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First published online 14 June 2006
doi: 10.1242/dev.02436

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calderón encodes an organic cation transporter of the major facilitator superfamily required for cell growth and proliferation of Drosophila tissues

¹ Icrea and Institut de Recerca Biomedica, Parc Cientific de Barcelona, Josep Samitier, 1-5, 08028 Barcelona, Spain.
² Centro de Biología Molecular `Severo Ochoa'-CSIC, Campus de Cantoblanco, 28049 Madrid, Spain.

View larger version (85K): [in a new window]   Fig. 1. calderón expression in embryonic and imaginal tissues. (A) Genomic organization of the cald locus. The P-element insertions cald-gal4 and EP1072 are shown as blue triangles. (B) Dorsal view of a cald-Gal4; UAS-lacZ embryo at stage 13. Note lacZ expression, visualized by histochemical staining for ß-gal activity, in the amnioserosa (black arrowhead) and in the central nervous system (CNS, white arrowhead). (C-G) Lateral view (anterior left, dorsal up) of embryos showing the distribution of CG13610 transcripts from stage 5 (C) to 13 (G). Early embryos (C) have uniform levels of cald expression. Gastrula embryos (D) have higher levels of cald transcripts in the most dorsal cells. From germ band extended (E,F) to germ band retracted embryos (G). cald expression is restricted to the amnioserosa and the CNS. (H) Dorsal view of an embryo at stage 13 showing cald expression in the amnioserosa and the CNS, which reproduces cald-Gal4 expression (compare with B). (I,K,M) cald-Gal4:UAS-GFP staining in wing (I), eye-antenna (K) and leg (M) imaginal discs. (J,L,N) Wing (J), eye-antenna (L) and leg (N) imaginal discs showing cald transcripts. cald transcripts reproduce the cald-Gal4 expression. (O) Alignment of Calderon and the human protein SLC22A4. Both proteins have 11 transmembrane domains with a Major Facilitator Superfamily domain (boxed amino acids) and a Sugar Transporter domain (red amino acids). Identical amino acids are identified by a star. Double dots label conserved substitutions and single dots semi-conserved substitutions. The two proteins are 36.4% identical.

View larger version (34K): [in a new window]   Fig. 2. calderón embryonic phenotype. (A-C) Lateral view (anterior left, dorsal up) of wild-type (A), caldR107/caldR107 (B), and caldEP1072/caldEP1072(C) first instar larvae. Mutant embryos exhibit the characteristic U-shaped phenotype due to a defective germ band retraction. (D) Lateral view of a caldR107 embryo driving the expression of GFP in the calderón pattern. (E-H) Lateral view of embryos showing the distribution of CG13610 transcripts. Note reduced expression levels in caldR107 (F) or caldEP1072 (G) mutant embryos, when compared with heterozygous caldR107 (E) or caldR107/caldEP1072 (H) embryos. Arrows point to the amnioserosa. Double in situ hybrdization and antibody staining against ß-Gal protein (brown in E) was performed to identify homozygous and heterozygous cald embryos.

View larger version (46K): [in a new window]   Fig. 3. calderón adult phenotype. (A) Graph depicting the developmental delay of caldR161 animals. The length of embryonic (E, green), larval (L, red) and pupal (P, blue) stages is shown for caldR161 and wild-type animals. (B) caldR161 animals are smaller than wild-type ones. (C) caldR161 and wild-type heads; note the caldR161 eye is markedly smaller. (D) caldR161 and wild-type wings. Note the caldR161 wing is smaller than the wild-type one. (E) Histogram plotting the size and cell density ratio of calderón/wild-type animals. Ratio (fly length): 0.9±0.04; ratio (eye size): 0.8±0.05; ratio (wing size): 0.85±0.04; ratio (cell density): 1.3±0.16. Number of scored flies: n (wt):7; n (cald):9. Number of scored eyes: n (wt):7; n (cald):9. Number of scored wings: n (wt):6; n (cald):6. Number of scored wings for cell density counting: n (wt):7; n (cald):9. (F,G) caldR107/caldEP1072 male (F) and female (G) animals are smaller than wild-type ones when raised at 18°C, but nearly wild-type at 25°C.

