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First published online 28 February 2007
doi: 10.1242/dev.02807

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The Zic family member, odd-paired, regulates the Drosophila BMP, decapentaplegic, during adult head development

Division of Cell and Gene Therapy, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892, USA.

View larger version (148K): [in this window] [in a new window]   Fig. 1. opa and dpp interact genetically and produce identical head capsule defects. Scanning electron micrographs of wild-type (A) and head-capsule defect heads in opa and/or dpp mutants (B-G). (B) dppTgR46.1/Df(2L)DTD2, P20 (a strong dpp head capsule mutant phenotype), (C) opa12.3/+; dppTgR46.1/+ and (D) opats125/opa12.3. (E) Missing palpus and disordered vibrissae from opa12.3/+; dppTgR46.1/+. (F) Enlargement of the palpus in C. (G) Enlargement of vibrissae region in D. dpps-hc homozygotes, dpp/+;opa/+, and opats125/opa12.3 have identical head capsule defects; eyes are smaller and rounder than control (compare the length of brackets), vibrissae are disrupted and clustered together (arrows), the maxillary palps are altered in shape and in number (circles).

View larger version (75K): [in this window] [in a new window]   Fig. 2. opa is spatially restricted in both peripodial epithelium and cells of the disc proper in eye/antennal discs. The expression of opa was examined by RNA in situ hybridization and immunohistochemistry, and analyzed by confocal microscopy. The antennal disc is up in both A and B. (A) opa RNA expression by fluorescent in situ hybridization, 2D projection of confocal optical sections through the eye/antennal disc. The bracket indicates non-imaginal disc cells (adepithelial cells or hemocytes) in the preparation that also hybridize with opa probe. The arrow indicates medial disc proper antennal ring; the arrowhead indicates lateral peripodial epithelium staining. The lateral side of the ring in the disc proper is obscured by the broad peripodial expression in this photo. (B) ß-galactosidase expression is directed by the opa enhancer trap opa3D246. The white line shows the area of z-section, and corresponds to the xz-image shown in (C). The arrows indicate the antennal ring in the disc proper. The arrowheads in B and C indicate peripodial epithelial staining. (D) Fate map of third instar disc. ANT, antennal disc; EYE, eye disc. (E) Schematic diagram of head structures. Shaded areas indicate position of primordia on adult head. ANT, antennae; AR, aristae; PAL, maxillary palps; VI, vibrissae.

View larger version (72K): [in this window] [in a new window]   Fig. 3. Eye/antennal imaginal disc expression from opa Lac-Z constructs. (A) Schematic diagram of the opa gene. Stippled boxes represent exons and thin lines represent introns. Positions of five opa constructs are indicated below the gene diagram. Position of the opa3D246 enhancer trap is indicated by the triangle. (B-D) ß-galactosidase expression from (B) opa02 lacZ, (C) opa03 lacZ and (D) opa04 lacZ, as detected by histochemistry. Lateral peripodial staining in B-D is indicated by arrowheads, and staining of the disc proper ring in D by an arrow. Note that B-D comprises the entire pattern seen in the opa enhancer trap opa3D246 shown in Fig. 2B.

View larger version (149K): [in this window] [in a new window]   Fig. 4. dpps-hc-lacZ expression is reduced in LOF opa mutants. (A) Wild-type expression of the dpps-hc-lacZ reporter construct, SH53, is seen on the lateral side of eye/antennal discs. Expression from this reporter construct is reduced, most notably in the middle region (arrow) in (B) opats125/opa12.3, (C) dppTgR46.1/+; opa12.3/+ and (D) Df(2L)DTD2, P20/+; opats125/+ (at 25°C) mutants.

View larger version (43K): [in this window] [in a new window]   Fig. 5. opa LOF clones fail to express dpps-hc-lacZ. Homozygous opa mutant clones were generated by using the FLP/FRT system. dpps-hc-lacZ construct, SH53, expression (in red) in non-recombination control disc (A), and in opa mutant tissue in single section images (B-E). The clonal areas are outlined in white in the merged panels (B-D) and are marked by absence of green fluorescence, which can be seen in the B'-E' green-only channel. The box in E indicates dpp reporter expression missing in a small clone, and its enlargement is shown in the white inset box in E and E'. The positions of clones relative to the entire lateral dpps-hc-lacZ expression pattern is shown by labeled brackets in A.

