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First published online March 24, 2005
doi: 10.1242/10.1242/dev.01738

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Functional analysis of the chicken 1-crystallin enhancer activity in Drosophila reveals remarkable evolutionary conservation between chicken and fly

¹ Department of Cell Biology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
² Institut de Génétique Humaine, Centre National de la Recherche Scientifique UPR 1142, 141 rue de la Cardonille, 34396 Montpellier, France
³ Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

View larger version (81K): [in a new window]   Fig. 1. Functional analysis of the chicken DC5(8x) enhancer in Drosophila. (A) Wild-type and mutant sequences of the DC5 enhancer used in this study. SOX and PAX6 binding sites are indicated. Altered nucleotides are shown in red. (B) Scheme of the construct used to test the functionality of the DC5 enhancer in Drosophila. The octamerized DC5 enhancer was cloned upstream of a minimal promoter (MP) and the EGFP reporter gene. (C-I) Activity pattern of the DC5(8x) enhancer in the adult Drosophila head. Enhancer activity was detected in the compound eye when the wild-type sequence was used (C). The mutant M4 and M7 enhancers failed to drive EGFP expression (D,E), although the transgenesis marker used (3xP3-DsRed1) was equally expressed in the three cases (F,G,H), indicating that the chromosomal insertion point of the different constructs did not affect enhancer functionality. (I) Enhancer activity is also detected in the adult antenna (red arrow), the maxillary palps (blue arrow) and the labial palps (yellow arrow). (J-L) During larval development, the DC5(8x) enhancer is active in the eye imaginal disc (J) and in Bolwig's organ (K,L).

View larger version (48K): [in a new window]   Fig. 2. The cells responsive to DC5 activity in Drosophila are the cone cells. The activity pattern of DC5(8x) in the adult compound eye (A) and in the larval eye imaginal disc (B) was compared with the activity pattern of the cone-cell-specific enhancer SME (C,D). SME is the minimal eye-specific enhancer of D-Pax2 (Flores et al., 2000). Fluorescence microscopy indicates that both activity patterns are identical, as shown in preparations of the larval eye imaginal disc (E,F,G) and in cryosections of the adult compound eye (H,I,J). When a signal-peptide-tagged EGFP was used as a reporter for the enhancer activity, the fluorescence signal was detected in the lenses of the compound eye (K). This experiment was done in a w+ genetic background to keep the ommatidium structure unaltered (L).

View larger version (44K): [in a new window]   Fig. 3. Cooperative binding of the Drosophila PAX6/2 and SOX2 homologues to the DC5 sequence in vitro. (A) Schemes of the Drosophila PAX6, PAX2 and SOX2 homologues and their variants with different molecular tags. DNA-binding domains are indicated: paired domain (PD), homeodomain (HD), N-terminal portion of the homeodomain (H) and high mobility group domain (HMG). The octapeptide sequence (O) present in D-PAX2 is also shown. (B) Binding of the Drosophila PAX6 and SOX2 homologues to the wild-type monomeric DC5 sequence. Five nanograms of the tagged variants of the different proteins were used in gel mobility shift assays. (C) Comparison of the binding affinities of D-PAX2-PD, EY-PD and TOY-PD for the monomeric DC5 sequence. (D,E,F) Cooperative binding of Drosophila PAX6/2 and SOX2 homologues to the wild-type monomeric DC5 sequence. Combinations of the different tagged variants were included in the binding reaction, and their ability to cooperatively bind to the DC5 sequence was analysed by gel mobility shift assays. In all the cases, the results were similar. The duplex complex SOX2 homologue-DC5 (red asterisks) migrated more slowly and became more intense upon addition of increasing amounts of the PAX6/2 homologues, giving rise to the triple complex SOX2 homologue-PAX6/2 homologue-DC5 (blue asterisks).

View larger version (37K): [in a new window]   Fig. 4. Synergistic activation of the DC5(8x) enhancer in vivo. (A) Cell culture co-transfection assays in Schneider 2 cells. Wild-type and mutant DC5 enhancers were tested for activation by exogenous Drosophila Pax6, Pax2 and Sox2 homologues. The structure of the reporter and effector plasmids is shown (A1). ß-gal activity of the reporter vector co-transfected with the empty effector vector was taken as 1 (column C). (B) Ectopic activation in Drosophila tissue. Wild-type DC5(8x) enhancer was tested for activation in the wing imaginal disc by ectopic expression of Drosophila Pax6, Pax2 and Sox2 homologues.

View larger version (62K): [in a new window]   Fig. 5. D-Pax2 and SoxN are expressed in the Drosophila compound eye and in the eye imaginal disc. (A) Genomic map of the SoxN locus and structure of the construct used to trace SoxN expression. The intronless SoxN gene consists of a translated region (TR) flanked by extensive 5' and 3' untranslated regions (5' UTR and 3' UTR, respectively). Regulatory sequences important for SoxN expression in the adult compound eye (B) and in the eye imaginal disc (C) are present in the promoter and the 5' untranslated region of SoxN (from –2939 to +869. PSoxN). D-Pax2 expression was monitored using its minimal eye-specific enhancer SME. This enhancer is active in the cone cells of the adult compound eye (E) and their precursors in the eye imaginal disc (F). Comparison of both expression patterns in the eye imaginal disc shows that they are coincident (D,G,H).

View larger version (63K): [in a new window]   Fig. 6. DC5(8x) activity in the Drosophila compound eye is abolished by depletion of either D-Pax2 or SoxN. (A) The DC5(8x) enhancer is inactive in Drosophila spapol mutant. This mutant is characterized by the absence of D-Pax2 expression in cone cells and primary pigment cells. Nevertheless, cone cells do still form in spapol mutant, as shown by means of the cone-cell-specific enhancer SME (B). (C-F) SoxN depletion was studied using the clonal analysis technique. Mutant clones in the adult compound eye were generated using the strategy depicted in panel E. SoxN– clones (red arrows in C,D,F) were identified by the lack of red pigment (w–) under white illumination (C). The use of UV light facilitated the identification of the mutant tissue (D). The activity of the DC5(8x) enhancer is abolished in the SoxN– clones (F).