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First published online January 13, 2009
doi: 10.1242/10.1242/dev.026054

Development 136, 473-481 (2009)
Published by The Company of Biologists 2009
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Fasciclin 2, the Drosophila orthologue of neural cell-adhesion molecule, inhibits EGF receptor signalling

Yanlan Mao and Matthew Freeman*

MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.


Figure 1
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  		Fig. 1. fas2 genetically interacts with EGFR pathway. (A) Control GMR-argos (GA)/+ eye is rough. (B) GA/+ rough eye was suppressed when heterozygous for the P-element allele fas2G0293. (C) Control sevrhomboid-1 (SR)/+ rough eye (grown at 18°C). (D,E) SR/+ rough eye was enhanced when heterozygous for fas2G0293 (D) and fas2eb112 (E) (grown at 18°C). (F,G) GMR-Gal4/+; UAS-EGFR/+ rough eye (F) was enhanced when one copy of fas2 was removed (G). (H,I) sevEP-Gal4/+; UAS-sprouty/+ rough eye (H) was suppressed when one copy of fas2 was removed (I).

 

Figure 2
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  		Fig. 2. Fas2 expression in the eye. In all images, anterior is towards the left, unless otherwise stated. (A) RNA in situ hybridisation of a third instar eye disc with a probe against fas2. (B) Third instar eye disc stained with antibodies against Fas2 (green) and Armadillo (Arm, red). (C,C') A higher magnification view of Fas2 expression in a third instar eye disc co-stained with anti-Arm and anti-Senseless (Sens, blue). (D) A projection of x/z sections of a third instar eye disc stained with anti-Fas2, anti-Elav (photoreceptor marker, blue) and anti-Arm (marks apical compartment). Fas2 is mostly expressed basolaterally. White and yellow arrowheads indicate where Fas2 is also expressed apically. (E) y/z section in the plane of the white arrowhead in D with apical (a) towards left, stained with Fas2 (green) and Arm (red) showing some Fas2 in the apical compartment, white arrowheads. (F) Y/Z section in the plane of the yellow arrowhead in D with apical towards the left, stained with Fas2 (green) and Arm (red) showing some Fas2 apically, yellow arrowheads. (G) Third instar eye disc of XA12, a β-gal reporter line strongly expressed in R7s, stained with anti-β-gal (red), Fas2 (green) and Elav (blue). x/z section shows Fas2 is elevated in R7 cells, arrowheads.

 

Figure 3
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  		Fig. 3. fas2 loss-of-function phenotypes in pupal retinas and adult eyes. (A-A'') Wild-type pupal retina stained with anti-Elav (blue; eight photoreceptors per ommatidium) and anti-Cut (red; four cone cells per ommatidium). (B-B'') Pupal retina of fas2eb112/fas2e76 grown at 18°C. Circles (red, excess cells; yellow, missing cells) indicate ommatidia with abnormal numbers of photoreceptors (B') and cone cells (B''). The strongly staining isolated Elav-positive cells in B' are interommatidial bristles not photoreceptors. (C) fas2eb112/fas2e76 eye is only mildly rough when grown at 20°C. (D) argosl{Delta}7/+ eye is wild type at 20°C. (E) Roughness of the fas2eb112/fas2e76 eye is enhanced by heterozygosity for argosl{Delta}7 (grown at 20°C). (F-F'') fas2eb112 clones marked by lack of β-gal (green). `Mini' ommatidia can be seen with Elav (circle, F') and Cut reveals cone cell defects (circle, F'').

 

Figure 4
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  		Fig. 4. fas2 loss-of-function phenotypes in third instar eye discs. All images in this figure are of fas2eb112 clones, marked by lack of β-gal (green) or GFP (green). (A) Anti-Arm (red) reveals ectopic clusters in mutant tissue (white arrows). (B-B'') Co-staining with antibodies against Arm (red) and Sens (blue) revealed that the ectopic clusters (red arrowhead, B') do not have R8 cells (missing Sens-positive cell, red arrowhead, B''). (C-D') Co-staining with anti-Elav (blue) and anti-Prospero (red) revealed frequent cases where two cells per ommatidium stained positive for both Elav and Pros (pink cells) in clones. (D,D') An enlargement of the area marked in C. Extra R7s can be seen within the clone (indicated by arrowheads); these were sometimes genotypically wild type (indicated by expression of GFP, lower two arrowheads in D'). (E,E') Co-staining with anti-Yan (red) showed Yan downregulation in fas2- clones. (F) An enlargement of the region outlined in red in E'. (G) An enlargement of the region outlined in green in (E'). (H) White rectangle marks the area used in the fluorescence intensity quantification in I. (I) Graph showing the averaged relative fluorescence (pixel) intensities of Yan staining (red) and β-gal staining (green). The top of the rectangle in H is 0 on the position axis. Intensities are averaged across the anterior-posterior axis of the marked rectangle. Cells within the clone, marked by the absence of β-gal, have reduced levels of Yan.

 

Figure 5
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  		Fig. 5. Fas2 in the notum and wing. In all images of wing discs, dorsal is towards the bottom. (A) In situ hybridisation of a third instar wing disc with a probe against fas2. (B) Optical section through a third instar wing disc stained with anti-Fas2 showing expression in the proneural cluster regions of the prospective notum (arrowheads). (C,C') rho-lacZ in third instar wing disc (red) marks wing veins and the wing margin; this colocalises with anti-Fas2 (green). (D) Wild-type adult notum with two dorsocentral (DC) bristles per heminotum (arrowheads). (E) fas2eb112 clones in the notum, marked by singed, resulted in extra DC bristles (arrowhead). (F) Hyperactivation of EGFR signalling by overexpressing a constitutively activated form of the EGFR in the notum also resulted in extra DC bristles (arrowheads). (G) Anti-Achaete (Ac) reveals the proneural cluster pattern in a wild-type late third instar wing disc. (H) Wing disc with fas2eb112 clones, marked by lack of GFP (green), and co-stained with anti-Ac (red). (H') Enlargement of the area highlighted in H. The DC proneural cluster, marked by Ac, expands to fill the fas2eb112 clones (outlined in green). (I) Wild-type late third instar wing disc stained with anti-Sens to mark the sensory organ precursors (SOP). A maximum of two SOPs will form in the DC region of a wild-type disc (arrowheads). (J,J') A fas2eb112 clone in the DC region of the notum of a late third instar wing disc. Three SOPs (arrowheads) have formed in the mutant DC region, as marked by the high accumulation of Ac (red in J, white in J').

 

Figure 6
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  		Fig. 6. Elevated Fas2 expression in the eye requires EGFR signalling. (A-A'') spitz null clones in third instar eye discs, marked by lack of GFP (green). Co-staining with anti-Elav (blue) and anti-Fas2 (red) revealed that Fas2 remains low in the absence of Spitz; note that non-R8 photoreceptors fail to differentiate in the clone. (B) An enlargement of the area marked in A''.

 

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