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First published online 16 August 2006
doi: 10.1242/dev.02524

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Organization of the peripheral fly eye: the roles of Snail family transcription factors in peripheral retinal apoptosis

Hui-Ying Lim^1,* and Andrew Tomlinson^2,

¹ Department of Pathology, Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, Room 1120, New York, NY 10032, USA.
² Center for Neurobiology and Behavior, Department of Genetics and Development, College of Physicians and Surgeons, Columbia University, 701 West 168th Street, Room 1120, New York, NY 10032, USA.

View larger version (46K): [in a new window]   Fig. 1. Enhancer trap lines showing marginal pigmentation in the fly eye. (A) Schematic depiction of the peripheral specializations of the eye. Residing immediately adjacent to the head capsule (blue) is the pigment rim (red) that completely circumscribes the eye. The dorsal rim ommatidia (green) lie next to the pigment rim only in the dorsal margin of the eye. The dorsal rim ommatidia and their ventral corresponding ommatidia, together with a number of interior rows of ommatidia (white), are devoid of bristles. The central field of ommatidia (light blue) bears bristles. Shown within the dorsal and ventral boxed areas are the photoreceptor-bearing ommatidia. The outer photoreceptors extend throughout the entire ommatidium, whereas the inner photoreceptor R7 and R8 each occupies half of the ommatidium (black bars). Inner photoreceptors of the dorsal rim ommatidia have enlarged rhabdomeres (pink bars) when compared with those of normal ommatidia. (B-D) Whole-mount views of the adult eyes of RR (B), Cir1 (C) and SK (D) showing marginal pigmentation pattern. The pigmentation deceptively appears to extend well into the body of the eye. This results from the cupped shape of the retina. Compare arrow in B with arrow in E. (E) Section through the anterior region of the eye of Rim Red showing pigment expression only in the pigment rim. The arrow indicates the pigment in the PR, and the corresponding position is indicated in B above. (F) Genomic map of the Snail region. The red arrowheads indicate the positions of the three P-element insertions.

View larger version (117K): [in a new window]   Fig. 2. Expression patterns of Escargot, Worniu and Wingless in the peripheral eye. (A-F) All micrographs are flat mounted pupal retinas of 32 hours APF. (A-A'') Pupal eye stained for Escargot (red) and Elav (green). Escargot is expressed in the HC, PR and peripheral 2°/3° pigment cells. (B-B'') Cir1-Gal4>UAS-lacZ eyes stained for lacZ (red) and Elav (green). Cir1 and Escargot show the same expression profile. (C-C'') Escargot (red) and Cut (green, specifically labels cone cells) are co-expressed in the marginal cone cells. (D-D'') Escargot (red) and Homothorax (green) co-expressed in the head capsule (arrow). Homothorax is also expressed in the central photoreceptors of the outer ommatidia (those that die) and an inner ring that will eventually form the DRO. (E-E'') wg-lacZ eyes stained for lacZ (green), Cut (red) and Elav (blue) showing Wg expression in the perimeter cone cells. (F-F'') wg-lacZ eyes stained for lacZ (green), Wg (red) and Elav (blue) showing the absence of both Wg expression in the perimeter photoreceptor cells. (G) Summary of the expression patterns of Esg and Wg. Top panel: a key to the cell types depicted below. Bottom panels: Schematic summary of Wg and Escargot expression in the eye periphery at 32 hours APF. Escargot is expressed in the HC, peripheral 2°/3° pigment and cone cells. Wg is expressed in the HC and peripheral cone cells.

View larger version (135K): [in a new window]   Fig. 3. Wg signaling regulates the expression of Snail family factors. (A) A clone of actin-wg in adult RR eye induces ectopic expression of the transcriptional reporter (arrow). (B-B") A clone of actin-wg (red) in Cir1 pupal eye (32 hours APF) showing ectopic expression of the transcriptional reporter (green). (C-C") An actin>Gal4; UAS-Arm clone in pupal retina (32 hours APF) marked by GFP (green) shows ectopic Escargot (red) non-autonomously. (D-D") A large dsh[V26] clone in the pupal eye periphery (32 hours APF) marked black by the absence of GFP. Peripheral Esg (red) is almost entirely abolished. The faint lattice of staining is the incipient Esg expression in the main retina 2°/3° pigment cells - this is not Wg dependent. (E-E") An arr[2] clone in the pupal eye edge (32 hours APF) marked black by the absence of GFP. Escargot expression (red) is lost in the clone. (E'') A merge of the two with an additional stain for Cut (blue). The arrowhead indicates a cone cell that would normally express Escargot but fails to do so in the clone.

