–GAL4 truncation constructs are shown in Z-projections of internal confocal sections. The three constructs with 486–465 bp of Or88a upstream DNA are expressed in the distolateral region of the antenna, but in fewer ORNs than Or88a−1.6kb. Constructs carrying 359 or 159 bp (not shown) do not drive detectable GFP expression in antennal ORNs. Antennae from flies heterozygous for both an Or88a–GAL4 driver and the UAS–GFP reporter were dissected 0–1 day post-eclosion. (C) Mean numbers of GFPlabeled sensilla per antenna (±SEM; 5 ≤ n ≤ 10 antennae) of 0- to 1-day males. Each bar represents data from an independent insertion of the indicated Or88a–GAL4 construct. 0 indicates that no GFP-labeled sensilla were detected. Abbreviations: P, proximal; M, medial; L, lateral; Di, distal.

Figure 7.—

The effect of age and gene dosage on the expression levels of different Or88a–GAL4 constructs. (A) Three independent lines (1–3) of Or88a−1.6kb show similar numbers of labeled sensilla in flies heterozygous for both the Or88a–GAL4 construct and the UAS–GFP reporter. These expression levels are the same at both 0–1 and 7–9 days post-eclosion. (B) Flies either heterozygous (“het”) or homozygous (“hom”) for both Or88a−1.6kb insertion 3 and the UAS–GFP reporter show similar numbers of labeled sensilla at both 0–1d and 7–9 d. (C) Flies either heterozygous (“het”) or homozygous (“hom”) for both an Or88a−486 truncation and the UAS–GFP reporter show an age-dependent increase in number of GFP-labeled sensilla. Bars indicate mean numbers of GFP-labeled sensilla per antenna (±SEM) dissected from male flies. For each bar, 6 ≤ n ≤ 10 antennae.

Figure 8.—

Mutation of two GCAATTA elements upstream of Or88a. (A) Constructs. Each Or88a promoter fragment extends to, but does not include, the ATG of Or88a. Hatched lines represent downstream Or88a promoter sequence; green arrows represent vector and GAL4 coding sequence. Mutated base pairs are in lowercase type and shaded, while the positions of three iterations of GCAATTA are underlined. (B) Confocal Z-projections of 0- to 1-day male antennae heterozygous for both the indicated Or88a–GAL4 construct and the UAS–GFP reporter. (C–F) Numbers of labeled sensilla in flies heterozygous for one of the Or88a–GAL4 constructs in A and the UAS–GFP reporter. 5 ≤ n ≤ 13 antennae for all lines except that for −486M4, line 1 and −465M1, line 1, n = 3. The scales in C and D are different from those in E and F. 0 indicates that no GFP-labeled sensilla were detected. Abbreviations: nd, not determined; d, days post-eclosion; P, proximal; M, medial; L, lateral; Di, distal.

Figure 9.—

Mutation of sequences near −472 of Or88a. (A) Constructs. Mutated bases are in lowercase type and shaded. Dots indicate deleted base pairs. Red letters denote a novel GCAAT sequence in Or88a−486M1 created by mutating GCAATTA at –472. The mutated subregions of Or88a−486 are indicated below the sequence (M1, M2, M3). (B–G) Z-projected interior confocal sections of 0- to 1-day male antennae heterozygous for both the UAS–GFP reporter and the indicated Or88a–GAL4 construct. (H–M) Z-projections of 0- to 1-day male antennal lobes heterozygous for both the indicated Or88a–GAL4 construct and the UAS–GFP reporter. Antennal lobes are immunostained with anti-GFP (green) and nc82 (magenta). (N) Mean numbers of GFP-labeled sensilla (±SEM; 5 ≤ n ≤ 11). (O) The putative repressor element GCAATTA upstream of Or88a, and similar sequence elements upstream of other trichoid Or genes. In each case the element lies within or very near a region with negative regulatory function. Occurrences of these elements are aligned and shown in blue type. For A and O, positions are relative to the predicted translation start sites of the indicated genes. Abbreviations: d, days post-eclosion; P, proximal; Do, dorsal; M, medial; L, lateral; Di, distal; V, ventral.