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First published online 18 May 2005
doi: 10.1242/dev.01841

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Identification of functional sine oculis motifs in the autoregulatory element of its own gene, in the eyeless enhancer and in the signalling gene hedgehog

Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland

View larger version (33K): [in a new window]   Fig. 1. Defining a minimal version of so7 important for ocellus development. (A) Genomic map of the last intron of the so locus between exon6 (E6) and exon7 (E7) (black boxes). The physical mapping is indicated as: A, AseI; S, SspI; E, EcoRI; K, KpnI. An enlargement of the region deleted in so1 shows relative positions of the enhancer fragments so7, so10 and so9. so10 contains EY/TOY- and TOY-specific binding sites (black boxes and black triangles, respectively). So9 harbours the SO-binding site. The deletion map illustrates the constructs that were tested for the expression pattern they mediate (X indicates introduced mutations). Nr, number. Twenty-seven bp fused to so10 (number 14) are sufficient to resemble the so7-mediated pattern including ocellus-expression (B, referred to as +++ expression in ocellus and +++ expression in compound eye). All constructs including so10 but missing the 27 bp (represented by number 21) just resemble the so10-pattern and show no expression in the ocellar region (indistinguishable from D). Constructs that are devoid of functional EY and TOY sites (number 17, 18, 19 and 21) but include the 27 bp of number 21, show an expression pattern identical to so9 (C, illustrated by the pattern mediated by number 21). Fragment number 21 is sufficient to recapitulate the expression pattern of so9; it is referred to as soAE in the article. Construct number 20 (no so10 and no number 21) does not mediate any expression at all (–). (B-E) Arrows indicate the ocellar region. (B) Expression pattern mediated by number 14, similar to wt so expression pattern and the so7-mediated pattern. (C) Number 21-lacZ: expressing only along the posterior margin of the eye disc similar to so9-lacZ. (D) so10-lacZ: resembling wt-expression despite the ocellar signal. (E) So7mut: so7 where the SO-binding site is mutated mediates only weak expression to the ocellar region (fragment number 22). (F) Vertex region of a wt fly showing three normal ocelli. (G) so driven by so10-soAE (number 14) rescues ocellus development of so1 mutant flies. The lateral ocelli appear almost normal (arrowhead). The size of the anterior ocellus is reduced (empty arrowhead).

View larger version (68K): [in a new window]   Fig. 2. soAE is a direct target of so. soAE (sine oculis autoregulatory element) corresponds to fragment number 21. Arrows indicate the ocellar region. (A-C) soAE harbours three putative transcription-factor-binding sites, which have been mutated. (A) Mutating the core of the HD recognition sequence (mutHD) or (B) mutating the GATA sequence (mutGATA) abolishes expression of the lacZ reporter in the ocellar region. (C) so10-mutPAX in which the putative Pax6-binding site is mutated, mediates expression indistinguishable from wt/so10-soAE-mediated expression. (D-F) Oligomerization of soAE boosts its expression. By contrast to soAE alone, which only mediates expression in the posterior margin of the eye disc (D), 4xsoAE drives expression posterior of the MF, within the MF and in some cells in front of the MF, but not in the ocellar region (E). This expression resembles wt so-expression despite the ocellar region. (F) 10xsoAE does not further amplify expression intensity but results in a more blotchy type of expression pattern. (G) Sequences of soAE and the mutated versions of it. Sequences TAA and GAT of soAE are important for the ocellus-specific expression of so10-soAE-lacZ. (F) Radiolabelled probes of mutHD and mutGATA are not shifted by SO in EMSA. By contrast, mutPAX is bound by SO and therefore shifted in EMSA.