View larger version (139K): [in a new window]   Fig. 4. Snail transcription factors mediate the effects of Wg signaling in peripheral eye development. With the exception of C, all micrographs are flat mounted pupal retinas of 40 hours APF. (A-A')A dsh[V26] clone at the eye margin marked black by absence of GFP showing ectopic cone cells stained with Cut (red, arrowhead). (B)A Df(3L)H99 clone in the eye margin marked black by the absence of GFP showing ectopic cone cells (red). The cone cells from more interior ommatidia are not visible at this focal plane. (C) Whole-mount views of adult eyes uniformly ectopically expressing Wg and Escargot under the GMR promoter. High level Wg and Escargot result in small heavily pigmented eyes. (D-D") A dsh[V26] clone at the eye margin marked black by absence of GFP and showing retinal protrusion caused by the inappropriate survival of the peripheral ommatidia stained for Elav (blue). The PR, highlighted by coracle staining (red) is significantly reduced in the clone (arrowhead) compared with the adjacent wild-type patch (arrow). (E) A dsh[V26] clone at the eye margin marked black by absence of GFP showing ectopic primary pigment cells stained with BarH1 (red). (F) A clone doubly mutant for esg and sna, and overexpressing two different worniu RNAi constructs (triple mutant clone) in the eye margin marked by GFP (green). (F',F") Ectopic photoreceptor cells stained for Elav (arrowheads, red) are present in the clone. The photoreceptors of the more internal ommatidia are not visible at this focal plane. (G) A triple mutant clone marked by GFP (green) at the eye margin. (G',G") Ectopic cone cells (red) are present in the clone (arrowheads). Cone cells of interior ommatidia are not fully visible at this focal plane. (H,H') A triple mutant clone in the eye margin marked by GFP (green) showing a significant reduction of the pigment rim (red, arrowheads). (I,I') A higher magnification view within a triple mutant clone (clone marker not shown) showing no effect on homothorax expression (green) in both ectopic (arrowheads) and standard DRO ommatidia.

View larger version (102K): [in a new window]   Fig. 5. The expression and function of Notum in the eye periphery. (A-A") A notum-Gal4, UAS-lacZ pupal retina (34 hours APF) doubly stained for lacZ (green) and Escargot (red). Notum and Escargot expression coincides in the HC (arrowheads), PR and some 2°/3° pigment cells. (B-B") A notum-Gal4, UAS-lacZ pupal retina (32 hours APF) stained for lacZ (green), Cut (red) and Elav (blue). Notum expression is detected only in the cone (green) and not photoreceptor cells (blue). The more interior cone cells (B') are not visible at this focal plane. (C-C") A notum clone in the pupal eye (32 hours APF) marked black by the absence of GFP (green) showing precocious expression of Escargot (red) in the cone cells of peripheral ommatidia (arrowheads). (D,D') Patterning defects in notum clones (absence of green in D'') of 40 hours APF retinas. At this stage, the ommatidial death is in process, and degenerate cone cells (blue) and photoreceptors (red) can be observed in the second interior row of ommatidia that would not normally be affected (arrows). (E,E') A notum clone in the pupal eye (32 hours APF) marked black by the absence of GFP (green) showing an expansion of the Wg protein expression domain (blue, arrowhead). (F) Schematic summary of Notum expression in the marginal eye structures when compared with Wg expression (see also a summary of Escargot expression in Fig. 2G).

View larger version (144K): [in a new window]   Fig. 6. Expression and function of Esg in late-stage differentiation of interommatidial pigment cells. (A-C') Flat mounted pupal retinas of 42 hour APF. (A,A') Wild-type pupal retina stained for Escargot (green), Elav (red) and Cut (blue), showing the selective expression of Escargot in the pigment cells surrounding the ommatidia. (B-B'') Pupal retina containing a dsh[V26] clone marked by the absence of GFP (green) stained for Escargot (red). Escargot expression is not perturbed in the interior interommatidial pigment cells. (C,C') Pupal retina containing a clone mutant for escargot (marked by the absence of green) is stained for Armadillo (red) that outlines the cellular profiles. The mutant pigment cells fail to undergo apical constriction. (D) Section through an adult eye in which escargot clones have been induced (marked by the lack of pigmentation). Interommatidial pigment cells are absent or defective, causing vacuolar structures (arrowheads) and ommatidial fusion (not shown).

View larger version (34K): [in a new window]   Fig. 7. Schematic description of how the pigment rim is formed. (A) Upper panel shows the presumptive retina at three stages of the third instar larva. Towards the left, the early retina is flanked by the Wg-secreting presumptive head capsule (HC, blue) and the Hh wave is incipient (green arrows). In the middle and to the right; as the wave sweeps the retina, the antagonistic Wg signal (blue t-stops) prevents ommatidia from differentiating close to the HC. (B) Lower panel shows a high power view close to the HC during the pupal phase. Left: the strip immediately adjacent to the HC is occupied by presumptive pigment cells because ommatidial differentiation was inhibited there. The outer two rows of ommatidia express Hth, thereby specifying them as DRO, but the most peripheral row also receives the Wg signal (blue arrows) that indirectly causes their apoptosis. Middle: the peripheral ommatidia die. Right: the pigment cells that surrounded the dying ommatidia now join the peripheral pigment cells to form the PR, and the most peripheral ommatidia are now the surviving DRO units.