View larger version (81K): [in a new window]   Fig. 4. calderón mutant cells are eliminated by cell competition. (A-C) Wing discs with caldR107 mutant clones marked by the absence of expression of the lacZ gene, as visualized with an antibody against the ß-Gal protein (grey). Twins were marked by the presence of two copies of lacZ (white). Larvae were dissected 24 (A), 48 (B) and 72 (C) h after clone induction. Twins were larger than mutant clones and 72 h after induction cald mutant cells disappeared. (D) Rescue of caldR107 mutant cells by expression of an UAS-cald transgene using the MARCM technique. Clones were visualized 72 h after induction by the expression of an UAS-GFP transgene (white). (E,E') caldR107 mutant clones marked by the absence of the GFP marker (green) and visualized 24 h after induction. Expression of the activated form of the Caspase-3 protein is shown in red. The upper panel shows an XY confocal section of the wing pouch. The right and lower panels show YZ and XZ sections of the same wing discs at the level of the white lines. (F,F') brinker (brk) expression in cald mutant cells. caldR107 mutant clones marked by the absence of the GFP marker (green) show an up-regulation of brk-lacZ expression (in red).

View larger version (69K): [in a new window]   Fig. 5. The role of calderón activity in Minute mediated cell competition. (A) Diagram depicting the induction of Minute+ cald mutant clones (upper cell) and their Minute- cald+ twins (lower cell) after a mitotic recombination event. (B-G,I) Clones are marked by the absence of the ß-Gal marker (grey). Twins are marked by the presence of two copies of the ß-Gal marker (white). (B,D,F) Minute+ control clones visualized 24 (B), 48 (D) and 72 (F) h after induction. Note all twin Minute- homozygous clones have already disappeared 48 h after induction. (C,E,G,I) cald Minute+ clones visualized 24 (C), 48 (E) and 72 (G,I) h after induction. Some twins (Minute- homozygous and wild type for cald function) are recovered even 72 h after induction (magnified in I). (H) Graph plotting the ratio of Minute-/Minute+ control clones (in black) and the ratio of Minute-/Minute+ cald mutant clones (in red). 24 h after clone induction: number of Minute+ clones: 522; number of Minute+ cald clones: 253. 48 h after clone induction: number of Minute+ clones: 507; number of Minute+ cald clones: 187. 72 h after clone induction: number of Minute+ clones: 58; number of Minute+ cald clones: 81.

View larger version (75K): [in a new window]   Fig. 6. calderón clonal phenotypes. (A,B) cald mutant cells are small in imaginal discs. Minute+ caldR107 mutant clones marked by the absence of the GFP marker (green) in the wing imaginal disc. Cell membranes are delineated by Phaloidin (red in A) and cell nuclei are labelled by DAPI (blue in B). dFOXO (red in B) remains cytoplasmic in cald clones. The white trace outlines the mutant clone in B. (C-F) cald mutant cells are small in the adult. Clones of Minute+ cald mutant cells marked by the absence of the P(yellow+) rescue construct in the notum (C,D), and in the wing margin (E,F). Each hair-like structure is a trichome emanating from a single epithelial cell. Note the reduced size and increased density of cald cells (red arrow) compared with surrounding yellow+ ones (black arrowhead).

View larger version (58K): [in a new window]   Fig. 7. calderón expression is regulated by the Insulin pathway. (A-E) Lateral view (anterior left, dorsal up) of embryos showing the distribution of cald transcripts in various genetic backgrounds. (A,C,E) Activation of the InR pathway up-regulates cald expression. Stage 13 embryos showing cald transcripts in wild-type (A), ptc-Gal4:UAS-dS6K (C) and Ubx-Gal4:UAS-dS6K (E) backgrounds. At this stage, wild-type embryos show cald expression only in amnioserosa and a group of cells of the CNS. Activation of InR pathway in ectodermal cells in ptc (C) and Ubx (E) domains up-regulates cald expression in the ectoderm. (B,D) Down-regulation of the InR pathway eliminates cald expression. Stage 11 embryos showing cald transcripts in wt (B) and Kr-Gal4:UAS-PI3K92E-Dp110DN (D) backgrounds. Note reduced expression of cald in amnioserosa cells in D (red arrow). (F) Down-regulation of InR pathway in amnioserosa cells phenocopies cald mutants. Lateral view of Kr-Gal4:UAS-PI3K92E-Dp110 DN first instar larva. These larvae exhibit the characteristic U-shaped phenotype of cald mutants (compare Fig. 2B with C). (G,H) Third instar wing discs showing cald transcripts in wild-type (G) and MS1096-Gal4:UAS-PI3K92E-Dp110DN (H) backgrounds. MS1096-Gal4 is expressed at higher levels in the dorsal compartment (d) of the wing pouch. The boundary between dorsal (d) and ventral (v) compartment is shown.

View larger version (13K): [in a new window]   Fig. 8. Mode of action of Calderón. Proposed model illustrating the role of cald in Drosophila growth. Genetic and/or biochemical relationships are depicted as arrows to indicate positive regulators or as bars to represent negative regulators of targets. The TOR and InR pathways integrate the nutritional information and exert a control on cell growth, in part, regulating cald expression. Cald activity is required for the control of growth: cell size and proliferation.