Positive and negative regulation of odor receptor gene choice in Drosophila by acj6

Abstract

Little is known about how individual olfactory receptor neurons (ORNs) select, from among many odor receptor genes, which genes to express. Abnormal chemosensory jump 6 (Acj6) is a POU domain transcription factor essential for the specification of ORN identity and odor receptor (Or) gene expression in the Drosophila maxillary palp, one of the two adult olfactory organs. However, the mechanism by which Acj6 functions in this process has not been investigated. Here, we systematically examine the role of Acj6 in the maxillary palp and in a major subset of antennal ORNs. We define an Acj6 binding site by a reiterative in vitro selection process. The site is found upstream of Or genes regulated by Acj6, and Acj6 binds to the site in Or promoters. Mutational analysis shows that the site is essential for Or regulation in vivo. Surprisingly, a novel ORN class in acj6 adults is found to arise from ectopic expression of a larval Or gene, which is repressed in wild type via an Acj6 binding site. Thus, Acj6 acts directly in the process of receptor gene choice; it plays a dual role, positive and negative, in the logic of the process, and acts in partitioning the larval and adult receptor repertoires.

Figure 1.

Role of acj6 in the maxillary palp. A, The wild-type maxillary palp contains three sensillum types, each housing a pair of ORNs. B, Expression of a subset of Or genes depends on acj6. In situ hybridization of Or genes to wild-type and acj6 maxillary palps is shown.

Figure 2.

Identification of an Acj6 binding site. A, Acj6 binding site consensus. Alignment of sequences from an in vitro binding selection, SELEX, is represented in a consensus logo, generated with WebLogo 3 (Crooks et al., 2004). The overall height of each stack indicates the sequence conservation at that position (measured in bits), whereas the height of nucleotide symbols within the stack reflects the relative frequency of the nucleotides at that position. B, All five Or genes that depend on Acj6 for expression in the maxillary palp have Acj6 binding sites with scores >6 within 500 bp upstream of their translational start sites. The proximal Acj6 site at Or85e lies at position −17 with respect to the translation start site and seems likely to lie in the 5′-untranslated region of the gene rather than in the promoter region. C, Acj6 binds to sites in Or gene promoters. EMSA was performed with biotin-labeled oligonucleotide probes representing the Acj6 site and its flanking sequences in the Or85e promoter. The amount of Acj6 protein is indicated as a multiple of the molarity of the labeled Or85e oligonucleotide probe used (0.02 pmol). Lanes 5, 7, and 9 contain a 40-fold excess of unlabeled competitor oligonucleotide (comp.) that contains the wild-type Acj6 site in Or85e, Or46a, and Or45b, respectively. Lanes 6, 8, and 10 contain a 40-fold excess of unlabeled competitor oligonucleotide in which the Acj6 sites were mutated. The competitor sequence in lane 11 contains an Or46a site flanked by sequences adjacent to an Acj6 site in the Or45b promoter, as described below. The single arrow indicates bands likely to represent binding of the site to an Acj6 monomer; the double arrow indicates a band likely to represent binding of an Acj6 dimer. Faint bands may represent binding of Acj6 degradation products. Unbound oligonucleotides are at bottom of gel.

Figure 3.

Acj6 sites are required for expression of Or genes. A, B, GFP expression driven by wild-type Or85e- and Or46a-GAL4 constructs (top panels) and constructs with mutations in predicted Acj6 sites (bottom panels). The proximal Acj6 site in Or85e, which is likely to lie in the 5′-untranslated region of the gene, is not shown. Data were from four independent transgenic lines for the mutant Or85e construct, and three lines for the mutant Or46a construct. C, The effects of mutations in Acj6 sites in Or42a. Data were pooled from three independent transgenic lines for each mutated construct. n = 11 for wild type; 30 < n < 37 for mutated constructs. *p < 0.05; **p < 0.01; ****p < 0.0001 (one-way ANOVA and Holm–Sidak test).

Figure 4.

Odor responses of a subset of antennal ORNs are lost in acj6⁶. A, Odor response profiles of large basiconic sensilla of wild-type and acj6⁶ antennae. 6 ≤ n ≤ 12. Error bars indicate SEM. Odorants were applied as vapors of a 10⁻² dilution. B, Fate changes of antennal large basiconic neuron classes in acj6⁶. ab1A and ab1B are absent or electrically silent in acj6⁶ and are depicted by dashed lines. Uncolored neurons depicted by solid lines yield spontaneous action potentials but show no response to tested odors.

Figure 5.

A subset of antennal Or genes require acj6 for expression. A, In situ hybridizations of Or genes to wild-type and acj6⁶ antennae. Cells are labeled in red. B, Effects of acj6⁶ on receptor expression shown by in situ hybridization (Or42b, Or92a, Or59b, Or85a, Or22a, Or85b) or inferred by response properties of antennal neurons (ab1C, ab1D). The number of Acj6 sites within 1 kb upstream of these genes is indicated.

Figure 6.

Acj6 represses the larval receptor gene Or45b in the adult maxillary palp. A, Response profiles of pb2C (in acj6⁶) and Or45b (in the empty neuron system). n = 6 for pb2C; n = 19 for Or45b. Error bars indicate SEM. B, In situ hybridization to Or45b mRNA in wild-type and acj6 maxillary palps. The arrowheads indicate labeled cell bodies. C, Effects of altering an Acj6 site on Or45b-GAL4-driven GFP expression. Or45b-GAL4 is not expressed in the wild-type maxillary palp but is ectopically expressed in the acj6⁶ maxillary palp. Mutation of the Acj6 site at −392 caused expression in the wild-type adult, mimicking the effects of the acj6⁶ mutation. Replacement of the Acj6 site at −392 with another Acj6 site, one that directs activation of Or46a, yielded the same expression pattern as that driven by Or45b-GAL4. Two independent lines were examined for each genotype. The Or46a site contains two Acj6 consensus sequences that overlap by 1 bp and are on opposite strands of the DNA. D, Model for dual mode of regulation. Acj6 activates transcription (left) or represses transcription (right) depending on the presence of other proteins that bind in the vicinity.