View larger version (72K): [in this window] [in a new window]   Fig. 6. opa LOF clones display severe head malformations. (A,B) Heat-shock induced clones in adult heads have small and round eyes, abnormal antennae (arrowhead), vibrissae defects (arrow) and missing palps (circle). (C) Clonal area in dorsal head, indicated by full-length bristles, is normal.

View larger version (114K): [in this window] [in a new window]   Fig. 7. Targeted misexpression of opa causes ectopic dpps-hc-lacZ expression. (A) dpps-hc-lacZ expression (green) in a wild-type disc. (B) ß-galactosidase expression (red) driven by the MS1096-Gal4. (C) Ectopic expression of dpps-hc-lacZ from MS1096-Gal4>Opa. Note boxed areas of ectopic expression from the dpps-hc-lacZ reporter on the medial side of the disc. The white arrow indicates the region of Opa expression where dpps-hc-lacZ is not expressed. Cytoplasmic ß-galactosidase expression (green) and Opa overexpression (nuclear) as detected by Opa antibody (red). (D,E) Higher magnification images of the areas boxed in C. (F) Cross section of area in E, showing that ß-galactosidase expression (green) and Opa antibody (red) are colocalized within the peripodial epithelium (arrow). (G) ß-galactosidase expression (red) driven by the c309-Gal4 driver. (H) Ectopic expression of dpps-hc-lacZ from c309-Gal4>Opa. A small area of ectopic ß-gal expression is boxed. (I) Higher magnification of the box in H. Nuclear Opa (red) and cytoplasmic ß-galactosidase expression (green) are colocalized. (J) Cross-section analysis of I. dpps-hc-lacZ and Opa expression are again limited to the peripodial epithelium (arrow). The nuclei of all discs are stained with DAPI (blue). The peripodial epithelium is oriented up in F and J, and lateral is to the left in all pictures.

View larger version (47K): [in this window] [in a new window]   Fig. 8. Opa misexpression causes severe head malformations. (A) Wild-type head. (B,C) Adult heads from ey-Gal4>Opa. Eyes are absent. Arrowheads indicate antennal and aristal duplications and arrows indicate ectopic maxillary palps. (D) Adult head from ey-Gal4>Dpp. Eyes are present, but reduced. Arrowhead indicates misplaced antenna, arrows indicate maxillary palps. Note duplicated palpus on right. Asterisks represent outgrowths. (E) Wild-type and (F) ey-Gal4>Opa third instar imaginal discs stained with antibody to Dachshund. Note duplicated antennal ring, and lack of eye field staining in the ey-Gal4>Opa disc. (G) dpps-hc-lacZ expression in ey-Gal4>Opa discs. Note the small amount of the remaining eye disc (asterisk), and bifurcated dpps-hc-lacZ expression. (H) dpps-hc-lacZ expression in ey-Gal4>Dpp discs. Staining no longer extends into the eye disc, and extends further medially in the posterior antennal disc. Compare with Fig. 4A. (I) ey-Gal4>Dpp discs stained with antibody to Dachshund. Note partial duplication of antennal ring, and the expansion of the retinal field anteriorly throughout the entire eye disc.

View larger version (44K): [in this window] [in a new window]   Fig. 9. ß-galactosidase expression directed by peripodial reporter constructs persists in adult heads. (A) Diagram showing front and back of adult head with relevant structures labeled. Modified from Bryant (Bryant, 1978). Prst indicates Proximal rostral sensilla. (B-F) Histochemical detection of ß-galactosidase activity in B, dpps-hc- lacZ, anterior view. (C) Same construct, posterior view. Arrows represent Prst. Expression of (D) opa02 lacZ, (E) opa03 lacZ and (F) opa04 lacZ.

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