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Differential interactions of eyeless and twin of eyeless with the sine oculis enhancer

Claudio Punzo^*, Makiko Seimiya^*, Susanne Flister, Walter J. Gehring and Serge Plaza

Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
^* The first two authors contributed equally to the work
Present address: Universite Paul Sabatier Centre de Biologie de Developpement, bar 4R3, 118 route de Narbonne, 31062 Toulouse cedex 4, France

View larger version (75K): [in a new window]   Fig. 1. so10-lacZ activity in different mutant backgrounds. (A-I) Eye discs of third instar larvae expressing so10-lacZ. (A) Expression in a wild-type eye disc. Arrowhead indicates the ocellus region where no expression is detected. Dorsal is top and posterior is left. (B-J) Expression in an ey2, eyJ5.71, so1, eya1, spapol and toyG7.39 mutant background, as indicated. G and H, I and upper box in I show a pair of the eye discs from a single larva of toyG7.39. L indicates left and R indicates right eye disc. (J) Leg disc where UAS-toy is ectopically expressed with dppblink-Gal4 in an ey2 mutant background. lacZ staining (arrow) shows that toy is able to activate this enhancer element in the absence of endogenous ey.

View larger version (105K): [in a new window]   Fig. 2. EY and TOY bind to the so10 enhancer through their paired domains. (A) Bandshift experiment performed with equal amounts of in vitro synthesised full-length EY and TOY on a 128 bp fragment from the 428 bp enhancer of so10. EY and TOY are both able to bind specifically to this fragment since binding is abolished with a 10-fold molar excess of cold competitor (compare lanes 3, 4 with lanes 5, 6). (B,C) Western blot analysis of the ectopically expressed EY and TOY proteins in third instar leg discs with an anti-TOY (B) and an anti-EY (C) antibody. Lanes 1: yw control eye discs; lanes 2: misexpression of full-length toy and ey; lanes 3: misexpression of toyPD and eyPD; lanes 4: misexpression of toyHD and eyHD. Asterisk indicates the deleted proteins. (D-I) Third instar wing discs in which different UAS-ey or UAS-toy constructs are misexpressed with dppblink-Gal4 in an ey2 mutant background. The X-gal staining reveals the ability of these proteins to induce so10. (D,G) Full-length EY and TOY, respectively, are able to induce so10. (E,H) Deletion of the HD in EY or TOY, respectively, does not abolish so10 activation. (F,I) Deletion of the PD of EY or TOY, respectively, completely abolishes so10 activation. Note that despite a lower level of HD proteins so10-lacZ is still significantly activated.

View larger version (94K): [in a new window]   Fig. 3. Mapping of EY and TOY binding sites on the so10 enhancer. (A) Sequence alignment of the so10 wild-type (wt) and mutated (mt) enhancer. The boxes indicate the protected regions revealed by the footprint experiment. The common EY- and TOY-binding sites are shaded grey and the TOY-specific binding sites are white. The bold letters on the wt so10 enhancer indicate the sequence most related to the Pax6 consensus sequence (P6CON), also shown in C. The 128 bp fragment used for the bandshift assay is underlined between the two arrowheads. (B) Footprint experiment on the wild-type (WT) and mutated (MT) so10 enhancer. Sites 1, 2 and 5 are protected by EY- and TOY-PD whereas sites 3 and 4 are TOY-PD specific. No binding was detected after mutagenesis (MT) of the five binding sites. (C) Sequence alignment of the five binding sites identified on so10 with the Pax6 consensus binding site (P6CON) (Epstein et al., 1994). The bases fitting with the consensus are shown in capital letters. (D) Wing discs where either EY or TOY were misexpressed by dppblink-Gal4 in flies carrying the lacZ reporter gene under the control of the so10EY+TOYmt enhancer. In both cases no ß-galactosidase expression was detected indicating that the mutated enhancer was not inducible anymore by TOY or EY.

