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doi: 10.1242/10.1242/dev.00522

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Two Pax genes, eye gone and eyeless, act cooperatively in promoting Drosophila eye development

Chuen-Chuen Jang^1,2, Ju-Lan Chao^1,2, Nikolas Jones³, Li-Chin Yao², Dmitri A. Bessarab², Yien M. Kuo³, Susie Jun⁴, Claude Desplan^4,*, Steven K. Beckendorf³ and Y. Henry Sun^1,2,

¹ Institute of Genetics, National Yang-Ming University, Taipei 111, Taiwan, Republic of China
² Institute of Molecular Biology, Academia Sinica, Taipei 11529, Taiwan, Republic of China
³ Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3204, USA
⁴ Howard Hughes Medical Institute, The Rockefeller University, New York, NY 10021, USA
^* Present address: Department of Biology, New York University, 1009 Main, 100 Washington Square East, 10003, New York, New York, USA

View larger version (127K): [in a new window]   Fig. 1. eyg loss-of-function mutant phenotype. (A-D) Scanning electron micrographs showing a range of eyg mutant phenotypes. (A) eyg1, (B) eygM3-12/eyg1, (C,D) pharate adults that failed to eclose: (C) eygEq-2-d2-2/In(3LR)gvu, most of the head structures were lost, but the antennae were present; (D) eygM3-12, a headless null mutant at higher magnification. (E-H) Eye-antennal disc. (E) DIC image and (F) Acridine Orange staining of the same disc from a mid-third instar eyg1 larva. (G) BrdU labeling of an eyg1 disc and (H) a wild-type disc from a late third instar larva. (I) Expression of P35 driven by the ey-GAL4 (abbreviated to ey>P35 in text) did not rescue the eyg1/eygM3-12 phenotype. The eye disc is strongly reduced with only a few ELAV-positive cells (red), as in eyg1/eygM3-12 mutants, while the antenna disc (with DAC expression, green) is of normal size. (J) eygM3-12 clones (marked by the absence of GFP staining; green) induced at 24-48 hours AEL were not detected in the eye disc. The heterozygous cells have nuclear GFP, while the wild-type twin-spots have a very strong GFP signal. ELAV (red) stains the photoreceptors.

View larger version (65K): [in a new window]   Fig. 2. eyg encodes a Pax protein. (A) The Lune cDNA encodes a Pax protein of 670 amino acids rather than the 523 amino acids previously reported (Jun et al., 1998). The corrected Lune sequence had been deposited into GenBank (AY099362). There are three potential start codons (indicated by arrows), translating to 670, 643 and 554 amino acids, respectively. It contains a truncated Paired domain (the N-terminal extent of the truncated PAI subdomain is indicated by a wavy line; RED subdomain, residues 167-220) and a Prd-type homeodomain (HD; residues 348-407). (B) Molecular map of the eyg-toe genomic region. The 5' end of the eyg transcripts are defined by 5'-RACE. Two splicing isoforms were identified by 5'-RACE that have identical 5' ends but differ in the presence or absence of a 67 intron (GenBank AY167573 and AY167574, respectively). The exon-intron structure of eyg is indicated. Exons are indicated by black boxes. The six exons are 310, 159, 321, 141, 584 and 1072 bp, respectively. The introns are 67, 1321, 680, 125 and 211 bp, respectively. Restriction sites indicated are: BamHI (B), BglII (Bg), EcoRI (E), HindIII (H) and PstI (P). The three potential translation start sites are indicated. The toe gene is located about 30 kb downstream of eyg and has the same transcriptional orientation as eyg. (C-G) eyg expression in wild type is detected by in situ hybridization in eye-antenna disc of late third instar (C) and late second or early third instar larva (D,E), and in wing disc (F) and leg disc (G) of late third instar larva. In this and all other figures, all eye-antenna discs are oriented with anterior to the right and dorsal to the top. (H) Heat shock induction of the hs-Lune transgene can partially (center) or fully (right) rescue the eyg1/eygM3-12 mutant phenotype, which is completely eyeless with complete penetrance (left).

View larger version (71K): [in a new window]   Fig. 7. eyg and ey can function independently. (A) In the ey2 mutant, E132>eyg can cause an ectopic ventral eye, similar to E132>eyg in the wild-type background. (B) In the eygM3-12 mutant, dpp>ey can cause ectopic eye formation on wings, legs and antennae, similar to the effect of dpp>ey in wild-type background.

View larger version (63K): [in a new window]   Fig. 3. Molecular analysis of eyg mutants. (A) eyg37-1, eyg22-2 and eyg94-4 are three eyg mutants independently derived from mobilization of the P[GawB]EM458 (see Materials and Methods). The three lines have the P[GawB] inserted at the same location, 124 bp upstream from the eyg transcription start site, but the extent of the deletion of the flanking genomic region varies. (B) eyg and toe are both expressed in the eye and antenna discs, as detected by in situ hybridization. In the three eyg mutants, eyg RNA is progressively reduced in both the eye and the antenna discs but toe RNA is not significantly affected. The three deletion mutants also show progressively stronger eye reduction. (C) The eygM3-12 mutant has a 16861 bp deletion beginning at 23 bp upstream of the eyg transcription start site and extending about 13 kb downstream of eyg. The P[lacW] insert is indicated (eygM3-12) with the arrow showing the transcriptional direction of mini-white and lacZ. The black triangle indicates the presence of an opus retrotransposon.

