1. Combgap Relays Wingless Signal Reception to the Determination of Cortical Cell Fate in the Drosophila Visual System 
Yuechun Song, Soohee Chung, Sam Kunes 
Molecular Cell 
Volume 6, Issue 5, Pages (November 2000)
DOI: /S (00)00112-X

2. Figure 1 Cortical Pattern Formation in the Drosophila Visual System
(A) Schematic illustration of the three cortical cell precursor populations of the Drosophila visual ganglia at the third instar larval stage. Two Wg-positive cellular domains (blue color) lie at the prospective dorsal (D) and ventral (V) margins of the cortices. The dorsal and ventral margins are adjacent to one another at this stage, and separate during pupal morphogenesis at a site indicated by the black arrow. The dorsoventral midline of the lamina cortex is indicated (M). Dpp-positive domains (red color) lie adjacent to the Wg domains. At this stage, photoreceptor receptor neurons differentiate in the eye imaginal disc (eye) and send their axons through the optic stalk (os) to retinotopic positions in the crescent-shaped lamina target field. The dorsal ommatidium shown sends axons to a dorsal cortical position. The ventral ommatidium sends axons to a ventral position.
(B) Micrograph of a late third instar visual system from an animal harboring the dpp-lacZ reporter construct. BS3.0 (red color, anti-β-galactosidase antibody). Dpp-positive domains are as indicated in (A). Lamina cortical cells and precursors express the transcription regulator Ci (blue color, anti-Ci antibody). The dorsal (D), ventral (V), and midline (M) of the cortices are indicated, as in (A). A white arrow marks the junction of the dorsal and ventral margins at this stage.
(C) A higher magnification view of dorsal cortex region of the specimen shown in (B).
(D) Micrograph of a late third instar dpp-lacZ (BS3.0; red color, anti-β-galactosidase antibody) specimen in which the Wg-positive cells are revealed by staining with anti-Wg antibody (blue color). The Wg-positive and Dpp-positive cell populations are nonoverlapping.
(E) Micrograph of a late third instar specimen stained to reveal the expression of Omb (red color; anti-Omb staining). Omb is expressed by both the Dpp-positive and Wg-positive populations. The Omb-positive cells are also Ci positive (blue color). Omb-positive glia (arrowhead) have migrated from the Wg domains into the lamina.
(F) Micrograph of a late third instar al04352 specimen to reveal expression of the al-lacZ reporter (red color). al-lacZ expression includes Dpp-positive, Wg-positive, Omb-positive, and Ci-positive cells. Ci expression (blue color) demarcates the lamina field.
(G) A high magnification view of the dorsal cortical region of the specimen shown in (F).
Scale bar in (B) (for [B] and [D–F]) is 20 μm. Scale bar in (C) (for [C] and [G]) is 10 μm.
Molecular Cell 2000 6, DOI: ( /S (00)00112-X)

3. Figure 6 cg Expression Is Under wg+ Control
(A) The pattern of Cg expression was examined with an anti-Cg polyclonal serum (blue color in [A], shown alone in [A′]). Cg expression is absent from the midline region of the cortex (left of the arrow) and reduced in domains adjacent to the wg-expressing cells (red color in [A], lacZ expression from the wg enhancer trap reporter 17en40).
(B) Nonautonomous induction of Omb by ectopic wg+. Omb expression (red color [B], shown alone in [B′]) was examined after the induction of ectopic wg+-expressing clones by the flp-out activation of P{tubαy+,CD2>wg+} (Zecca et al. 1996). The flp- out clone was visualized by the loss of CD2 expression (blue color in [B], outlined in [B] and [B′]). Ectopic Omb expression extends beyond the boundary of the wg+ clone.
(C) Downregulation of Cg expression by ectopic wg+. The activity of somatic wg+ clones, produced as in (B), was monitored by Omb expression (red color in [C], arrowheads). Ectopic Omb expression correlated with a reduction of Cg expression (blue color in [C], shown alone in [C′]).
A white bar indicates the position of the dorsal (D) and ventral (V) margins. Scale bar in (A) (for [A–C]) is 15 μm.
Molecular Cell 2000 6, DOI: ( /S (00)00112-X)

