Effect of Apterous overexpression on ORN populations.

<p>(A) Model depicting the changes to the combinatorial code in <i>rn</i>^{<i>89GAL4</i>} UAS-ap flies. (B) and (C) Rn-EGFP (green), Bar (red), and Dachshund (blue) staining on antennal discs from wild type (B) and <i>rn</i>^{<i>89GAL4</i>} UAS-ap flies (C) shows expansion of Bar to R(4) and decreased Dac staining in R(2/3) (brackets). Individual channels are shown on the right. Boxed areas are shown below at a higher magnification. (D) qRT-PCR of OR genes as a readout of ORN populations in antennal samples from <i>rn</i>^{<i>89GAL4</i>} UAS-ap flies. The receptors that show the same phenotype as in <i>rn</i> mutants are blocked in green shade. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Intercalation of prepatterning genes inside and outside the central fold.

<p>(A) A schematic model showing 7 concentric domains in the antennal disc determined by unique combinations of transcription factors. Four rings are placed outside the central fold (CF), and three are inside. (B-F) Expression analyses of Rn, Bab and Dac (B); Rn, Bab, and Bar (C); Rn, Bar and <i>ap</i> (D and E); Bab and <i>ap</i> (F) demarcate 7 different rings in the antennal discs based on combinations of these factors as well as Bab concentration gradient. Middle confocal sections were shown for (B-D), and superficial sections were shown for (E) and (F). Bab expression is highest near the central fold and decreases towards outermost and innermost regions. Bab was labeled with anti-Bab2. Rn was labeled with tagged EGFP in Rn-EGFP. Dac was labeled with a monoclonal anti-Dac antibody. Bar was labeled with anti-B-H1. <i>ap</i> was labeled with anti-β-gal in <i>ap</i>^<i>rK568</i>. Individual channels are shown on the right of each image. Boxed areas are shown below in higher magnification. Rn expression is seen as a circle (arrow) inside the central fold in the middle sections (C). Central fold is denoted by a dashed line. Each ring is numbered and marked by a bracket.</p

Effect of Bar overexpression on ORN populations.

<p>(A) Model depicting the changes to the combinatorial code in <i>rn</i>^{<i>89GAL4</i>} UAS-BarH1 flies. The fate conversions among the rings are represented by arrows. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.s013" target="_blank">S1 Text</a> for explanations. (B) <i>rn</i>^{<i>89GAL4</i>} UAS-GFP (green), Bar (red), and Dachshund (blue) staining on antennal discs from control flies. (C) <i>rn</i>^{<i>89GAL4</i>} UAS-BarH1^M13 flies shows expansion of Bar up to the Dac expression boundary (bracket). Some Bar is detected inside of the Dac expressing region, but this expression is weak and only present in a small number of cells. Central fold is missing in the overexpressing line (arrowhead). (D) qRT-PCR of OR genes as a readout of ORN populations in antennal samples from <i>rn</i>^{<i>89GAL4</i>} UAS-BarH1^M13 flies. The receptors that show the same phenotype as in <i>rn</i> mutants are blocked in green shade. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Relationship between <i>BarH1/2</i> and <i>rn</i>.

<p>(A) Expression of Or47b from at4 sensilla as detected by Or47b::mCD8-GFP reporter in control, <i>rn</i> single mutant and <i>rn</i>/<i>Bar</i> double mutant flies. Or47b expression is expanded into an anterior zone (right panel, arrowhead) in the <i>rn</i>-/- antenna. When mutant clones for <i>BarH1/2</i> have been introduced in the <i>rn</i>-/- background, the expansion of Or47b expression in the anterior zone is no longer obvious (arrowhead). (B) Quantification of the numbers of Or47b expressing neurons from (A). There is a significant decrease in the number of Or47b neurons in <i>Bar/rn</i> double mutants as compared to <i>rn</i> single mutants. ANOVA revealed a statistically significant change in the number of Or47b neurons (p<0.001, Fcrit = 3.25, df = 39). Statistics displayed represent post-hoc Student’s T-tests. n = 15. **<i>p</i><0.01, ***<i>p</i><0.001. (C) Or47b neurons in wildtype and <i>Bar</i> MARCM clones. Or47b neurons were labeled using Or47b-GAL4 to drive UAS-CD8 GFP. Clones were generated using ey-FLP FRT19A TubGAL80. (D) There was no detectable difference between the numbers of Or47b neurons in wildtype and mutant clones. Quantification showed that the average number of cells observed was approximately one fifth of the total number of Or47b neurons in (B), suggesting that MARCM occurred in a small portion (20%) of the antennal disc. (E) ChIP-qPCR using anti-EGFP antibodies to pull down endogenous Rn-EGFP from third instar antennal discs and test the binding of Rn to <i>Bar</i>/<i>bab</i> regulatory regions. T13 enhancer sequence upstream of <i>bab2</i>, which was previously reported to bind to Rn <i>in vitro</i>, showed enrichment for Rn-GFP in the ChIP assay. Primers spanning different regions of BarH2 upstream its TSS (BarH2-399, BarH2-728, and BarH2-1588) also showed binding to Rn-EGFP. Or82a, which is expressed in ORNs that develop from <i>rn-</i>positive ORNs did not show binding as previously reported [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.ref038" target="_blank">38</a>]. **<i>p</i><0.01.</p

Model depicting the PD network that determines the precursor identities of the rings and the ORN populations.

