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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01443

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Spalt transcription factors are required for R3/R4 specification and establishment of planar cell polarity in the Drosophila eye

¹ Howard Hughes Medical Institute, Strang Laboratory of Cancer Research, The Rockefeller University, Box 252, 1230 York Avenue, New York, NY 10021, USA
² Brookdale Department of Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029, USA

View larger version (86K): [in a new window]   Fig. 1. Salm is expressed in R3 and R4 when planar polarity is established. Anterior is to the left and dorsal is up. (A) Third larval instar eye imaginal disc, stained with anti-ELAV (red) and anti-ß-galactosidase (blue), in which lacZ is expressed under sev-Gal4 control to show the R3/R4 position (lower staining levels are also observed in R1, R6 and R7) and reveal the rotation of the developing ommatidia. Close to the morphogenetic furrow (MF, to the left) the R3/R4 pair is perpendicular to the DV midline, the equator (yellow line). By row six, ommatidia have rotated 45°, in a clockwise or counter-clockwise direction in the dorsal and ventral halves, respectively. In the posterior part of the eye disc, the 90° rotation of the ommatidia is almost complete, and the R3/R4 pair is parallel to the equator. (B) Schematic illustrating ommatidial rotation in the imaginal disc. (C) Tangential section of an adult eye (left) and corresponding schematic drawing (right). The section is at the level of R7. R8 is not visible as it is localized below the R7 plane. Ommatidia in the adult eye are arranged as two opposite chiral forms separated by the equator (yellow line), as a consequence of R3/R4 specification and the following 90° rotation. Ommatidia in the dorsal half are represented with black arrows and in the ventral half with red arrows. (D) Magnification of one dorsal (top) and one ventral (bottom) ommaditium; arrows as in C. Numbers indicate the identities of the photoreceptors. (E) Eye imaginal disc stained for Salm (blue) and svp-lacZ (green). The initiation of Salm expression in R3/R4 precedes svp-lacZ by one row. Posterior to row seven, Salm expression in R3/R4 starts to fade, whereas svp-lacZ continues to be expressed in R3/R4 and also, at lower levels, in R1/R6. (F) Salm (green) expression in R3/R4 precedes the onset of m0.5-lacZ (red) expression in R4 by one to two rows. Scale bars: 10 µm.

View larger version (77K): [in a new window]   Fig. 2. sal is required in R3 for the establishment of correct chirality. (A) Tangential sections of adult eyes containing sal null mutant clones [in all experiments we used a small chromosomal deficiency – Df(2L)32FP5 – covering only salm and salr (Barrio et al., 1999)]. sal mutant (sal–) cells are shown by the absence of the pigment (w) marker (dark dots at the base of each rhabdomere and in pigment cells). In schematic drawings, black arrows represent dorsal and red arrows ventral orientation. Green arrows represent ommatidia where it is possible to identify R1/R6 and R7, but not R3 or R4. Black circles represent ommatidia where it is impossible to score orientation, because R7 or R8 are transformed into outer PRs, or they contain extra photoreceptors. Top panel: note that in the ommatidium with (wrong) ventral chirality, only the presumptive R3 precursor is sal– and has acquired an R4 fate. (B) Statistical analysis of mosaic ommatidia that always present correct chirality. sal– cells are represented as white circles and non-mutant cells as black circles. The number of ommatidia is indicated below each configuration. The inset at the top right corner represents the common feature of these configurations, which is that R3 always has the sal+ genotype. The numbers inside the circles represent the identity of each PR. (C) Statistical analysis of mosaic ommatidia exhibiting chirality inversions. The common feature of these configurations is that the cell in the R4 position always has the sal– genotype. This R4 mutant cell corresponds to a R3 precursor that made the wrong chiral choice.

