Two conserved regions within OR463 are fundamental for wing development.

A. Illustration of ap upstream intergenic region. In light blue: enhancer sequences relevant during wing development (Bieli, Kanca, Requena, et al., 2015). In black: OR463 sequence conservation among related insects. B. Different regions in which OR463 was divided for mutation analysis. In dark grey, sequences with highly conserved TF binding sites. C. Percentage of wild-type wings when each of the fragments was deleted in homozygosis and (D) hemizygosis (over the apDG3 allele). E. Control wing (Re-integrated WT OR463 in apR2 landing site). F. Loss of wing displayed by ΔOR463 mutants. G. Loss of wing displayed by Δm3 mutants. H. Representative wing phenotype derived from Δm1 deletion in homozygosis. Anterior compartment presents correct venation, while P compartment presents an outgrowth. Venation pattern in this outgrowth is disturbed. Inset 1: Detail of the A compartment bristles. Inset 2: bristles of A identity in the P compartment. I. Representative wing phenotypes derived from Δm1 deletion in hemizygosis. P compartment venation pattern is disturbed. No outgrowth is formed but the P compartment present severe venation problems, with some veins position perpendicularly to its normal direction. Inset 1: Detail of A bristles. Inset 2: Detail of A bristles in the P compartment. Note that transformation of the P margin into A is not complete. I’. Wing phenotype also present in Δm1 hemizygous flies. In these, the P compartment is severely reduced and mostly A bristles are present in the margin.

Mirror image duplications arise due to changes in DV and AP boundary position.

A. Anti-Ap and anti-Wg immunostaining of control wing discs (Re-integrated WT OR463). A’. Anti-Ap and anti-Wg staining of wing discs in homozygous Δm1 mutants. The DV boundary is distorted in the P compartment, where it is extended into the presumptive wing hinge (arrowheads). A’’. Anti-Ap staining of Δm1 hemizygous wing discs. The DV boundary is further deformed in the P compartment (arrowhead). B. Quantification of the relative P size (P Area/Total Area) and relative PD size (PD Area/Total Area) in different mutants (p value<0.0005, control: n=13, N1/DG3: n=16, Δm1/DG3: n=10, Δm1m2m4/DG3: n=9). C, C’ and C’’. Relative position of AP and DV boundaries as revealed by immunostaining with anti-Ap and anti-Ptc in wing discs of control, Δm1/DG3 and ΔN1/DG3. Different quadrant maps are then subtracted and the AP and DV boundaries represented with dashed lines (red and blue, respectively). Asterisk depict the intersections of AP and DV boundaries. Scale bars: 100μm.

Spatiotemporal characterization of OR463 via localized dCas9 expression.

A. Schematic representation of the method. Upon localized expression, dCas9 would bind to DNA displacing or interfering with the binding of TF. B. Target sites of the gRNAs present in the U6-OR463.gRNAx4 transgene. C. Wings of animals expressing dCas9 under the en-Gal4 driver in the presence of the control gRNA at 23° and 25° degree. D. Wings of animals expressing dCas9 under the en-Gal4 driver in the presence of U6-OR463.gRNAx4 at 23° and 25° degree. Arrowhead indicates the P outgrowth. Notice the presence of A bristles in the P edge (dashed box). E. Control wing discs, expressing dCas9 under the en-Gal4 driver in the presence of control gRNAs at 23°. E’. Anti-Ap immunostaining showing the normal Ap expression pattern in late wing discs. E’’. Quadrant map subtracted from panel E. Notice the single perpendicular intersection between AP and DV boundaries. F. Anti-Ptc and Anti-Ap immunostaining of wing discs expressing dCas9 under the en-Gal4 driver in the presence of U6-OR463.gRNAx4 at 23°. Notice the P outgrowth (arrowhead). F’. Anti-Ap immunostaining exhibiting the extended posterior pattern in the posterior compartment. F’’. Quadrant map subtracted from panel F. Notice reduce size of PD quadrant. AP and DV contact twice, once perpendicularly as in control discs and once tangentially in the P outgrowth. G. Anti-Ptc and Anti-Ap immunostaining of wing discs expressing dCas9 under the ptc-Gal4 driver in the presence of U6-OR463.gRNAx4 at 23°. Notice lack of Ap signal along the central wing disc (asterisk). G’. Anti-Ap immunostaining of the disc in G. G’’. Quadrant map subtracted from panel F. Notice the lack of contact between AD and PD quadrants. Scale bars: 100μm.

Pnt and Hth are required for apE activity via m1.

A. Schematic representation of m1 and m4 predicted binding sites and the generated deletions within m1. B. Phenotypic penetrance of the different deletions within m1 in homozygosis, and hemizygosis (B’). C. Projected area of the different quadrants of control (WT/apDG3, Δm1/ apDG3, Δm1.2/ apDG3 and Δm1.3/ apDG3 wing discs, based on the immunostaining against Ap and Ptc in real wing discs. D. Effect on the Ap expression domain by the expression of hthRNAi and pntRNAi in the P domain using en-Gal. In both cases the PD domain is reduced. In green, the UAS-CD8:GFP reporter marks the P compartment. E. Adult wing phenotypes upon pntRNAi expression via en-Gal4. Left wing: Example of a wing outgrowth resembling Δm1 phenotype. Middle wing: Example of a partial A mirror-image transformation. Inset 1: campaniform sensillae of the A compartment. Inset 2: ectopic campaniform sensillae formed in the P compartment upon pntRNAi expression. Notice the presence of A bristles within the P compartment. Right wing: Example in which P compartment is reduced. F. Effect of the expression of hthRNAi and pntRNAi in the P domain on anti-Ap localization and apE-LacZ reporter (visualized with anti-ßGal) during L2 stage. In control discs, anti-Ap can be detected in the nucleus of P cells (marked with UAS-CD8:GFP) (arrowheads). anti-ßGal (in white) is detected in the dorsal compartment in both A and P compartments. Upon pntRNAi or hthRNAi, no anti-Ap nor anti-ßGal signal was detected in P cells (arrowheads). Scale bars: panels C and D, 100μm; panel E, 25μm

High resolution genetic analysis suggests a HOX-GATA complex important for m3 activity.