View larger version (108K): [in a new window]   Fig. 4. Mutant rescue by targeted expression of eyg and ey. Expression pattern of E132-GAL4 (A) and ey-GAL4 (C) in late third instar eye-antennal disc were visualized by inducing UAS-lacZ expression and staining with X-gal (insets show expression in early third instar disc). (B) The eyg1/eygM3-12 mutant phenotype (completely eyeless, see Fig. 2H) can be partially or completely rescued by targeted expression of eyg driven by E132-GAL4 and by ey-GAL4 (not shown). (D) The eygM3-12 mutant phenotype (headless, see Fig. 1D) can be rescued by ey>eyg with complete penetrance. (E) ey>ey can partially rescue the eyg1/eygM3-12 mutant phenotype. (F) ey>eyg can partially rescue the ey2/eyD eyeless phenotype.

View larger version (90K): [in a new window]   Fig. 5. Ectopic eye formation due to ectopic eyg and ey expression. (A-D) Misexpression of eyg can induce ectopic eye formation. (A,B) Adult phenotypes resulting from dpp-GAL4-induced UAS-eyg expression. (C,D) dpp>eyg eye-antenna discs double stained for dpp-lacZ (blue) and mAb22C10 (brown). (C) dpp-lacZ is expressed in the morphogenetic furrow (black arrow) and in a small dorsal region in the antennal disc (black arrowhead). Local overgrowth (white arrowheads) almost always occurred in the ventral and dorsal region of the eye disc adjacent to the antenna disc. These were often, but not always, accompanied by ectopic MF and photoreceptor formation (white arrow), frequently in the ventral pole and only occasionally in the dorsal pole. (D) Higher magnification showing that the axons from the ectopic photoreceptor neurons can sometimes correctly sense the direction of Bolwig's nerve (arrow), but sometimes project in the wrong direction (arrowhead). Occasionally multiple MF formation can be seen (not shown). (E-H) Synergistic effect of eyg and ey coexpression. (E,F) dpp>ey. (E) Ectopic eyes occurred at the base of the antennae, wings and multiple spots on the legs. (F) Ectopic MF (dpp-lacZ, blue) and photoreceptors (22C10, brown) can be detected in the antennal disc but not in the eye disc. (G,H) dpp>ey+eyg coexpression. (G) All ectopic eyes were larger than in dpp>ey. (H) Ectopic MF (dpp-lacZ, blue) and photoreceptor (22C10, brown) were enhanced in the antennal disc, and extended more anteriorly in the ventral margin of the eye disc. Multiple sites of ectopic MF formation in the eye disc and in the antenna disc sometimes led to fusion of the eye fields.

View larger version (79K): [in a new window]   Fig. 6. eyg and ey are transcriptionally independent. (A-D) eyg and ey expression in stage 17 embryos. In wild-type embryos, eyg (A) and ey (C) were expressed in the eye-antennal disc primordium (arrow). (B) In ey2 mutant embryos, eyg expression in eye-antennal disc primordium appeared normal. (D) In embryos homozygous for the C1 deficiency (null for eyg), ey expression in the eye-antennal disc primordium appeared normal, although the head was deformed. (E) eyg expression in an ey2 mutant eye-antennal disc. The ventral expression was reduced, while the dorsal expression was not significantly affected. (F,G,J,K) Ectopic dpp>eyg induced eyg expression in the dpp domains in eye (F, black arrowheads), antenna (F, arrow) and wing (J, black arrowheads) discs. The induced eyg expression was patchy along the eye disc lateral margins and did not occur in the posterior margin (F). The induced expression was much stronger than the endogenous eyg expression in the anterior notum in wing discs (J, white arrowhead) and in the antenna discs (F, white arrowhead), which is barely detectable with this staining condition. dpp>eyg did not induce ey expression in the eye-antennal (G) and wing (K) discs. (H,I,L) dpp>ey induced ey expression in the eye-antennal disc (I, arrowheads) and in wing disc (L, green), but did not induce eyg expression in the eye-antennal disc (H) and wing disc (L, red). Note the outgrowth of the antennal disc (H,I) that corresponds to the ectopic eye induced by dpp>ey. The expressions were detected by RNA in situ hybridization.

View larger version (67K): [in a new window]   Fig. 8. eyg can suppress wg expression. (A) wg-lacZ expression in a wild-type eye-antennal disc. (B) The expression in the dorsal and ventral poles of the eye disc were suppressed in dpp>eyg, and in dpp>ey (not shown). Note the posterior margin and the dorsal overgrowth express wg-lacZ. (C) The wg-lacZ expression domain expanded to the posterior margin in the eyg1 mutant eye disc. The eye disc was significantly reduced. (D) wg-lacZ expression in a wild-type wing disc. The expression in dpp>eyg (E) was suppressed in a region where the wg expression domain intersects the dpp expression domain (*). The dpp-GAL4 expression domain was visualized by dpp>lacZ (F). wg-lacZ was similarly suppressed by dpp>ey in wing disc (not shown).