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First published online 17 July 2008
doi: 10.1242/dev.006189

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Cell-type-specific transcription of prospero is controlled by combinatorial signaling in the Drosophila eye

¹ Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA.
² Wyeth Research, Department of Oncology, 401 N. Middletown Road, Pearl River, NY 10965, USA.

View larger version (13K): [in this window] [in a new window]   Fig. 1. The pros enhancer and eye development. (A) Schematic of the developing third instar eye-antennal disc complex. Anterior, top; posterior, bottom. The So/Eya expression domain is colored red. Positions of the morphogenetic furrow and PPN zone are shown. Black dots represent differentiating photoreceptor and cone cells, and show the progressive differentiation of cells from the furrow to the posterior disc margin. (B) Molecular analysis of the pros enhancer and transcription factor-binding sites. The original pros enhancer reporter is shown at the top, as a fusion of four tandem copies with hsp27-lacZ. Various constructs used in this study are shown below, including the full-size enhancer fragment fused to pros-lacZ and hsp70-lacZ. Deletion mutants of the fragment are presented below those. The transcription activity of each enhancer construct is presented as a qualitative scale from a β-gal pattern that completely mimics the Pros pattern (+++), to a β-gal pattern that exhibits variegated expression in R7 and cone cells (+ and ++), to no expression (-). Multiple independent transgenic lines were assayed for each construct. Colored triangles indicate putative transcription factor-binding sites based on their similarity to consensus-binding sequences. Their identities and locations in the pros enhancer region are indicated. Putative sites for which there are experimental binding data that confirm their binding-site activity (this paper) (Xu et al., 2000) are marked by an asterisk.

View larger version (63K): [in this window] [in a new window]   Fig. 2. Alignment of the pros enhancer from four Drosophila species. CLUSTAL alignment of sequences from the core-enhancer and the mini-enhancer used in experiments. Sequences conserved in all four species (D. melanogaster, D. simulans, D. pseudoobscura, D. virilis) are marked below the alignment by red asterisks. Long stretches of sequence found in only one species are not included, and their lengths are indicated by numbers of bases and a species identifier, below the alignment. Putative factor-binding sites are colored. These are: Su(H), green; Yan/Pnt, red; So/Eya, orange; Gl, purple; Lz, blue; Svp, cyan.

View larger version (79K): [in this window] [in a new window]   Fig. 3. So binds to the pros enhancer and activates transcription. (A) The three putative So-binding motifs are highlighted in red within the core-enhancer. These are also present in the mini-enhancer. (B) Alignment of a consensus So-binding site and the three putative So-binding sites. Essential residues are colored red; conserved residues are colored blue. (C) A mobility-shift gel showing a complex that contains So protein and labeled SoAE DNA. SoAE is a So-binding site identified in the so gene autoregulatory element. Each reaction also contained a 50-fold molar excess of an unlabeled competitor DNA as indicated, except for the reaction loaded into the leftmost lane, which did not contain competitor. The SoAE(mut) competitor has a mutated So-binding site; the Lozenge competitor contains a So-target sequence identified in the lz enhancer. The Pros competitors contain the three putative So-binding sites from the pros enhancer. (D,E) Transgenic core-enhancer-β-gal reporter expression (green) in eye discs counterstained for Pros (purple). (D-D'') The wild-type core-enhancer induces β-gal expression in the same cells that express Pros. (E-E'') The core-enhancer with mutant So sites does not induce β-gal expression. (F) Schematic of the approach to make so(ey) and eya(ey) mutant eye discs. The expression patterns of So/Eya in wild-type (left), so/eya mutant (middle) and so(ey)/eya(ey) (right) mutant eye discs are shown in red. In so(ey) or eya(ey) eye discs, So/Eya expression is limited to cells anterior to the MF (black line). (G-J) Expression of neural-specific Elav (purple) and Pros protein (green) in a wild-type eye disc (G-G''), an eya(ey) disc (H-H''), a so(ey) disc (I-I''), and an Lz-GAL4/+; UAS-soDN/+ disc (J-J'').

View larger version (113K): [in this window] [in a new window]   Fig. 4. Lz is expressed in so(ey) and eya(ey) eye discs. Elav protein (purple) and Lz protein (green) in wild-type (A-A''), eya(ey) (B-B'') and so(ey) (C-C'') eye discs.