View larger version (50K): [in a new window]   Fig. 3. SO acts upon the soAE motif in vivo and in S2 cells. (A-C) 4xsoAE is ectopically induced in wing discs by EYA and EYA+SO protein but not by SO protein alone. (A) dpp-GAL4 driving UAS-so does not induce 4xsoAE-lacZ in wing discs. (B) Ectopic expression of 4xsoAE-lacZ is induced in spots along the AP boundary by dpp-GAL4:UAS-eya. (C) Co-expression of so enhances eya-mediated reporter gene activity. (D) Ocellus-specific expression of so10-soAE is lost in so2 mutant flies (arrow). so2 is a regulatory mutant that displays an ocelliless phenotype. (E) EYA protein is detectable in the ocellar region of so2 mutant flies (arrow). (F-I) so is necessary for soAE activation: in so3 clones, 4xsoAE-lacZ expression is lost although EYA is present in the cells. Clones are delimited by a dashed line. (F) EYA expression is shown in red and marks the non-dying cells within the clone. (G) so3 clones are negatively marked by the absence of GFP expression (in green). (H) 4xsoAE-lacZ reporter gene expression (blue) is lost in so–/–cells. (I) overlay of G and H. (J) ß-Galactosidase reporter assays in Drosophila S2 cells. Reporter plasmids containing the lacZ gene under the control of different enhancer fragments were transfected into the cells. Co-transfection of so or eya alone together with the 10xsoAE-lacZ reporter plasmid does not exceed basal activity (lanes 2, 3). By contrast, co-expression of so+eya with the 5xsoAE or 10xsoAE reporter leads to a strong induction of ß-galactosidase (lanes 4, 5). The mutated versions of the 5xsoAE reporter (5xmutHD, 5xmutGATA) still show some amount of induction when co-transfected with so+eya (lanes 6, 7). ß-Galactosidase values were normalized by co-transfecting 5 ng of plasmid expressing luciferase as an internal standard. The results represent an average ß-galactosidase activity taken from transfections done in triplicates (±s.d.) and are illustrated as the X-fold activation over the basal activity found for the reporter plasmid alone.

View larger version (53K): [in a new window]   Fig. 4. Identification of nucleotides important for SO-DNA (soAE) interaction. (A) SO protein is shifted by soAE (SO + hot probe) in EMSA. Double-stranded probes bearing a single point mutation (6-22) or a stretch of mutations (2-5) were used as cold competitors (100 x molar excess) and compared to soAE (1) for their ability to compete for SO-binding. Nucleotides important for protein-DNA interaction are highlighted in red (very important) and orange (important) according to their competing potential. As a control, mock transfected reticulocyte lysate was incubated with p32 marked soAE (TNT mock). (B) DNA probes of sequences resembling soAE taken from other genes were used as cold competitors as well. A sequence out of the eye-specific enhancer of the ey gene is a strong competitor. The fragment loses its binding property when the GAT sequence is mutated to CCC (eyeless, eyeless mut). Out of the lozenge gene, only one of the previously described SO-binding sites shows a strong competition potential in EMSA (lz first, lz second). Strong competing sequences are also found in the first intron of the hh gene (hh first, hh second). SO binding is furthermore strongly competed by the well-described AREC3/Six4-binding site. (C) Upper half: sequences of the probes that were used as cold competitors in Fig. 4B. Based on these sequences and the results shown in A, a consensus binding sequence for SO was proposed. Lower half: previously described binding sites for the vertebrate Six1,2,4,5. These sequences appear to be related to the SO-binding sequence.

View larger version (62K): [in a new window]   Fig. 5. The eye-specific enhancer of ey contains a functional SO-binding site. (A) Genomic map of the ey locus between exon 2 (E2) and exon 3 (E3) (black boxes). The previously described eye-specific ey-enhancer is indicated by brackets. Relative positions of transposable elements that interfere with eye development are indicated by their allelic name (eyR, ey2). The sequence of the eye-specific ey enhancer is given below (ey enhancer). B4M: the putative Pax6-binding sites (boxed) were mutated accordingly to Hauck et al. (Hauck et al., 1999) to get rid of toy-mediated signal. B4M SOmut: the putative Pax6-binding sites and additionally the putative SO-binding site (grey shaded) were mutated for comparison to B4M. (B) lacZ expression mediated by the wt ey-enhancer fragment in a third instar eye imaginal disc. (C) B4M-lacZ expression: without an influence of TOY protein, due to the mutated sites, the so-mediated expression is restricted to a portion of the posterior margin (similar to so9/Nr21 in Fig. 1C). (D) B4M SOmut-lacZ: mutating the Pax6 sites and the so site, expression is reduced to a weak spot in the centre of the posterior margin.

View larger version (41K): [in a new window]   Fig. 6. hh contains functional SO-binding sites. (A) Genomic map of the hh locus. The physical mapping is indicated as: X, XhoI; S, SacI; K, KpnI. The hh1 (bar-3) deletion is mapped according to Lee et al. (Lee et al., 1992). Two SO-binding sites are located within the region deleted in hh1 (sequences are given in Fig. 4C: hh first, hh second). One additional SO-binding site is found upstream of the hh1 deletion. (B) hh1-lacZ: lacZ expression posterior to the MF is mediated by 1.4 kb genomic DNA from the region deleted in hh1. (C) hh1 SOmut: expression is hardly detectable when the two SO-binding sites are mutated. (hh first, GAG is changed to CCC; hh second, GAT is changed to CCC). (D) hh1 5'-lacZ: no expression is detected from a reporter construct containing only one mutated SO-binding site

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