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First published online 21 November 2006
doi: 10.1242/jcs.03294

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The Cdi/TESK1 kinase is required for Sevenless signaling and epithelial organization in the Drosophila eye

¹ Departament de Genètica, Facultat de Biologia, Universitat de Barcelona, Diagonal 645, 08028 Barcelona, Spain
² Institute for Molecular Systems Biology, ETH Zürich, c/o Zoologisches Institut, Universität Zürich, Winterthurerstr. 190, 8057 Zürich, Switzerland
³ Institute of Biotechnology and Department of Biological and Environmental Sciences, University of Helsinki, FIN-00014 Helsinki, Finland

View larger version (67K): [in this window] [in a new window]   Fig. 1. The EP(33-077), EP(44-004) and EP(34-165) suppression of the rough eye phenotype when Sev RTK signaling is activated. (A) Rough eye phenotype caused by the constitutively activated Sev receptor (sevS11/+). (B) Suppression of the rough eye phenotype due to the overexpression of a double-headed EP line identified in the screen (sev-Gal4 sevS11/EP). Note that the regular pattern of ommatidia is rescued. (C) The double-headed EP transposable element, showing the 5' and 3' UAS sites (black arrowheads). Activation of the Gal4 drives expression of the genes adjacent to 5' and 3' UAS sites. The loxP sites flanking the 5' UAS (red boxes), will remove the 5' UAS site when in the presence of the recombinase cre. Note that flies lacking the 5' UAS site will be distinguished by the lack of the yellow+ marker and named EP(y-). (D) Summary of the suppression of the SevS11 construct by the three EP lines. (E) Genomic region and insertion of EP(33-077), EP(44-004) and EP(34-165). There are three genes in this region: ATPsyn-d, mRpL55 and cdi. Black arrows indicate the transcription starts and orientation of the three genes. The EP(33-077) is 1.800 bp upstream from 5' UTR of mRpL55. They are orientated with the 3' UAS site (small blue arrows) towards the 5' end of cdi and mRpL55. The blue bar represents the 2.077 bp deletion of the cdiR47 allele. (F) Molecular characterization of the gene expression induced by the EP lines. RT-PCR from hs-Gal4 EP larvae after heat shock induction indicates that both cdi and mRpL55 transcripts are overexpressed. EP(34-165y-) and EP(44-004y-) are shown as examples, and rp49 expression as a control. C lanes show expression without heat shock. E lanes show expression after heat shock. (G) Wing disc from en-Gal4/+; EP 34-165/+ larvae stained with an antibody against Cdi. Cdi protein is strongly expressed in the posterior compartment due to EP activation. (H) en-Gal4/UAS-cdi wing discs stained with anti-Cdi show abundant localization of Cdi in the posterior compartment.

View larger version (38K): [in this window] [in a new window]   Fig. 2. cdi as a suppressor of the activated Sev pathway. (A) Semi-thin section from a control eye (left) where UAS-cdi is not overexpressed (UAS-cdi/UAS-cdi; sevS11/+) and an experimental eye (right) where the UAS-cdi is overexpressed by sev-Gal4 (UAS-cdi/UAS-cdi; sev-Gal4 sevS11/+). The histogram shows distribution profiles of the number of R7 photoreceptors per ommatidium for each genotype represented. The statistical analysis reveals significant differences between the genotypes (P<0.001). (B) Semi-thin section of control eye (left), where UAS-Boss is expressed under sev-Gal4 (UAS-Boss/+; sev-Gal4/+) and experimental eyes (UAS-cdi/UAS-Boss; sev-Gal4/+) are shown. The histogram quantifies the number of R7 per ommatidium with and without cdi overexpression (P<0.001). The inset below shows two ommatidia from a UAS-cdi/UAS-cdi; sev-Gal4 fly, one with R7 (right) and one without (left). R7 rhabdomeres are colored green.

View larger version (120K): [in this window] [in a new window]   Fig. 3. cdi expression pattern in the eye imaginal disc. (A-D) Cdi pattern (red) of a disc co-stained with prospero (green). The confocal image was captured at the level of the R7 cells to discriminate from the prospero-stained cone cells. Upper panels: cross-section. Lower panels: transverse section. Arrowheads point to the localization of R7 cells. The merged figure in A also shows nuclear staining with Sytox-green (blue) to show distribution of nuclei. Note that in the transverse XZ sections (lower panels) the localization of Cdi is more abundant in the apical region of the epithelium (top). (E-G) Co-localization of F-actin (green) and Cdi (red) in the apical constrictions. Arrowheads point to apical constrictions. Upper panels: optical section taken through the most apical domain of the eye disc. Lower panels: transverse sections. (H-J) High magnification of an ommatidium of the previous image, showing Cdi (red) in the F-actin enriched apical constriction (green). (K) High magnification of a transverse section of a pupal photoreceptor to show cytoplasmic localization of Cdi. Apical: top. Arrowhead points to the nucleus. (L-M) Two confocal planes of developing ommatidia of a cdiBA01 early pupal disc stained with anti ß-galactosidase. Labeling is extensive throughout the ommatidial cells. Bars, 10 µm.

