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First published online May 28, 2004
doi: 10.1242/10.1242/jcs.01123

Journal of Cell Science 117, 2777-2789 (2004)
Published by The Company of Biologists 2004
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A screen for modifiers of RacGAP(84C) gain-of-function in the Drosophila eye revealed the LIM kinase Cdi/TESK1 as a downstream effector of Rac1 during spermatogenesis

Karine Raymond*, Evelyne Bergeret, Amélie Avet-Rochex, Ruth Griffin-Shea{ddagger} and Marie-Odile Fauvarque{ddagger}

CEA-Grenoble, 17 rue des Martyrs, Département de Réponse et Dynamique Cellulaires, UMR 5092, 38054 Grenoble CEDEX 9, France



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  		Fig. 1. Eye phenotypes caused by misexpression of the RacGAP(84C) protein or its GAP domain. (A,C,E,G,H) External aspects viewed with a dissecting microscope. (B,D,F) Semi-thin tangential histological sections. (A,B) GMRGal4/UAS-lacZ control flies. (A) Eyes exhibit a regular array of about 800 repeated ommatidia. (B) Normal ommatidial architecture characterised by a trapezoidal array of outer photoreceptor cells R1-6 and central R7 cell, surrounded by accessory cells. (C,D) GMRGal4/UAS-RacGAP(84C). (C) Eye-directed expression of RacGAP(84C) induces rough eyes with black patch in the anterior-most part. (D) RacGAP(84C)-expressing retina shows a reduced number of randomly dispersed ommatidia and vacuolated material (arrows) associated with abnormal numbers of photoreceptor cells and misshapen rhabdomeres (arrowheads). (E,F) GMRGal4/UAS-Rac1N17. (E) Eye-directed expression of Rac1N17 induces rough eyes. (F) Rac1N17-expressing retina shows loss of rhabdomeres (open arrowhead), abnormal photoreceptor cells (black arrowheads), vacuolated material (arrows), and polarity defects (bar). (G) GMRGal4/+; UAS-GAP17/+ eye expressing the isolated GAP domain of RacGAP(84C) (weak strain). (H) Eye-directed expression of RasN17 induces reduced and rough eyes (RasN17/Y; GMRGal4/+).

 


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  		Fig. 2. Modifiers of the reference eye phenotype induced by the GAP domain of RacGAP(84C). Heads from female flies raised at 25°C (A-L) or 20°C (M-P) were viewed by scanning electron microscopy. B,D,F,H,J,L,N,P are higher magnifications of parts of A,C,E,G,I,K,M,O, respectively. (A,B) GMRGal4/UAS-lacZ control flies. (A) The eye surface is convex and of characteristic size and shape. (B) Each ommatidium is uniform and round and there are regular polarised inter-ommatidial bristles. (C,D) GMRGal4,UAS-GAP17/+ flies. (C) Reference rough eye phenotype. (D) Ommatidia are flattened, inter-ommatidial bristles are disoriented, of variable length, and occasionally absent. (E-H) Suppressor lines showing a more wild-type eye in size, shape, roundness and surface texture. Ommatidial architecture is basically restored, and inter-ommatidial bristles are nearly normal in number, length and polarity. (E,F) GMRGal4,UAS-GAP17/EP(2)386 flies misexpressing MESR4. (G,H) GMRGal4,UAS-GAP17/+; EP(3)3319/+ flies misexpressing Cdi/TESK1. (I-P) Enhancer lines. (I,J) GMRGal4,UAS-GAP17/EP(2)2072 flies misexpressing AKAP200. (I) Strong defects are apparent posteriorly corresponding to a black patch when viewed by optical microscopy. (J) Ommatidia are collapsed with distinct borders, except in rare cases of fusion. (K,L) GMRGal4,UAS-GAP17/EP(2)587 flies misexpressing the kinase Grapes. (K) Reduced eye size. (L) Fusion of ommatidial units and loss of bristles. (M,N) GMRGal4/EP(2)2299 flies misexpressing Beach at 20°C exhibit slightly rough eye surface and occasional fusion of ommatidia. (O,P) GMRGal4/EP(2)2299; rn20/+ flies have strongly affected eyes with a rough surface, reduced size (O) and fused ommatidial units (P) (whereas rn20/+ flies appear normal; not shown).

 


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  		Fig. 3. Specificity of Cdi and Beach towards Rac and Ras signalling. Eyes of flies raised at 20°C viewed by scanning electron microscopy. Coexpression of EP lines with Rac1N17 (A-F) or RasN17 (G-L). (A,B) Control flies expressing only Rac1N17 (GMRGal4/+; UAS-Rac1N17/+) show moderately rough eyes, occasional ommatidial fusion and bristle polarity defects. (C,D) GMRGal4/+; UAS-Rac1N17/EP(3)3319 flies. The LIM kinase Cdi is a strong suppressor of the dominant inhibition of Rac1, restoring normal eye size and ommatidial patterning. (E,F) GMRGal4/EP(2)2299; UAS-Rac1N17/+ flies. Beach is a strong enhancer of the dominant inhibition of Rac. Eye size is further reduced and the ommatidial surface is totally fused with a high density of randomly-oriented bristles. (G,H) UAS-RasN17/Y; GMRGal4/+ control flies expressing RasN17 exhibit rough eyes with frequent fusion between ommatidia. (I,J) UAS-RasN17/Y; GMRGal4/+; EP3319/+ flies expressing Cdi do not exhibit modification of the eye defects induced by RasN17. (K,L) UAS-RasN17/Y; GMRGal4/EP(2)2299 flies expressing Beach exhibit strong enhancement of RasN17, further reducing eye size and leading to completely fused ommatidia and almost complete loss of bristles.

 


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  		Fig. 4. Partners of RacGAP(84C) encode testis-specific genes. (A) Ethidium bromide-stained agarose gel showing RT-PCR amplified fragments after electrophoretic separation. Lane 1: cdi (498 bp); 2: TMS1d (426 bp); 3: RacGAP(84C) (274 bp); 4: Fragment size marker (Gibco-BRL); 5: Fragment size marker (Invitrogen); 6: Rac1 (856 bp); 7: RacGAP(84C) (274bp); 8: Rac1 amplification without reverse transcriptase (negative control). (B) Northern analysis. Total RNA from: lane 1, 300 isolated testes; lane 2, 200 adult flies and lane 3, 200 isolated ovaries. Detection of cdi transcripts with a 32P-random-labeled probe shows that the 6.0 kb transcript is specific for testes and the 6.8 kb transcript is specific for ovaries. The third form of 5.7 kb described by Matthews and Crews (Matthews and Crews, 1999Go) was detected only in total adult RNA after longer exposure of northern blot (not shown). (C) Detection of ß-gal activity in testis from the enhancer trap line cdiP242. Staining is present in the apical part of the germarium with dividing spermatogonia (arrowhead), and in the distal part where the final stages of differentiation occur (arrow). (D-E) Dissection of testes and seminal vesicles of 5-day-old males. (D) Control w1118 males have transparent testes (arrow) and seminal vesicles full of sperm (arrowhead). (E) w; Rac1j11,FRT2A,cdiR47/Rac1j11,FRT2A males exhibit an accumulation of dense material in the distal region (arrow) and seminal vesicles full of sperm (arrowhead). (F-G) Isolated testis of Rac1j11,FRT2A,cdiR47/Rac1j11,FRT2A separated from its seminal vesicle. (F) Before crushing, no sperm are detectable (arrows). (G) After crushing, in addition to uncharacterised material, we observe motile sperm issuing from the distal extremity of the testis (arrows).

 

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© The Company of Biologists Ltd 2004