<p>(A) Wild type antennal disc showing the 7 rings corresponding to subsets of sensilla subtype fates created by the combinatorial expression of the PD network components, as well as the Bab gradient. The cross-regulatory network that operates within the antennal disc is shown below. The origin of ab3 (with a question mark) is unclear. (B) Scheme showing the changes to the overlapping domains within the antennal disc in <i>rn</i> mutants. R(6) and R(1) are expanded at the expense of R(2–5). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.s013" target="_blank">S1 Text</a> for explanations. (C) qRT-PCR of OR genes as a readout of ORN populations in antennal samples from <i>rn</i> mutant flies. *<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001.</p

Rn controls the expression of <i>BarH1/2</i> and <i>bab1/2</i> in the antennal disc.

<p>(A) Expression levels of <i>B-H1/2</i> and <i>bab1/2</i> between the control and mutants in different stages. Normalized expression data by DESeq2 was used. † <i>p</i><0.1 * <i>p</i><0.05 ** <i>p</i><0.01 *** <i>p</i><0.001. (B) Expression patterns of PD genes in the third instar larval antennal disc. They are expressed in concentric rings along proximodistal axis. Anti-B-H1 (T. Kojima), anti-Bab2 (F. Laski), anti-Dac were used to visualize Bar, Bab and Dac. <i>ap</i>^<i>rK568</i> (stained with anti-β-gal) and CRISPR tagged Rn-EGFP [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.ref049" target="_blank">49</a>] were used to visualize Ap and Rn. The antennal disc is the upper portion of the eye-antennal disc complex in the bright-field image. Images were taken from different animals. (C) Confirmation of RNAseq results on Bar expression. Antibodies to B-H1 (red) were used. Expansion of B-H1 outside CF (arrow) is apparent in <i>rn</i> mutants. The expansion of Bar is restricted within the distal boundary of Dac (blue). In the expanded zone outside the central fold, Bar expression overlaps with <i>rn</i> reporter expression (lower panel). (D) Bab2 antibody staining (blue) shows concentration gradients in wild type. In these composite images, two circles (arrow heads) on the ridges of the central fold show the highest concentrations, but the high level of expression is continuous along the central fold (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.s003" target="_blank">S3C Fig</a>). Bab2 level is weaker in <i>rn</i> mutants, although the overall pattern is unchanged. The <i>rn</i>^{<i>89GAL4</i>} reporter labels cells that have active <i>rn</i> promoter (green). CF, central fold (arrow). (E) Quantification of Bab2 levels in (F). n = 10, *** <i>p</i><0.001.</p

Expression pattern of PD network components in ORNs.

<p>Antennal lobe innervation of ORNs expressing GFP driven by <i>Bar</i>^{<i>NP4099</i>} (A), <i>ap</i>^<i>md544</i> (B), or <i>bab1</i>^{<i>Pgal4-2</i>} (C). For <i>bab</i> and <i>Bar</i> expression, analysis was done on adults. For <i>ap</i>, 50–75 hour APF pupal brains were examined. Top rows show anterior confocal slices and the bottom rows show the posterior slices. Also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.s004" target="_blank">S4 Fig</a> for additional analysis and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.t001" target="_blank">Table 1</a> for summary of the results.</p

Regulation of GRN patterns by the PD network.

<p>GRN and ORN specification may be regulated by the same molecular network. In <i>rn</i> mutant, tarsal segment (ta3) is missing, and other segments are fused. Ectopic expression of Gr61a and Gr58c can be seen in the distal region of the second tarsal segment, suggesting a duplication of 4s/5b sensilla. Neurons labeled by the red asterisk in the pictures are the ectopic neuron only seen in the mutant. Different sections of confocal images are shown when neurons are not on the same focal plane. All legs are either from the mid or from the hind leg. Cartoon illustrating the <i>rn</i> phenotype in GRNs is shown below. The ectopic sensillum is labeled in red. The scheme for GRN classes and the receptors expressed in the 5b/4s and 5v sensilla are modified from [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.ref032" target="_blank">32</a>]. The GR that distinguishes 5b from 5v is circled in yellow (also see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005780#pgen.1005780.s009" target="_blank">S9 Fig</a>). Receptors that are analyzed in the figure are circled in green. In the wild type, there is a pair of sensilla for each sensilla type, although only one for each pair is drawn in the cartoon.</p