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First published online 5 October 2005
doi: 10.1242/dev.02061

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Pointed regulates an eye-specific transcriptional enhancer in the Drosophila hedgehog gene, which is required for the movement of the morphogenetic furrow

Edward M. Rogers, Catherine A. Brennan^*, Nathan T. Mortimer, Summer Cook, Andrea R. Morris and Kevin Moses^,

Department of Cell Biology, Emory University School of Medicine, Atlanta GA 30322, USA

View larger version (72K): [in a new window]   Fig. 1. hedgehog mutations form an allelic series. (A) The hedgehog locus and the transgenic reporter constructs used. The lesions of four hedgehog alleles is indicated (see text). Seven transgenic constructs are drawn to a larger scale (indicated). The checks and crosses indicate the constructs that express or do not express lacZ reporter posterior to the morphogenetic furrow. The gray box indicates the minimal region required for eye-specific expression. The white triangles indicate Pointed-binding sites (this paper) and the black triangles indicate Sine oculis (So)-binding sites (Pauli et al., 2005). (B-I) Adult eyes, anterior rightwards and dorsal upwards. Genotypes are indicated below the panels. (J) Histogram of female facet counts for different hedgehog genotypes as indicated. Bars show mean facet counts; number of individuals counted indicated in bar. Error bars show the 95% confidence limits. Letters above some bars refer to panels of same genotype. Scale bar: 50 µm in B for B-I.

View larger version (110K): [in a new window]   Fig. 2. hhbar3 phenotypes in the developing eye. Eye-antennal imaginal discs, shown anterior rightwards and dorsal upwards. Black arrowheads indicate the morphogenetic furrow. Genotypes are indicated below each panel. (A-F) Hedgehog antigen (red in C and black in the other panels). Boss is in green in C. (G-I) lacZ driven by the BS3.0 dpp enhancer (Blackman et al., 1991). an, antennal disc; oc, ocellar domain. In hhbar3, Hedgehog antigen is undetectable in the presumptive eye field (compare at white arrows in A and D) but is normal in the ocellar region and in the antennal disc (black arrows in A,D). Hedgehog is first expressed in only two cells per ommatidium (white arrowheads in B) and that these are not Boss-expressing R8 cells (white arrows in C). A low level of anti-Hedgehog stain is seen at the posterior margin near the time of furrow initiation (white arrows in E,F). dpp:lacZ expression is reduced in the furrow in hhfse and eliminated from the central furrow in hhbar3. Scale bar in A: 50 µm for A,D,G-I; 10 µm in B; 1 µm in C; in F, 50 µm for E,F.

View larger version (70K): [in a new window]   Fig. 3. ß-Gal expression from hedgehog enhancer constructs. Third instar eye-antennal imaginal discs, stained for ß-gal antigen, and shown anterior towards the right and dorsal upwards. Arrowheads mark the morphogenetic furrow. Transgenic lacZ lines are named as shown in Fig. 1A and indicated below the panels, see text. Constructs containing the three adjacent Pointed-binding sites have robust ß-gal expression posterior to the morphogenetic furrow (white arrows in A,C,F) but not in the antennal disc or the ocellar region (black arrows in A,C,F). The 203 bp fragment bar3L2 is sufficient to drive expression of lacZ reporter in the eye (F). Mutation for all three Pointed-binding sites eliminates this reporter expression (G). Six tandem Pointed-binding sites are not sufficient to drive expression of ß-gal (H). Scale bar: 50 µm.

View larger version (84K): [in a new window]   Fig. 4. The hedgehog eye enhancer drives expression in all photoreceptors except for the R8 photoreceptor. Third instar eye imaginal discs, anterior rightwards. The fields shown are just posterior to the furrow. A-D and E-H are the same fields in the same eye discs. Genotypes are shown on the left; antigens are shown below; colors as indicated. Elav marks all photoreceptor nuclei (A,D,E,H;, Senseless (Sens) marks only the R8 photoreceptor nuclei (B,D,F,H); lacZ reporter (C,D,G,H). Black arrows indicate a Sens positive R8 nucleus in a single ommatidium. White arrows (B-D) mark R8, 2 and 5 as indicated. Both the 1.9 kb full-length bar3:lacZ (A-D) and the minimal 203-bp bar3L2:lacZ (E-H) have indistinguishable lacZ expression patterns. There is no LacZ expression in the R8 photoreceptor. Scale bar: 10 µm.

View larger version (113K): [in a new window]   Fig. 5. pointed regulates the expression of hedgehog from an eye specific enhancer. (A-D) Third instar bar3:lacZ eye imaginal disc. The field shown is several columns posterior to the morphogenetic furrow, anterior towards the right. The disc contains a mosaic clone that is homozygous for the null mutation pntdelta88. (A,D) GFP negatively marks the outlined clone. (B,D) Elav marks all the photoreceptors. (C,D) lacZ reporter. lacZ reporter expression is absent from within the pointed clone (a wild-type example outside the edge of the clone is indicated with a white arrow). The single R8 cells are Elav positive in the clone (black arrow). Scale bar: 5 µm.

View larger version (74K): [in a new window]   Fig. 6. Pointed P2 protein binds specifically to the hedgehog eye specific enhancer. Electrophoretic mobility shift assay (EMSA) gel for GST-Pointed binding. Competitor DNA is indicated at the top, with the rising level indicated by ramps. The presence or absence of GST-Pointed is indicated by + or –. Lane numbers are indicated. Lane 1, probe alone; lane 2, no competitor; lanes 3-7, 203 bp Bar3L2 competitor, ranging from 15 to 240 times molar excess; lanes 8-12, 203 bp Bar3L2(deltaETS) competitor, ranging from 15 to 240 times molar excess; lane 13, cold probe as competitor and lane 14 Ets site mutated cold probe as competitor. The probe sequence is shown below gel with the three Ets consensus sites highlighted. The nine mutations made in Bar3L2(deltaETS) are shown in white boxes.

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