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First published online June 1, 2005
doi: 10.1242/10.1242/dev.01869

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Dual regulation and redundant function of two eye-specific enhancers of the Drosophila retinal determination gene dachshund

¹ Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
² Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
³ Department of Pathology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
⁴ Human Genome Sequencing Center, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
⁵ Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
⁶ Department of Neuroscience, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA

View larger version (58K): [in a new window]   Fig. 1. Conserved non-coding sequences in the dac locus uncover two eye enhancers. (A) The dachshund genomic locus, with 5' and 3' eye enhancers indicated (see text for details). (B) AVID/mVISTA representation of the 5' eye enhancer (5EE). The numbered peaks indicate four areas of significant conservation. A small inversion in D. willistoni masks the first two peaks. (C) AVID/mVISTA representation of the 3' eye enhancer (3EE). The numbered peaks indicate six areas of significant conservation and two sub-fragments tested are shown. 3EE850 bp contains all six CNCS blocks and is expressed only posterior to the MF. 3EE659 bp contains the first four CNCS blocks and is expressed both anterior and posterior to the MF. The smallest active enhancer fragment identified is 194 bp (3EE194 bp) and contains CNCS blocks 3 and 4. Regions of significant conservation are indicated in pink (B,C), and the predicted Sine oculis-binding site is highlighted in bright blue and is within CNCS block 3. (D,E) Representative third instar eye discs from 3EE850 bp-GFP (D) and 3EE659 bp-GFP (E) larvae triple labeled with GFP (green, D,E), Sens (magenta, D',E') and Dac (blue, D'',E''). GFP expression in 3EE659 bp eye discs is detected anterior to the earliest Sens expression and overlaps with anterior Dac expression (E',E''). By contrast, GFP expression in 3EE850 bp eye discs is not detected anterior to the anterior-most Sens-expressing column (D').

View larger version (91K): [in a new window]   Fig. 2. The 3' eye enhancer is dispensable for dac activation in vivo. (A) dac7 is associated with a deletion, beginning at exon 9, that uncovers the entire 3' genomic region of dac. (B-D) Scanning electron micrographs (SEMs) of adult eyes from wild-type (B), dac7 (C) and dac3 (D) animals. (E-G) Third instar eye imaginal discs from wild type (E), dac7 (F) and dac3 (G) animals stained with a monoclonal Dac antibody, mab2-3. dac7 eye discs show relatively normal Dac protein expression when compared with wild-type eye discs (compare F with E). dac7 adult eyes are rough and disorganized but still contain 50% of the normal number of ommatidia (compare C with B). By comparison, dac3 homozygotes express no Dac protein (G) and develop with no eyes (D). (H,I) A first instar (H) and second instar (I) eye disc of wild type shown for comparison with J and L. (J-M) Late first (J) and third (K) instar eye imaginal discs from 5EE-lacZ and second (L) and third (M) instar eye imaginal discs from 3EE-lacZ transgenic larvae. ß-Galactosidase activity is detected in both first instar and third instar 5EE-lacZ eye discs, primarily at the posterior margin (J,K). The earliest 3EE-lacZ reporter expression is observed in second instar eye discs and this ß-galactosidase activity persists in the third instar eye disc (L,M).

View larger version (82K): [in a new window]   Fig. 3. Synergistic activation of 3EE-GFP by eya, so and dpp. (A-C) Each set of three panels shows the same wing disc stained with anti-Dac (magenta) and anti-GFP (green) or a merge of the two channels. (A) Wing imaginal discs in which the expression of UAS-ey is driven by 30A-Gal4 show ectopic expression of 3EE-GFP and Dac in two regions at the anteroposterior (AP) compartment boundary. (B) Wing imaginal discs expressing a combination of eya and so driven by 30A-Gal4 can strongly induce 3EE-GFP and Dac at the AP compartment boundary where their expression coincides with endogenous dpp (white). (C) A combination of dpp, eya and so driven by 30A-Gal4 synergistically induces the expression of 3EE-GFP and Dac in the entire ring around the wing pouch.

