Wingless mediated apoptosis: How cone cells direct the death of peripheral ommatidia in the developing Drosophila eye

Abstract

Morphogen gradients play pervasive roles in development, and understanding how they are established and decoded is a major goal of contemporary developmental biology. Here we examine how a Wingless (Wg) morphogen gradient patterns the peripheral specialization of the fly eye. The outermost specialization is the pigment rim; a thick band of pigment cells that circumscribes the eye and optically insulates the sides of the retina. It results from the coalescence of pigment cells that survive the death of the outermost row of developing ommatidia. We investigate here how the Wg target genes expressed in the moribund ommatidia direct the intercellular signaling, the morphogenetic movements, and ultimately the ommatidial death. A salient feature of this process is the secondary expression of the Wg morphogen elicited in the ommatidia by the primary Wg signal. We find that neither the primary nor secondary sources of Wg alone are able to promote ommatidial death, but together they suffice to drive the apoptosis. This represents an unusual gradient read-out process in which a morphogen induces its own expression in its target cells to generate a concentration spike required to push the local cellular responses to the next threshold response.

Fig. 1

Peripheral features of the fly eye. (A) Scanning EM image of adult fly eye. The array of facets and bristles are the most prominent features. The head capsule (HC) circumscribes the eye (white arrow), and the outermost ommatidia lack bristles (red arrow). (B) Schematic cross section through the eye highlighting: the head capsule (HC grey); the pigment rim (PR pink), and DRO (purple). The region of bald ommatidia is white, and the array of standard ommatidia throughout the main body of the eye is blue. (C) Schematic longitudinal section through the eye. (D) Phase contrast image of the dorsal peripheral eye. The head capsule is seen as a grey strip adjacent to the pigment rim. The first row of ommatidia are DRO (purple arrows) indicated by the large R7 rhabdomeres. In the second row, the R7 rhabdomeres are normal (blue arrows). (E) Schematic longitudinal section through the peripheral dorsal eye. The head capsule secretes Wingless (Wg) which diffuses (grey triangle) into the retina and directs the specializations.

Fig. 2

Features of wild type and GMR.wg pupal eyes. (A–C) The location of the nuclei in a 32 h APF wild type eye. (A) The cone cells are apical and express Cut (blue) and Pax2 (red). (B) Below them lie the photoreceptors labeled with Elav (green) flanked by the 1° PCs expressing Pax2 (red). (C) Basally lie the 2°/3° PCs (unlabeled) and the bristle group cells. (D–F) Cone cells in 32 h APF GMR.wg eye. (D) The cone cells express Cut (blue), (E) esg.lacZ (red), (F) and wg.lacZ (blue). (G, H) 36 h APF GMR.wg eye. (G) The cone cells fall to the photoreceptor layer. (H) The 1° PCs move more basally. (I, J) 42 h APF GMR.wg eye. (I) Death (Caspase) begins in the cone cells, followed by (J) the photoreceptors and 1° PCS. (K) Summary diagram.

Fig. 3

Features of pros-arm* eyes. (A) pros.Gal4 is expressed in the cone cells (GFP-blue). (B–L) Features of pros-arm* eyes. (B) All cone cells express esg.lacZ (red) and (C) Wg (green) at 32 h APF. (D) At 36 h APF the cone cell nuclei fall to the photoreceptor layer. (E–G) Images of 42 h APF retinas. (E) Wg expression (green) persists in the cone cells. (F) D/E-Cadherin staining (red) highlights the apical junctions and the profiles of the four cone cells (asterisks) are stained indicating intact adherens junctions. (G) Caspase staining (red) indicates a broad swath of apoptosis at the periphery (white bar). (H–L) Phase contrast images through pros-arm* eyes. (H) An expanded pigment rim (white bar) is evident. (I) A longitudinal section shows many photoreceptors (arrows) below the basal lamina of the retina. (J) Lens units are disrupted and lack 1° PCs. Inset shows a similar image from the posterior region of the same eye where 1° PCs are present (arrow). (K) The photoreceptor array is disrupted (asterisks indicate ommatidia completely or partially lacking photoreceptors). (L) A longitudinal section showing the continuous sheet of lens-like material overlying the lens units (arrow), and arrowhead indicates the protrusion into a pseudocone.