View larger version (89K): [in this window] [in a new window]   Fig. 4. Overexpression of cdi in eye discs. Clones of cells overexpressing cdi are labeled with GFP (green). (A-C) Clone anterior to the morphogenetic furrow where cells overexpressing cdi concentrate more F-actin (red) in the cell cortex. Upper panels show a cross-section, lower panels a transverse section. (D-G) Detail of a confocal cross-section at the apical region of developing ommatidia posterior to the morphogenetic furrow stained with phalloidin (red) and anti-ß-catenin (blue). Note the F-actin accumulation in the cdi-overexpressing cells (F). Arrows point to a cdi-overexpressing ommatidial cluster and arrowhead to a wild-type cluster. Localization of ß-catenin in cdi-overexpressing cells is displaced basally (G). Left panels: cross-sections. Right panels: transverse section through a mutant ommatidial cluster adjacent to a wild-type cluster. (H-K) Apical cross-section through ommatidial clusters stained with Fasciclin III (blue) and DE-cadherin (red). Note that the DE-cadherin of the closely apposed photoreceptor membranes in wild-type cells (arrowhead) is absent in the cdi-overexpressing ommatidial cluster (arrow). However, in a transverse section containing a cdi-overexpression cluster (arrow) next to a wild-type cluster (arrowhead), DE-cadherin is found more basally and in an abnormal arrangement (right panels). Fasciclin III, used to mark the cell contour, denotes that the cdi-overexpressing cluster is not shrunk or smaller. Diagonal lines in D and H indicate the region represented in the transverse sections. Bars, 10 µm.

View larger version (125K): [in this window] [in a new window]   Fig. 5. Loss of cdi function in the developing eye. (A-C) Clones of cdiR47 (lack of GFP in A and B) show a lack of Cdi expression as assayed by anti-Cdi (red in A and C). The confocal image was taken at the level of inner photoreceptors of a late third instar larva where a transient accumulation of Cdi is found. (D-F) Mitotic clones of homozygous cdiR47 cells (lack of GFP). All these clones were supplied with UAS-mRpL55 under GMR-Gal4 driver. Actin organization is severely altered in mutant ommatidia (arrow). Clones containing both wild-type and mutant cells appear normal (arrowhead). (G-J) Clone of cdi mutant cells (lack of GFP) counterstained with anti-ß-catenin (blue) and anti DE-cadherin (red). A single confocal cross-section (upper panels) taken at the level of the adherens junctions of the wild-type cells. Note that in this level of the mutant sector, both proteins are absent. Transverse section (lower panels) where ß-catenin and DE-cadherin are aberrantly positioned. (K-N) Clones of cdiR47 cells (lack of GFP) complemented with the double transgene UAS-mRpL55 and UAS-cdi under the GMR-Gal4 driver and stained for phalloidin (red), and ß-catenin (blue). (O) Transverse section of another clone (lack of GFP) as in K stained with DE-cadherin (red). Top, merged; middle, GFP; bottom, DE-cadherin. (P) Eye of a fly showing cdiR47 mutant clones (white sectors, arrow) in Minute+ background. (Q) Semi-thin section of a mosaic eye, showing a clone of cdiR47 mutant ommatidia (absence of inter-ommatidial pigmentation; arrow points to mutant cells). (R-T) Clones of cdiR47 supplied with UAS-mRpL55 and stained with anti-caspase-3. Thin lines represent the plane of transverse sectioning. Bars, 10 µm.

View larger version (60K): [in this window] [in a new window]   Fig. 6. ssh suppression of the activated Sev pathway. (A) Rough eye phenotype due to the sevS11 construct (sev-Gal4 sevS11/+). (B) Suppression of the sevS11 rough eye phenotype when UAS-RNAi-ssh is activated (UAS-RNAi-ssh/+; sev-Gal4 sevS11/+). (C) The activation of the transgene (UAS-RNAi-ssh/+; sevGal4) does not alter eye morphology. Upper panels: SEM images. Lower panels: tangential semi-thin sections with R7 photoreceptors colored green. (D) The histogram shows distribution profiles of the number of R7 photoreceptors per ommatidium for each genotype represented. The statistical analysis reveals significant differences between the genotypes (P<0.001).

View larger version (101K): [in this window] [in a new window]   Fig. 7. Sev RTK localization in cdi gain- and loss-of-function clones. (A-D) Upper panels show a cross-section confocal image of cdi gain-of-function clones labeled with GFP (green) and F-actin (red). Lower panels show a transverse section of the same preparation taken through the region indicated with the white line in A. Sev RTK protein (blue) localization is extended to a more basal position in the mutant ommatidia (arrowheads). (E-G) Loss-of-function clone marked by the absence of GFP. Note that in some mutant ommatidia (arrow) Sev RTK (blue) staining is lower than in the wild-type. (H) Transverse sections of a loss-of-function clone where the Sev RTK staining (blue) of the apical membrane (arrow) is reduced. Bars, 10 µm.