View larger version (52K): [in a new window]   Fig. 4. Expression pattern of the different so10 enhancers. (A) Schematic drawing of the enhancer with the different EY and TOY binding sites indicated by five boxes. The different mutated so10 enhancers used are represented using crossed boxes. All panels in a column refer to the same so10 enhancer. (B-E) Early third instar eye discs and (F-I) late third instar eye discs. During larval stages the lacZ transgene was detected by staining for ß-galactosidase. For all eye discs, posterior is left and dorsal is towards the top. During early third instar larval stage the expression is lost in the central part of the eye disc when the EY binding sites are mutated (D), in contrast to mutations of the TOY binding sites which show no obvious defects at this stage (C). At late third instar stage mutations of the TOY binding sites restrict expression to the posterior edge of the eye disc (G) whereas mutations of the EY binding sites do not alter the expression pattern from second to third instar larvae (D,H). Expression is completely abolished in the eye primordium and in the eye disc when all five binding sites are mutated (E,I).

View larger version (100K): [in a new window]   Fig. 5. Rescue of the so1 mutant by the different mutated so10 enhancers. (A) Schematic drawing of the Gal4 system used for the rescue experiment. Flies expressing the Gal4 gene under the control of the different so10 enhancers were crossed to flies carrying the UAS promoter controlling the so cDNA. The crosses were all carried out in a so1 mutant background. (B,C) Rescue of the so1 mutant eye phenotype with so10 and so10TOYmt, respectively. (D) Rescue obtained with the so10EY/TOYmt enhancer. The eye size shown is the largest obtained. The variations in size were reminiscent of those observed in an ey2 mutant with one eye missing. (E) No rescue was observed with the so10TOY+EYmt. (F) Schematic drawing of the Gal4 system used to rescue the ey null mutant. (G,H) Rescue of the ey null mutant eyJ5.71 with the ey-enhancer Gal4. Both UAS-ey (G) and UAS-toy (H) were able to rescue the eyJ5.71 mutant when driven with ey-Gal4. In both cases (G,H), the eyes were almost normal in size for all of the flies analysed. The eyJ5.71 null mutant showed an eye reduction of between 50-75% for 20% of the flies (I), and 75%-100% for 80% of the flies (J) (Callaerts et al., 2001).

View larger version (106K): [in a new window]   Fig. 6. Rescue of the so1 mutant phenotype by the 1.6 kb element so7. (A) Schematic representation of the different so7 drivers in combination with the previously described so10 mutated elements. (B-E) Expression pattern of the different so7 drivers that have the extended so9 fragment. All panels show third instar eye discs with posterior to the left and dorsal to the top. (B) so7 shows additional expression in the ocellus region (arrow). (C) so7EY/TOYmt completely loses expression in the eye disc but still has an extended expression in the ocellus region (arrow). (D) so7TOYmt has normal expression in the eye disc but not in the ocellus region. (E) so7EY+TOYmt only shows expression reminiscent of the additional 1 kb element at the posterior margin of the eye disc. (F-I) Rescue of the adult eye with the so7 drivers. The wild-type so7 and the so7TOYmt show full rescue of the compound eye (F,H), whereas the removal of the EY binding sites does not rescue, or only partially in very rare cases (G). so7EY+TOYmt never shows any kind of rescue (I). (J-M) Rescue of the ocelli with the so7 drivers. In contrast to the compound eyes, the TOY binding sites are absolutely required for ocelli development. (J,K) Rescue of the ocelli (arrowheads) with so7 (J) and so7EY/TOYmt (K). No rescue is observed with so7TOYmt (L) and so7EY+TOYmt (M).

View larger version (17K): [in a new window]   Fig. 7. Eye specifying gene hierarchy depends on the cell type and the stage of development. The network of regulatory genes specifying eye development is modified based on this study and other recently published results. (A) In the embryonic eye precursor cells, toy directly activates ey through the ey-enhancer (Czerny et al., 1999) without ey activating its downstream genes so/eya (Kumar and Moses, 2001). (B) In the eye disc of late L2 and early L3 ey and toy directly regulate so in a complex network by using different sites on the same enhancer element in different sets of cells to direct the development of the compound eyes and the ocelli.