A. anti-Ap and anti-Wg immunostaing in control and Δm3 third instar wing discs. In contrast to control wing discs, Ap can only be detected in a small group of cells in the anterior hinge in Δm3 mutants (arrowhead). The territory within Wg inner ring is totally missing (asterisk). No Wg stripe is detected in the pouch. B. X-Gal staining of control apE-LacZ and apEΔm3-LacZ in third instar wing disc. apEΔm3-LacZ only showing minimal X-Gal staining in the P hinge. C. Summary of the base pair substitutions generated within m3.1. Each row corresponds to a different allele containing the changes labelled in red. D. Scoring of wild-type wings across the library of Δm3 mutants. In black, the percentage of WT wings. Between 80 and 250 wings were scored in each case. Asterisks denote mutants that gave rise to no wings with different penetrance. E-E’’’. Wing phenotypes of the control, and three of the mutants of the library. F. Phenotypic penetrance scoring of control animals and individuals in which the linker between GATA and HOX binding sites was extended or contracted. G. Percentage of flies with wings upon deletion of m3, or deletion of m3 and simultaneous contraction of m2 linker. Scale bars: 100μm

the GATA TF Grain is fundamental for wing development.

A. Phenotypes produced upon UAS-grnRNAi expression driven by en-Gal4 in the presence of UAS-dicer. Reduction of the P size and partial P to A transformation (evidenced by the campaniform sensilla in the P territory (arrowhead)). A’. Mirror image duplication of an anterior proximal rudimentary wing structure. A’’. Thorax defects observed in some of the adults. B. Anti-Ap and anti-Wg immunostaining of control wing discs and P knockdown of grn (grnRNAi driven by en-Gal4). In both cases, UAS-GFP was used to mark P cells and UAS-dicer was included to increase knockdown efficiency. B’. Example of wing disc in which the P compartment (arrowhead) was located close to the DV boundary. In these cases, the pouch was specified and grew to some extent, as revealed by the space within the inner ring of Wg (asterisk). B’’. Example of wing disc in which the remanent of the P compartment (arrowhead) is located close to the notum. Here, the notum primordium grew until a considerable size and presented the characteristic Wg band, indicating, to some extent, correct patterning. The pouch (asterisk) is completely absent. C. anti-Ap immunostaining upon tissue-specific knock-out of grn in the P cells using en-Gal4, UAS-Cas9 in the presence of U6-grn.gRNAs. UAS-CD8:GFP labelled posterior cells. Notice the total loss of P compartment in mid L3 wing discs (illustrated by the complete absence of GFP signal). D. L2 wing disc of the same genotype as in C. Immunostaining of apE-LacZ using anti-ßGal4, as well as anti-Ap reveals total lack of signal in the P cells. Scale bars: panels B and C, 100μm; panel D, 25μm

The Hox gene Antp is fundamental for early wing development and ap expression.

A. Scheme of the experimental setup to delete Antp during early stages of wing development. sna1.7-Gal4 driver is used to express Cas9 in the embryonic precursors of wing and haltere. Cas9 is targeted to the Antp locus by three gRNAs (labelled with an arrow). Position of the homeodomain within Antp sequence is also indicated. B. Anti-Antp and anti-Ap immunostaining in control third instar wing discs. Notice the distribution of Antp throughout the disc, with its highest levels in the A compartment and hinge, and the faint band along the DV boundary. C. Example of wing disc derived from sna1.7-Gal4 driven Cas9 in the presence of U6-grn.gRNA. In this case Ap presented a rather normal distribution in the tissue. Antp could be still detected in the wing discs, amid a reduction of its levels. C’. Wing disc of the same genotype as C presenting severe problems in the Ap expression pattern. Ap was detected in the pouch only in two groups of cells (marked with an arrowhead). C’’. Example of a wing disc of the same genotype as C and C’’ in which no immunoreactivity against Antp is detected. Note the complete lack of the anti-Ap signal. In grey, DAPI. These discs lack all recognizable structures (no notum, and no pouch). D. Adult phenotype arising from the same genotype as B (control). The haltere is marked by an asterisk. E. Example of a severe case in which Antp was knocked out from the wing primordia (same genotype as in C, C’ and C’’). In this case, only a small portion of the right notum in still present, with the total loss of the right wing (arrowhead). Left wing presents severe morphological defects and forms a balloon-like structure. Further analysis of this wing revealed the presence of campaniform sensillae and A bristles in the P compartment (data not shown). Notice that the halteres were unaffected (asterisk). Scale bars: 100μm

Working model for OR463 regulatory inputs.

During late embryonic or early larval stages, the HOX input in m3, possibly Antp, would be responsible of OR463 activation. During this early phase the enhancer is not yet functional and the HOX could be priming the enhancer, permitting the later action of the other factors. Grain would participate in this process but its requirement is less critical than that of the HOX. Pnt and Hth would then, during L2 larval stage, activate apE first in the proximal area of the wing disc. Grn could also play a role in this early activity, confining the activity of ap to the dorsal hinge.