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First published online 24 September 2003
doi: 10.1242/dev.00773

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Egfr signalling defines a protective function for ommatidial orientation in the Drosophila eye

Katherine E. Brown and Matthew Freeman*

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK



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  		Fig. 1. Rotational defects in eyes overexpressing Keren. (A-D) Sections through adult eyes (A,C), with schematics shown below (B,D). (A,B) In wild-type eyes, all ommatidia are orientated at precisely 90° to the equator. Dorsal and ventral ommatidia are of opposite chiral forms. (C,D) Eyes in which full-length keren is misexpressed under the control of sev-Gal4 (referred to as UAS-keren) show severe defects in rotational angle. All ommatidia, however, are of correct chiral form. In B and D, and all subsequent schematics, black trapezoids represent correctly orientated ommatidia, green trapezoids show underrotations, and blue trapezoids overrotations. The red line marks the position of the equator. In this and all images, anterior is to the left. (E) Confocal image and schematic of rotation in the third larval instar disc. (Left) Confocal projection of svp-lacZ/+ disc stained with {alpha}-Elav (green) to mark photoreceptors and {alpha}-ß-galactosidase (red) to highlight R1,3,4 and 6. The R3/R4 pair is initially parallel to the morphogenetic furrow (MF, arrowhead); ommatidia then undergo a fast 45° rotation, followed by a second, slower turn to 90°. Rotational positions are shown by white arrows. (Right) Schematic of the events occurring in left panel, showing rotation of, and recruitment of further photoreceptors to, the five-cell cluster.

 


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  		Fig. 2. Perturbing Egfr signalling disrupts ommatidial rotation. Upper panels show sections through adult eyes, lower panels are schematics of these images. (A,B) aosw11 clone (note, in this section, the equator can not be seen, but runs left to right as in all other images); (C,D) S5671/+; (E,F) ru1; (G,H) DN-Egfr expressed under the control of HS-Gal4; (I,J) pnt1277/pnt{Delta}88 (position of equator cannot be accurately determined because of the large proportion of mis-specified ommatidia). In all cases, misrotated ommatidia can be seen. (K) Graph showing rotational angles in wild type (blue), sev-Gal4, UAS-keren (red) and ru1 (green) eyes. Data is plotted as percentage of ommatidia at each angle. In each case, 5-600 ommatidia were scored from 5-6 eyes. UAS-keren and ru1 have qualitatively similar effects on rotation. (L-Q) roulette is allelic to argos. (L,M) rlt1 mutants show similar phenotypes to Egfr pathway mutants (compare L with other images in Fig. 2). (N,O) The rlt1 mutant fails to complement aosw11. (P,Q) rlt1 phenotype can be rescued by overexpression of one copy of the sev-argos transgene. Colour coding in schematics is as for Fig. 1 (black, correctly orientated; green, underrotated; blue, overrotated; red line, equator). Black circles indicate misspecified ommatidia in this and all subsequent Figures.

 


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  		Fig. 3. The role of Spitz in rotation and evidence that the Egfr acts directly. (A,B) Section through adult eye of spiscp1 hypomorph. Many ommatidia show under-recruitment defects; misrotations, however, are very rare (green trapezoid). Colour coding in schematics is as for previous Figures (black, correctly orientated; green, underrotated; blue, overrotated; black circles, mis-specified ommatidia; red line: equator). (C) Quantification of rotational defects in spiscp1 versus ru1 and S/+ versus spiscp2. `No.' indicates the number of eyes scored for each genotype. In spiscp1, very few misrotations are seen relative to the proportion of misrecruitments; the converse is seen in ru1. However, spiscp2 dominantly enhances rotational defects of S/+, suggesting Spitz plays some role in the control of rotation. (D-F) S/+; svp-lacZ/+ 40 hour pupal retina stained with {alpha}-cut (D; red in F), {alpha}-lacZ (E; blue in F) and {alpha}-Elav (green in F). Ommatidial orientation and cone cell number are not correlated: ommatidia with too few cone cells may be either correctly (solid circle) or incorrectly (broken circle) orientated, and incorrectly orientated ommatidia may also have the correct number of cone cells (dotted circle).