View larger version (81K): [in a new window]   Fig. 4. eya, so and dpp signaling are required for regulation of 3EE-GFP. (A-C) Each set of three panels shows the same eye disc stained with anti-Dac (magenta in A-C) and anti-GFP (green in A'-C') or a merge of the two channels (A''-C''). (A-A'') Wild-type eye imaginal discs stained with GFP and Dac reveal the normal expression of endogenous Dac (A) and 3EE-GFP (A'). (B-C') so1 (B,B') and eya2 (C,C') eye imaginal discs have drastically reduced levels of Dac (B,C) and completely lack 3EE-GFP (B',C'). (D-D''') Each set of four panels shows the same eye disc stained with anti-ß-galactosidase (magenta in D), anti-Dac (green in D'), anti-GFP (green in D''), or a merge of the three channels (D'''). Posterior margin mad mutant clones, negatively marked by the lack of ß-galactosidase, block Dac (D') and GFP (D'') expression. (E,F) An eye-antennal disc from a w; UAS-ey, 3EE-GFP; dpp-Gal4 third instar larva stained with an antibody against GFP alone (E) or GFP and Dac (F). Ectopic ey expression in the antenna driven by dpp-Gal4 can strongly induce 3EE-GFP (E) and Dac (F) in the ventral antenna (arrows). (G,H) Eye-antennal discs from w; eya2; dpp-Gal4, 3EE-GFP/UAS-ey (G) and w; so1; dpp-Gal4, 3EE-GFP/UAS-ey (H) larvae co-stained with antibodies against GFP and Dac (both panels show a merge of the two channels). Ectopic ey expression in the antenna (arrows) driven by dpp-Gal4 cannot induce 3EE-GFP expression (green in G and H) but retains the ability to induce Dac expression in the ventral antenna (magenta in G and H).

View larger version (69K): [in a new window]   Fig. 5. 5EE-lacZ is regulated by ey, eya and so. (A-J) All panels show wing (A-F) or eye-antennal (G-J) discs from 5EE-lacZ transgenic lines stained to reveal ß-galactosidase reporter activity. (A) A wing imaginal disc from a 5EE-lacZ third instar larva shows weak enhancer activity (present in multiple transgenic lines). (B,C) Wing imaginal discs from 5EE-lacZ third instar larvae in which the expression of UAS-ey alone (B) or a combination of UAS-dpp, UAS-eya and UAS-so (C) is driven by 30A-Gal4. (B) ey alone, but not (C) a combination of UAS-dpp, UAS-eya and UAS-so, is capable of inducing 5EE-lacZ in the ring around the wing disc. (D-F) Wing imaginal discs from 5EE-lacZ third instar larvae in which the expression of UAS-ey is driven by dpp-Gal4 in wild-type (D), so1 (E), eya2 (F) or mutant backgrounds. Ectopic ey expression is able to induce ß-galactosidase reporter expression via 5EE at the AP boundary in all three cases. The activity is stronger in so1 mutant wing discs than eya2 mutant wing discs. (G,H) Eye-antennal imaginal discs from so1 (G) or eya2 (H) mutant 5EE-lacZ third instar larvae stained for ß-galactosidase activity. No reporter activity is detected in these mutant eye discs. (I,J) Eye-antennal imaginal discs from w; so1; dpp-Gal4, 5EE-lacZ/UAS-ey and w; eya2; dpp-Gal4, 5EE-lacZ/UAS-ey third instar larvae. Strong induction of 5EE-lacZ is seen in the ventral antenna in both so1 and eya2 mutants (arrowheads in I and J, respectively). In addition, ß-galactosidase activity is restored at the posterior margin of so1 mutant eye discs (arrow in I).

View larger version (55K): [in a new window]   Fig. 6. Mutating the putative So binding sites in 3EE abolishes enhancer activity in vivo. (A) A multiple sequence alignment of the 40 conserved bases in the 3' eye enhancer. Mismatched bases are shown in grey and the two putative So-binding sites are shown in green. Mutated So-binding sites are shown in red. (B-E) Each panel shows a single eye disc co-stained with anti-Dac (blue) and anti-GFP (green). Mutating each putative So-binding site individually (C,D) results in the dramatic reduction of GFP reporter expression from the 3EE enhancer when compared with the wild-type version of same enhancer (B). When both So-binding sites are mutated, enhancer activity and GFP expression is completely abolished (E). Endogenous Dac expression is unaffected in all cases.

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