View larger version (83K): [in this window] [in a new window]   Fig. 5. Gl regulates pros transcription. (A) Gl protein binds specifically to a sequence present in the mini-enhancer. (Left) The sequence is aligned with a characterized Gl-binding site in the Rh1 promoter. Conserved nucleotides are highlighted in red. A mutated version of the pros site is shown, with altered nucleotides in blue. (Right) A mobility-shift gel showing a complex that contains Gl protein and labeled Rh1 DNA. Each reaction also contained a 100x molar excess of an unlabelled competitor DNA, as indicated at the top, except for the leftmost lane, which did not contain any competitor. (B-B'') gl60j mutant cells are marked with cytoplasmic GFP (green), whereas Gl+ cells are unmarked in a genetically mosaic larval eye disc. Nuclear Pros protein was visualized by antibody (purple). Gl+ cells that normally express Pros appear as non-ringed purple, whereas gl60j cells that normally express Pros appear as white or purple, ringed in green. gl60j cells that express undetectable or very low levels of Pros appear as hollow green rings or green rings with a weak white signal (see B''). (C-C'') A large clone of gl60j mutant cells is marked by GFP (green) in the pupal eye. Cell morphology is visualized by the β-catenin protein Arm (purple). Arrowheads indicate the morphologies of wild-type cone cells. Arrows indicate gl60j cone cells that exhibit altered sizes and morphologies. (D-D'') Transgenic mini-enhancer-lacZ reporter expression (green) in an eye disc counterstained for Pros protein (purple). The mini-enhancer has its Gl-binding site mutated, which induces very weak or no β-gal expression in Pros-positive cells.

View larger version (72K): [in this window] [in a new window]   Fig. 6. Notch signaling activates pros expression. The expression of Pros protein (green) and neural-specific Elav protein (purple) are shown in eye discs. Co-expression is seen as white images in the merged panels. (A-A'') A wild-type eye disc from an animal raised at 25°C. (B-B'') A Nts3/Y; UAS-NECN/+; sev-GAL4/+ eye disc from an animal raised at 25°C. (C-C'') A Lz-GAL4 UAS-H/UAS-H eye disc. (D-D'') A sev-Nact/+ eye disc at a focal plane where only photoreceptor cells are visible. Two to three photoreceptors express Pros protein in each ommatidium. (E-E'') A high magnification view of one sev-Nact/+ ommatidium showing ectopic Pros in R1/R6 photoreceptors (arrows). (F-F'') A high magnification view of one Lz-GAL4/+; UAS-Su(H)-VP16/+ ommatidium. Ectopic Pros in R1/R6 is seen (arrows).

View larger version (79K): [in this window] [in a new window]   Fig. 7. Svp regulates the pros enhancer. (A) Alignment of the consensus Su(H)-binding sequence with the S1 site in the E(spl)m4 gene and the ten putative-binding motifs found in the core-enhancer. Mismatched residues are colored in red. Two of these motifs (pros7 and pros10) are also present within the mini-enhancer. (Right) A mobility-shift gel showing the complex that contains Su(H) protein and labeled E(spl)m4S1 DNA (m4S1). Each reaction contained a 100x molar excess of an unlabeled competitor DNA as indicated, except for the reaction loaded into the leftmost lane, which did not contain competitor. (B-C'') Transgenic mini-enhancer-β-gal reporter expression (green) in eye discs counterstained for Pros protein (purple). (B-B'') β-gal expression induced by the wild-type mini-enhancer is weakly variegated. (C-C'') β-gal expression induced by the mutated pros7 mini-enhancer is not variegated but is still restricted to R7 and cone cells. (D-D'') Transgenic core-enhancer-β-gal reporter (green) which has 10 mutated Su(H)-binding sites in a sev-Nact/+ mutant eye disc. sev-Nact induces ectopic β-gal expression in R1/R6 cells. Endogenous Pros protein (purple) is also induced ectopically in these cells. (E) Sequence alignment of a Svp-binding site in the pros mini-enhancer from D. melanogaster and D. simulans. Response elements correspond to two half-sites (consensus AGGTCA) spaced one bp apart as a direct repeat (Zelhof et al., 1995). The position of the three tandem half-sites in the enhancer is shown with a bar over each half-site. (F,G) Transgenic mini-enhancer-β-gal reporter expression (green) in eye discs counterstained for Pros protein (purple). (F) A wild-type eye disc. (G) An eye disc carrying four copies of the sev-Svp2 transgene. Although some cells are positive for both markers, other cells are positive for one marker but not the other.

View larger version (24K): [in this window] [in a new window]   Fig. 8. Model for cell-type-specific transcription of pros. At the top is a schematic of the pros mini-enhancer arrayed with the different transcription factors on their binding sites. Several factors act downstream of Notch and Egfr signals. Notch acting through Su(H) inhibits production of the Svp repressor (red). Svp might repress the enhancer directly, or it might function through an intermediary. Egfr signaling inhibits the Yan repressor (red) and stimulates Pnt (green). Several factors work cooperatively to increase the transcription activity of the enhancer, and these are colored yellow. They are Lz, Gl and So/Eya. At the bottom is a model in which the enhancer becomes competent prior to its activation of pros transcription. (1) The inactive enhancer is in a repressive chromatin environment when neither Notch nor Egfr signals are received. (2) It transits towards competence when either Egfr or Notch alone is active. (3) It is competent when both Notch and Egfr signals are received by a cell. Competence may be expressed as a relaxation of chromatin structure, or the ability of the enhancer to connect to the promoter and facilitate transcription, or both. The formation of a cooperative enhancer complex (yellow ovals) composed of Lz, Gl and So/Eya is required for the enhancer to set the level of transcription. Competence may enable the cooperative enhancer complex to form (left). Alternatively, the enhancer complex may form on a non-competent enhancer but may only connect with the promoter when competence is achieved (right).

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