 


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  		Fig. 4. Eye discs misexpressing Keren are indistinguishable from wild type (WT). All images show eye discs taken from crawling third instar larvae. In all cases, green is {alpha}-Elav, marking photoreceptors. Upper panels show the red channel alone; lower panels are merges. (A D) m{Delta}0.5-lacZ staining (red) in WT (A,B) and UAS-keren (C,D) discs. m{Delta}0.5-lacZ highlights the R4 cell and acts as a marker for chirality; R4 determination is normal in UAS-keren discs. (E-H) {alpha}-Bar staining (red), highlighting R1 and R6 in WT (E,F) and UAS-keren (G,H) discs. Rare misrotations can be seen in both WT and mutant discs (arrowheads). (I,L) svp-lacZ (red) staining in WT (I,J) and UAS-keren (K,L) discs. seven-up is expressed strongly in R3 and R4 (outer pair in each ommatidium) and weakly in R1 and R6 (inner pair). Note the similarity between WT and UAS-keren with all three markers.

 


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  		Fig. 5. Rotational defects arise during pupal eye development. All images are confocal projections of svp-lacZ/+ retinae stained with {alpha}-Elav (green) and {alpha}-ß-galactosidase (red). Upper panels show the red channel only; lower panels are merges. svp-lacZ highlights R1,3,4 and 6. (A-D) Discs taken from wild type (WT) (A,B) and UAS-keren (C,D) flies at 6 hours post-pupariation. At the back of the WT disc, ommatidia are arrested at 90°. UAS-keren discs show no disruption in rotation, even at the back of the disc where ommatidia are oldest. (EH) Retinae taken from WT (E,F) and UAS-keren (G,H) flies at 30 hours post-pupariation. By this stage, rotational defects are clear in the UAS-keren retinae (compare E with G).

 


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  		Fig. 6. Genetic interactions with other rotational genes. (A-F) Disrupting Egfr signalling has no effect on the nemoP1 phenotype. Top panels show sections through adult eyes; bottom panels are schematics of these images. (A,B) nemoP1. All ommatidia are arrested at approximately 45°. (C,D) sev-Gal4, UAS-keren/+; nemoP1. (E,F) ru1, nemoP1. Conditions of both overactive (C) and underactive (E) Egfr signalling fail to modify the nemoP1 phenotype. (G,H) sca1 mutants show relatively minor defects in ommatidial rotation in the adult eye. Most correctly specified ommatidia are orientated at 90° to the equator, with only a few being misrotated. Colour coding in schematics is as previously (black, correctly orientated; green, underrotated; blue, overrotated; black circles, mis-specified ommatidia; red line, equator).

 


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  		Fig. 7. E-cadherin interacts genetically with Egfr signalling. (A,B) Rotational defects in S5671/shgIG29 adult eye sections. Rotational defects of S5671/+ eyes are significantly enhanced by halving the dose of E-cadherin (compare Fig. 7A,B with Fig. 2C,D). (C) Table showing interactions between Star and shotgun, wing blister and myospheroid. `No.' indicates the number of eyes scored for each genotype. Only shotgun alleles significantly enhance the rotational defects of the Star heterozygote. Significant differences from S/+ are indicated in bold type (P<0.05).

 


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  		Fig. 8. Timeline showing significant events during Drosophila eye development. Larval time (blue) is scaled as number of ommatidial rows; pupal time (green) as hours post-pupariation. Events directly concerning rotation are shown in red above the line; all other events are in black below the line. CC, cone cells; IOC, interommatidial cells; MF, morphogenetic furrow; PC, pigment cells; PRC, photoreceptor cells; SMW, second mitotic wave. For precise timings of pupal events, see main text.

 

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© The Company of Biologists Ltd 2003