Fig. 4

The effects of wgRNAi. (A–C) Demonstrations of the efficacy of wg RNAi. (A) Shows a longitudinal section through a GMR.wg eye in which the entire retina appears as pigment rim. (B) When wg RNAi is simultaneously expressed (GMR.Gal4; UAS.wg/UAS.wgRNAi) there is a rescue of the eye back to wild type structure. (C) Image of a Wg-stained (green) 32 h APF pros.Gal4 UAS.arm* UAS.wg-RNAi, showing dramatically reduced Wg expression (compare with Fig. 3C). (D–F) Images of adult pros.Gal4 UAS.arm* UAS.wg-RNAi eyes. (D) The most peripheral ommatidia survive (black circles) bearing disrupted photoreceptor arrays. (E) The lenses are disrupted but 1 °PCs are present (arrow). (F) The photoreceptors array is disrupted, but individual cells appear healthy.

Fig. 5

The effects of esgRNAi, wgRNAi and ectopic Esg expression. (A–B) Confocal images of a 42 h APF pros-arm* UAS.esgRNAi eye periphery. (A) The cone cell array (Cut-blue) is normal with no evidence of cell death. (B) Below the cone cell layer, multiple rows (white bar) of dying photoreceptors (Caspase-orange) are evident. (C–F) Phase contrast cross-sectional images through pros.Gal4 UAS.arm* UAS.esg-RNAi eyes. (C) Peripheral ommatidia are present lacking photoreceptors (asterisks) (D) Normal lenses with 1° PCs (arrow) are present. (E) Apically many ommatidia contain rhabdomere-like material (black arrows). (F) Deeper down this is no longer evident. Green and orange circles indicate ommatidia with healthy photoreceptors. (G) Staining of a 42 h APF pros-arm* UAS.esgRNAi eye showing persistent Wg expression (green) in the cone cells. (H–J) Phase contrast images through pros.Gal4 UAS.arm* UAS.esg-RNAi UAS.wg-RNAi eyes. (H) Wild type lens units, with 1° PCs (arrow). (I) Surviving peripheral ommatidia are present (white circles). (J) A normal photoreceptor array is formed. (K) Phase contrast image of a 28 h APF pros.Gal4 UAS.esg eye showing the cone cell nuclei (blue) prematurely fallen to the level of the photoreceptors (green).

Fig. 6

Wg from different sources triggers ommatidial death. (A–C) Phase contrast images through dorsal tub > stop > wg; GMR.flip eyes. (A) Normal lenses are formed. (B) Healthy photoreceptors at the R7 level (the enlargement of DRO R7 rhabdomeres is present but difficult to observe). (C) The R8s show a mixture of DRO (purple arrows pointing to R8s with large rhabdomeres) and wild type ommatidia (white arrows). (D) When pros-arm* is introduced, a pigment rim-like retina results. (E–G) Schematic summary of the effects of Wg secreted from different sources. The image shows the effects on the dorsal eye, and the indication that ommatidia are specified as DRO before they are undergo apoptosis is inferred from previous work (Tomlinson, 2003).

Fig. 7

Activation of the Wg pathway in the photoreceptors. (A, B) Confocal images through a 32 h APF Elav-Gal4; UAS.GFP retina. (A) Staining is seen in the photoreceptor nuclei, but clear weaker staining is evident in the 1° PCs (white arrows) and (B) 2°/3° PCs. (C, D) Sections though an adult APC^Q8 retina. (C) Normal lenses form containing 1° PCs (arrow). (D) In the main retina the 2°/3° PCs lattice lacks photoreceptors. (E) Confocal image through pupal 32 h APF APC^Q8 retina shows R7/8s expressing Hth (purple). (F–H) Confocal images of a 42 h APF APC^Q8 retina. (F) A normal cone cell array is evident (Cut-blue). (G) The photoreceptors (Elav-green) express (H) high levels of Caspase (red).