4. Figure 2 The combgap Phenotype in the Visual Cortex
Wild-type (A–D) and cgk11504 (E–H) specimens stained to reveal photoreceptor axon projections, and dorsoventral cortical cell-type markers.
(A and E) Late third instar stage specimens stained to reveal photoreceptor axon projections (brown color; Mab24B10 staining). In the wild-type (A), photoreceptor axons form a crescent-shaped projection pattern in the lamina. In a cgk11504 specimen (E), photoreceptor axons establish an aberrant pattern, often straying into the medulla cortex region.
(B and F) Confocal micrographs of mid-third instar specimens stained to reveal the expression of the BS3.0 dpp-lacZ reporter (red color) and Wg (blue color). The wild-type specimen (B) displays Wg and dpp-lacZ expression in dorsal (D) and ventral (V) domains. In the cgk11504 specimen (F), dpp-lacZ is expressed in large domains that extend toward the midline (M), but Wg expression appears normal.
(C and G) Micrographs of mid-third instar specimens stained to reveal the expression of al-lacZ (red color). In the wild-type (C), al-lacZ expression diminishes in a graded fashion with distance from the dorsal (D) and ventral (V) margins. High-level al-lacZ expression extends toward the midline in the cgk11504 specimen (G).
(D and H) Micrographs of late third instar specimens stained to reveal Omb expression (red color). In the wild-type (D), Omb expression is restricted to domains adjacent to the dorsal and ventral margins, and to glia that migrate toward the midline (M) of the lamina. In cgk11504 (H), larger domains of Omb expression extend toward the midline region.
Scale bar in (A) (for [A] and [E]) is 30 μm. Scale bar in (B) (for [B–D] and [F–H]) is 20 μm.
Molecular Cell 2000 6, DOI: ( /S (00)00112-X)

5. Figure 3
A Cell-Autonomous Requirement for combgap in the Developing Optic Lobe and Wing
Somatic cgk11504 homozygous clones were generated using the FLP/FRT system and marked by arm-lacZ ([A and B] and [F–H]) or Myc-epitope reporters (C–E). Homozygous mutant cells lack reporter expression (blue color).
(A and A′) cgk11504 clones (outlined) do not affect the pattern of Wg expression (red color in [A], shown alone in [A′]).
(B and B′) cgk11504 clones (outlined) in the dorsal and ventral medulla cortex (arrowheads) display ectopic Omb expression (red color in [B], shown alone in [B′]). Clones near the midline (thick arrows) do not display ectopic Omb expression.
(C and C′) cgk11504 clones (outlined) express ectopic Dpp in a subset of cells localized to a band within the clone (red color in [C], shown alone in [C′]).
(D and D′) al-lacZ (red color in [D], shown alone in [D′]) is expressed at elevated levels in the cgk11504 clone (outlined) located in a dorsal region (arrowheads). al-lacZ expression is normal in a clone located near the midline.
(E and E′) combgap is not required for Wg signaling in the wing. Dll expression (green color in [E], shown alone in [E′]) forms a gradient in the wing pouch of a late third instar larvae, with highest levels at the dorsoventral margin (indicated by the thick bar). Dll expression is normal in a homozygous cgk11504 clone (indicated by arrows).
(F and F′) combgap controls Cubitus interruptus expression. cgk11504 clones in the anterior compartment (see arrowhead for example) display reduced Ci expression (blue color in [F], shown alone in [F′]), while clones in the posterior compartment (indicated by the arrow) express a low level of Ci. Ci is normally not detectable in the posterior compartment. Wg expression (green color in [F]) is not affected by cg loss of function.
(G and G′) combgap is required for Engrailed expression at the anterior–posterior compartment boundary. Homozygous cgk11504 clones that include the thin stripe of Engrailed-positive cells (green color in [G], shown alone in [G′]) that extend into the anterior lineage compartment (arrow; see Blair 1992) are deficient in Engrailed expression and Ci expression (blue color in [G]).
(H and H′) Dpp expression at the anterior–posterior compartment border is reduced by loss of combgap function. Dpp expression (green color, shown alone in [H′]) is reduced in cgk11504 clones (indicated by arrow).
Scale bar in (A) is 20 μm. Scale bar in (B) (for [B] and [C]) is 25 μm. Scale bar in (D) is 8 μm. Scale bar in (E) (for [E–H]) is 15 μm.
Molecular Cell 2000 6, DOI: ( /S (00)00112-X)

