Figure 1.
Maxillary Palp Organization and Conserved, Gene-Specific Elements
(A) Representation of fly head, labeled with Or22a-GAL4 (blue), Or47a-GAL4 (yellow), Or23a-GAL4 (magenta), and Or71a-GAL4 (green) drivers, each used to drive UAS-GFP. The image is a merge of four different compressed Z-stacks of four different flies, with the GFP pseudo-colored differently for each receptor. The antenna is tricolored; the maxillary palp is green.
(B) Nuclei of the ∼120 neurons of the maxillary palp, stained with anti-Elav antibody.
(C) The three types of sensilla. The pb1, pb2, and pb3 sensilla are represented by green, yellow, and red respectively, as described in the Materials and Methods.
(D) Map of the gene-specific conserved motifs in the upstream sequences of the maxillary palp Or genes. Names assigned to the different motifs include the unique part of the gene name and a number indicating relative proximity to the translation start site.
(E) Sequence alignments of the most highly conserved motifs for each gene. In some cases the indicated sequences are the reverse complement of those shown in (D).
Figure 2.
Functional Analysis of Gene-Specific Elements 46a1, 71a3, and 59c1
Expression of GFP driven by the wild-type promoter constructs (A, D, and G), mutated constructs (B and E), and minimal promoter constructs (C, F, and H).
(A–C) 46a1; (D–F) 71a3; (G and H) 59c1. All flies contained one copy of the Or-GAL4 constructs and two copies of UAS-GFP. At least eight independent transgenic lines were tested for each mutant construct, and at least two for each minimal promoter construct.
Expression of GFP in an acj66 background driven by the wild-type Or-GAL4 constructs (I, L, and O) and the minimal promoter constructs (J, M, and P).
Dependence of elements on Acj6 (K, N, and Q). The arrow between acj6 and the large triangle in (Q) could reflect an effect on either expression or function. Images are Z-compressions of confocal stacks.
Figure 3.
Neuron-Specific Repression Refines Expression of Or42a
(A) Z-compression of confocal stacks showing expression of GFP driven by the wild-type Or42a-GAL4 promoter; and (B) the same promoter with a mutation in the 42a4 element.
(C) Numbers of GFP-positive cells per maxillary palp for the line shown in (A) and for two independent lines in which the 42a4 element is mutated; each value represents n = 9 maxillary palps.
(D–G) Optical micrographs of (42a4)-GAL4/UAS-GFP;UAS-GFP/+ maxillary palps labeled with anti-GFP antibody and the indicated RNA in situ hybridization probes. Arrowheads in (F) indicate paired cells; arrowheads in (G) indicate colabeled cells.
(H) Optical sections of 46a1-GAL4/UAS-GFP;UAS-GFP/+ maxillary palps labeled with anti-GFP antibody and the indicated RNA in situ hybridization probes. Arrowheads indicate colabeled cells.
Figure 4.
Neuron-Specific Repression of Or59c Acts via the Downstream Region
(A, D, and E) Confocal micrographs showing cells labeled by anti-GFP antibody (top) driven by the indicated flanking sequences. The corresponding middle panels show double-labeling with an anti-GFP antibody and an Or59c RNA in situ hybridization probe. Graphs (below) indicate the numbers of GFP+ cells, Or59c+ cells, and GFP+Or59c+ cells, for eight individual maxillary palps of each genotype. For each palp, the three indicated values are plotted in a vertical column. Each arrowhead in (A) indicates labeling of paired cells in a sensillum.
(B andC) Optical sections from 59c5′-GAL4/UAS-GFP;UAS-GFP/+ maxillary palps labeled with an anti-GFP antibody and the indicated RNA probes.
Figure 5.
Scalloped-Mediated Repression of Or59c
(A) Predicted Sd binding site downstream of Or59c.
(B and C) Expression of a sd enhancer trap line, sdETX4, visualized by X-gal staining (B) and anti-βGAL antibody staining (C).
(D) RNA in situ hybridizations to maxillary palps of wild type and sd1, a hypomorphic allele.
(E) Numbers of Or RNA positive cells per maxillary palp (n = 10; SEM).
(F and G) Optical sections of sdETX4 maxillary palps labeled with anti-βGAL antibody (green) and RNA probes (red) for Or59c (F) and Or42a (G). Arrowheads in (F) indicate βGAL+ cells paired with Or59c+ cells.
(H) Optical sections of double-label in situ hybridizations to maxillary palps of two sd mutants. Arrowheads indicate Or85d+Or59c+ cells.
Figure 6.
Conservation of Regulatory Logic in Other Systems
Confocal micrographs showing expression of GFP driven by the wild-type Or42a promoter (A) and a mutant (42a4)-GAL4 promoter (B) in larval olfactory neurons and the adult labellum.
Figure 7.
The Expression Program Is Conserved and Enhances ORN Sensitivity
Electrophysiological analysis of the maxillary palp neurons from D. melanogaster and D. pseudoobscura. On the left is a receptor-to-neuron map of the D. melanogaster maxillary palp. The bar graphs on the right represent the responses in spikes per second, of different neurons, to a diagnostic set of odors that can be used to distinguish the functional expression of the different Or genes. Error bars = SEM; n = 10. For each sensillum type, the light-colored bar represents the A cell; the darker bar represents the B cell. The responses are measured as the change in action potential frequency following the onset of odorant stimulation. The map and the data for D. melanogaster are from [21]. We note that the maxillary palps of both species contain a small number of neurons that have not been well characterized yet.
Figure 8.
Model for Combinatorial Coding of Odor Receptor Gene Choice
Conserved gene-specific regulatory elements, the genes that contain them, and the maxillary palp ORNs in which the genes are expressed, are depicted. Green elements are positive regulatory elements; red, pink, and orange elements are negative. Solid green arrows connect genes to the ORNs in which they are expressed in wild-type. These arrows originate from regulatory elements in cases where the elements have been shown to act positively, but do not imply that such elements alone are capable of directing proper expression. By contrast, 42ax has not been tested functionally and its arrow does not originate from this element. Dashed green arrows indicate ectopic expression driven by the indicated elements in the context of a minimal promoter, expression that in wild-type is repressed through the agency of other indicated elements, as represented by the curved red, pink, and orange lines. The pink and orange elements and their positions in Or46a and Or59c, respectively, have not been defined. The prefix “pb” has been deleted from the ORN designations. In the ORNs are ovals representing predicted transcription factors, color-coded according to the elements upon which they are presumed to act. For simplicity, a single factor (dark green; left column, second row), expressed in pb1A, pb3A, and pb3B, is proposed to act on 59c1, and a single factor (light green; left column, top row) expressed in both pb2B and pb1A is proposed to act on 46a1; more complicated models in which these elements are acted upon by multiple factors are also possible. Promoters are not drawn to scale. AMC, antenno-maxillary complex, which contains the dorsal organs.