The location of the binding site upstream of the OR dictates Xbp1 function.

<p>(A) Motif density plot, showing motifs found upstream of OR genes that did not require the matching TF (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001280#pbio.1001280.s007" target="_blank">Table S3</a> for statistics). (B) Bar graph depicting the total number of motifs located upstream or downstream the TATA box for ORs that either require the TF (“essential”) or not (“nonessential”) for expression. (C) Double in situ labeling of <i>Or98a</i> and <i>Or56a</i> in wild type (Wt) and <i>xbp1-IR</i> antennae revealed ectopic <i>Or98a</i> expression next to <i>Or56a</i>. The RNAi phenotypes are summarized as a matrix (grey, wild-type expression; red, ectopic; and black, loss of expression). (D) One Xbp1 motif (purple) was found next to the TATA box (green) of <i>Or98a</i>. The <i>Or98a</i> promoter construct produced expression in a single domain (light blue oval, black expression). Whereas, the same <i>Or98a</i> promoter construct with a mutated Xbp1 motif (red) produced a distal expansion of the expression.</p

A regulatory matrix for <i>Drosophila</i> OR expression.

<p>(A) The regulatory matrix represents in situ hybridizations for 32 ORs/TF-IR, indicated as wild-type levels (gray dots) and lost (black dots) OR expression. Trichoid ORs marked in orange. (B) Bar diagram, representing number of ORs that required each TF for expression. Trichoid ORs marked as orange insets in each bar. (C) Number of ORs regulated by 0–7 TFs depicted as bar graphs. (See <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001280#pbio.1001280.s006" target="_blank">Table S2</a> for statistics and see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001280#pbio.1001280.s003" target="_blank">Figure S3</a> for domain and sensilla arranged matrix).</p

An RNAi screen identifies seven TFs required for OR expression.

<p>(A) Whole mount preparations of antenna from the two screening rounds (GFP in black). In the first round, expression of <i>Or98a-CD8::GFP</i> and <i>Or23a-CD8::GFP</i> in two mid-antennal domains (light blue and orange oval) were analyzed. In the second round, <i>Or92a-CD8::GFP</i> expression in the most proximal (dark blue oval) antenna domain and <i>Or47b-CD8::GFP</i> expression in the most distal (red oval) antenna domain were analyzed. (B) Statistics from the screen is depicted as a graph, summarizing the number of IR lines that did not affect OR expression (Wt, white), led to lethality (Lethal, grey) or lost OR expression (Loss of OR expression, Green). (C) Phenotype summary for the seven TF-IRs and the analyzed OSN classes, wild-type OR expression (grey dots) and loss of OR expression (black dots). (D) Antenna from each TF-IR with representative OR expression phenotypes. (E) Whole mount antennal lobe with the Or92a-CD8::GFP OSN projections shown in green and the synaptic marker, nc82, delineating the glomeruli of the antennal lobe, in magenta. The boxed region indicates the antennal lobe area in the right panel, which compares the RNAi and mutant phenotypes of <i>acj6</i>, <i>sim</i>, <i>xbp1</i>, <i>zf30c</i>. Note the loss of <i>Or92a</i> in both the mutant and RNAi lines.</p

Expression of OR gene regulators in the adult <i>Drosophila</i> antenna.

<p>(A) The identified TFs belong to different protein families as indicated by their protein domain organization. (B) In situ hybridizations and immunohistology on wild-type antenna sections showing the expression pattern of each TF (red) counterstained with the nuclear marker DAPI (blue). (C,D) RNAi-mediated reduction of Acj6 (C) and Xbp1(D) does not affect the overall expression pattern of the other six TFs. (E) Expression of the TFs (magenta) in either <i>Or47b-CD8::GFP</i> or <i>Or92a-CD8::GFP</i> (green) expressing OSNs. Note, that the Or47b expressing OSNs lack expression of onecut and zf30c (arrows).</p

Transcriptional activation and repression are required for correct expression of each OR gene to one OSN class.

<p>(A) Double in situ labeling of <i>Or23a</i> and <i>Or43b</i> in wild type (Wt) and <i>E93-IR</i> antennae, the <i>Or43ba</i> expression phenotypes are further depicted schematically and summarized as a matrix (grey, wild-type expression; red, ectopic; and black, loss of expression). (B) Double in situ hybridization labeling of <i>Or67a</i> and <i>Or67b</i> expression in wild type (Wt) and <i>acj6-IR</i> antenna. The resultant phenotypes are further summarized as a schematic and a matrix summary. Note the new pair of <i>Or43b</i> and <i>Or23a</i> when <i>E93</i> is knocked down (A), and OR coexpression generated in <i>acj6</i> knock-downs (B). (C) Model depicting how activation and repression of OR expression can specify an OSN class. Activation of OR gene expression (left box); different combinations of a limited set of TFs bind a proximal upstream region and produce OR expression in a broad antenna region. Repression of OR gene expression (right box), distal located repressors together with the dual function of the TFs determined by binding site location or possibly cofactor use, restrict OR expression. The combined sum of OR gene activation and repression produce expression to one single OSN class.</p

All seven TFs are continuously required for OR expression.

<p>(A) Schematic of the TARGET experiments. Flies were shifted at late pupal stage from 18°C to 29°C (red line), or from 29°C to 18°C (green line); the RNAi was induced specifically at 29°C. (B–D) <i>Or92a</i> and <i>Or47b</i> in situ hybridizations (red) counterstained with DAPI (blue). (B) With the suppression of RNAi at 18°C, the OR was expressed in all genotypes (red staining). (C) The TF knock down at the end of pupal development (shift from 18°C to 29°C) fully suppresses OR expression. (D) Developmental TF knock down (shift from 29°C to 18°C) does not affect OR expression except for <i>zf30c-IR</i>.</p