View larger version (90K): [in a new window]   Fig. 3. sal is required for the correct expression of R3/R4 specification and polarity markers. All panels represent third instar eye discs where sal– clones were induced by Flipase-mediated mitotic recombination and are labeled by the absence of ubi-GFP staining (green). Anterior is to the left and the equator is at the top. The blue channel shows Ro in A and ELAV in all other panels. (A) BarH1 (red) stains R1/R6 and allows the visualization of the progressive ommatidial rotation. White and yellow bars indicate ommatidia with correct and incorrect rotation, respectively. (B) In wild-type tissue, Fmi (red) localizes in the equatorial side of R3 and R4 (arrows). In sal– ommatidia, Fmi is present in all sides of the apical membrane of R3 and R4 (arrowheads). High magnification images of sal– (top, right) and wild-type (bottom, right) ommatidia (asterisk) show the localization of Fmi in the R3 and R4 apical membrane (dashed line). (C) Dl expression (red), visualized with the enhancer trap line Dl-lacZ1282, is transiently upregulated in R3 (arrows) in two to three rows. In sal– tissue, most ommatidia show low levels of Dl expression in both cells of the R3/R4 pair (arrowheads). In some ommatidia, the cell in the R4 position has stronger staining than R3 (+), or both cells in the pair have high levels of Dl expression (asterisk). (D) The expression of m0.5-lacZ (red) in R4 is lost in 91% (n=218) of sal– ommatidia. Some residual expression is still observed in 9% of the cases. In mosaic ommatidia where R3 but not R4 (arrows, n=29), or R4 but not R3 (arrowhead, n=21) is sal–, m0.5-lacZ expression is reduced. (E) The expression of svp-lacZ (red) is lost in R3/R4, but not R1/R6, in sal– clones. Scale bars: 10 µm.

View larger version (97K): [in a new window]   Fig. 4. sal acts upstream of svp during R3/R4 specification. All panels represent third instar eye discs where svp– clones (A-C, svpe22 – transcript null allele) or sal– clones (D,E) were induced by Flipase-mediated mitotic recombination and are labeled by the absence of ubi-GFP staining (green). Anterior is to the left and the equator is at the top. The blue channel shows ELAV. (A) Salm (red) expression in R3/R4 is not repressed after row seven in the svp– area. In wild-type ommatidia, Salm expression is progressively repressed in R3/R4 after row seven (arrows). In svp– ommatidia, Salm expression persists in R3/R4 in more posterior rows (arrowheads). (B) In svp– clones, Fmi (red) is present in all sides of the apical membrane of R3/R4 (arrowheads). In wild-type ommatidia, Fmi is localized in the equatorial side of R3 and R4 (arrows). High magnification of svp– (top, right) and wild-type (bottom, right) ommatidia (asterisk) show the localization of Fmi in the R3 and R4 apical membrane (dashed line). (C) In svp– clones, the expression of m0.5-lacZ (red) is lost in R4. (D) In sal– ommatidia, sev-svp rescues m0.5-lacZ (red) expression in one cell of the pair, in many cases the one in the R4 position. In the wild-type ommatidia, sev-svp leads to m0.5-lacZ expression in both R3 and R4. (E) sev-Nact induces m0.5-lacZ (red) in R3 and R4, both in sal– and non-mutant ommatidia. Scale bars: 10 µm.

View larger version (98K): [in a new window]   Fig. 5. sal is required for the transformation of R3/R4 into R7 in svp mutants. (A-C) Tangential cross-sections of adult eyes containing clones for svp– (A), sal– (B) and svp–/sal– double (C) mutants. The mutant cells are visualized by the absence of the w marker, and the svpe22 transcript null allele was used. (D) Quantitative analysis of the number of photoreceptors with large or small rhabdomeres in svp–, sal– and svp–/sal– mutants. The numbers under the columns represent the number of large (diagrams on the left) or small (diagrams on the right) rhabdomeres observed for each individual ommatidium. The number of ommatidia with a particular number of large or small rhabdomeres is indicated as a percentage of the total number of ommatidia analyzed (116 in svp–, 127 in sal– and 79 in svp–/sal–).

View larger version (18K): [in a new window]   Fig. 6. Model of the roles of sal and svp in the specification of R3/R4 versus R7 [based on our present findings and on Domingos et al. (Domingos et al., 2004)]. sal is expressed in R3/R4 from row three to row seven, after which it is progressively repressed. sal expression in R7 starts from row seven to nine. Expression of svp in R3/R4 starts in row four. From row three to row seven, sal is required for svp expression in R3/R4, for R3/R4 specification and for PCP establishment. After rows seven to nine, sal is necessary and sufficient for R7 differentiation. Repression of sal by svp in R3/R4 is necessary for the maintenance of R3/R4 identity and the inhibition of R7 fate.

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