6. Figure 4 Molecular Analysis of the combgap Locus
A 3.2 kb cDNA clone isolated from the combgap locus was subjected to DNA sequence analysis and aligned to the genomic DNA sequence AE (GenBank). combgap encodes a protein with 650 amino acids that contains ten C2H2-type zinc fingers and a polyglutamine stretch, features that are consistent with a role as a DNA binding transcriptional regulator. Top, schematic structure of the Combgap protein. The ten C2H2-type zinc fingers are indicated by purple shading. Conserved residues within each zinc finger are marked in red. A polyglutamine stretch is indicated by turquoise shading. Bottom, alignment of a cg cDNA with genomic DNA to indicate the position of intron/exon boundaries. Cg is encoded in nine exons. The site of the cgk11504 PlacW is within the first intron. Three dTCF consensus binding sites are also located within the first intron, adjacent to the second exon border. A primary structure of the Cg protein is shown to indicate the relative positions of the zinc finger domains.
Molecular Cell 2000 6, DOI: ( /S (00)00112-X)

7. Figure 5
Combgap Is Sufficient to Repress wingless Target Gene Expression
Animals harboring the P{UAS-cg+}, P{hsflp}, and P{tubα1y+,CD2GAL4} transgenes were subjected to a brief heat shock in the first instar stage to induce GAL4+ clones. In the late third instar specimens shown in (A) and (B), GAL4+ clones were marked by the lack of CD2 expression (red color).
(A and A′) A GAL4+ clone (outlined) includes the Wg-positive domain. Dpp expression (blue color in [A], shown alone in [A′]) in the dorsal domain is not affected by ectopic cg+ expression in the adjacent Wg-positive domain.
(B and B′) A GAL4+ clone (outlined) overlaps the dorsal Dpp domain. Dpp expression (blue color in [B], shown alone in [B′]) is absent within the clone. Though Dpp expression is lost from its normal site, a low ectopic level of Dpp expression occurs outside of the GAL4+ clone in the adjacent Wg-positive domain.
(C and C′) Ectopic cg+ expression can totally eliminate Omb (red color in [C]) and Dpp (blue color in [C], shown alone in [C′]) expression. The location of the GAL4+ clone is not marked.
Scale bar in (A) (for [A–C]) is 27 μm.
Molecular Cell 2000 6, DOI: ( /S (00)00112-X)

8. Figure 7
cg Function Is Downstream of Axn, a wingless Pathway Negative Regulator
Specimens were dissected from (A) wild-type, (B) omb-GAL4; UAS-Axn, and (C) omb-GAL4; cgk11504, UAS-Axn/ cgk11504 animals, and combined in a single tube for labeling with anti-Omb (red color in [A–C]; shown alone in [A′–C′]), anti-Dpp (blue color in [A–C]; shown alone in [A′′–C′′]), and anti-HRP (green color in [A–C]). Micrographs were collected using the same parameters so as to permit accurate comparison of the relative levels of labeling. In the wild-type (A), Omb and Dpp exhibit strong expression in overlapping dorsal (D) and ventral (V) domains. With the presence of omb-GAL4 and UAS-Axn, the level of Omb and Dpp expression is dramatically reduced (B); their expression is not detectable in the dorsal region. Photoreceptor axons project abnormally in the dorsal region (open arrow). In cgk11504, the presence of omb-GAL4 and UAS-Axn (C) reduces the normally high level of Omb expression in the dorsal and ventral domains (compare with the cgk11504 specimen shown in [2H]), but ectopic Omb and Dpp expression are nonetheless observed. Thus cg loss of function is epistatic to Axn gain of function.
Scale bar in (A) (for [A–C]) is 20 μm.
Molecular Cell 2000 6, DOI: ( /S (00)00112-X)