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First published online 12 October 2005
doi: 10.1242/dev.02082

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Restricted teashirt expression confers eye-specific responsiveness to Dpp and Wg signals during eye specification in Drosophila

¹ Centro Andaluz de Biología del Desarrollo, Universidad Pablo de Olavide-Consejo Superior de Investigaciones Científicas, Seville, 41013, Spain
² Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, 4150-180, Portugal

View larger version (85K): [in a new window]   Fig. 1. tsh expression in the eye disc starts during L2 and is restricted to the columnar ME. In all images, anterior is towards the left. (A) Dorsal view of a late ey-Z embryo. The eye disc primordium, visualized with an ey-Z reporter (anti-ß-galactosidase, red) does not express tsh (anti-Tsh, green). (B-E) vertical optical sections of L1 (B), L2 (C) and early L3 (D,E) eye discs; the PE is marked by an arrow. (D,E) Region where the retina is already differentiating is marked by the curved line. Merged images and separate channels are shown. Ey (anti-Ey, red) is expressed in both eye disc layers during the whole development of the disc (B,C,E). tsh, which is monitored by a tsh-Z reporter (anti-ß-galactosidase, green), is not expressed in L1 discs (B), but becomes upregulated in L2 discs in the columnar layer (C; arrowhead) and maintained there in early L3 discs (E). (D) Tsh expression overlaps Eya in the ME (anti-Eya, red). (F,G) Confocal images through the ME (F) or PE (G) layers of early L3 discs, stained for Hth (blue), Tsh (green) and Eya (red). (F) tsh is expressed only in the main epithelium (F), where it is co-expressed with Hth in an anterior subdomain (cyan) and with Eya in a posterior one (yellow-orange). In the scheme on the right, the domain of tsh expression is outlined in green, and the anterior and posterior subdomains are colored in cyan and orange, respectively. The approximate domains of wg and dpp expression at this stage are depicted as the blue and red bars, respectively. (G) In the PE of a similar stage disc, neither tsh nor eya is expressed. This is represented in the scheme on the right. (H) Idealized vertical cross-section through the line in F and G. The color codes as in F and G. pe, peripodial epithelium; me, main epithelium. (I) tsh-expressing clones in the disc margin or PE induce the columnarization of the cells. Tangential confocal section through a disc, showing a tsh+ clone (marked with anti-Tsh, green) in the dorsal PE. The membranes are labeled with Arm (red). tsh+ cells overproliferate and exhibit a columnar morphology, contrasting with the more squamous morphology of neighboring, Tsh non-expressing, cells. The broken line in the merged image delineates the tissue.

View larger version (68K): [in a new window]   Fig. 2. Overexpression of ey or toy is not capable of altering PE fate. Anterior is towards the left in all panels, and dorsal is upwards (except for z-sections in B and D). (A,B) tsh-expressing PE clones (arrows) upregulate Ey expression (A) and Eya (B). In some tsh-expressing clones, the upregulation of ey is not strictly cell-autonomous, as some cells adjacent to the expressing clone also increase Ey signal (A, arrowhead). (B) Confocal z-section through the white line is shown, demonstrating the PE location of the tsh+ clone (arrow), the cell-autonomous activation of Eya (green) and the repression of Hth (blue). Normal Eya expression is seen in the ME (me). (C) An ey-expressing clone in the peripodial epithelium (red), though, does not activate eya expression (green) or alter the morphology of the peripodial cells. (D,E) toy-expressing PE clones (toy+; arrowheads), marked with lacZ (blue). (D) toy-expressing PE cells (blue) upregulate Ey expression, while that of Eya remains absent. Separate channels of a z-section through the clone marked with the arrowhead are shown on the right. Ey and Eya are detected normally in the ME (me). The white line indicates the approximate location of the section. (E) In PE toy-expressing clones (arrowheads), Tsh is not upregulated.

View larger version (170K): [in a new window]   Fig. 3. tsh induces competence to respond to dpp and wg in peripodial cells. (A-C,F-H,J) PE clones. Anterior is towards the left, and dorsal upwards. (A) Clones expressing an activated tkv receptor (tkvQD+, red) do not activate Eya expression (green) or induce cell-morphological changes (arrows); in addition, Hth expression remains unchanged (blue). (B) Posterior tsh-expressing peripodial clones (Tsh+) induce Eya expression (arrows), but anterior ones do not (arrowheads). (C) In tsh-expressing clones simultaneously mutant for the dpp signal transducer Mad (Tsh+ Mad-; arrows) eya is never induced. (D,E) ME clones (arrows): (D) tkvQD-expressing clone (red) in anterior regions of the disc de-represses Eya (green; the coexpression is seen in yellow). These clones lose Hth expression (blue). (E) Conversely, a Mad– clone (marked by the absence of lacZ, in red) shows a strong reduction of Eya signal. (F) Axin-expressing clones (lacZ, red; arrows) grow normally in the PE, and do not affect Hth expression (green; overlap in yellow). (G) A tsh-expressing PE clone (tsh+; marked with Tsh in red; arrow) shows overgrowth, with more compact nuclei that strongly express Hth (overlap in yellow). (H) Cells in a clone coexpressing Axin and tsh (tsh+ Axin+, marked with Tsh in red; arrow) lose Hth expression and do not overproliferate. (I) An anterior ME Axin-expressing clone (lacZ in red; arrow) lying within the tsh domain (blue) downregulates Hth (green). Hth levels decrease towards the posterior of the clone, as Axin+ cells are farther away from the anterior wg expression domain. In these Axin+ clones, Tsh expression is maintained (co-expression seen in magenta). (J) PE clones expressing simultaneously tsh, Axin and tkvQD (marked with Tsh, red) activate Eya expression both in posterior (arrow) and in anterior (arrowheads) locations.

View larger version (105K): [in a new window]   Fig. 4. tsh expression induces ato. (A-C) tsh-GAL4; UAS-GFP L3 discs (tsh>GFP, red) stained with anti-Ato (green) and 22C10 (neural marker, blue). (A) In the eye disc, tsh>GFP expression extends up to the MF (arrowhead) and overlaps ato. Posterior to the MF, 22C10 marks differentiating photoreceptors. (B,C) tsh-GAL4; UAS-GFP is expressed in proximal domains in both leg (B) and wing (C) discs. (B) Leg disc showing Ato expression in a cluster of chordotonal organs (asterisk in B and E). (C) Wing disc showing a small cluster of Ato+ cells in the ventral anterior hinge region (asterisk). (D-F) In tsh-expressing clones (tsh+, red) some cells turn on Ato expression in the eye disc (D), leg (E) and wing (F) discs (arrows). The arrowheads in D indicate the ato expression at the endogenous MF.

View larger version (51K): [in a new window]   Fig. 5. Transient expression of tsh in the PE results in ectopic eye development. Anterior is towards the left in all images. Except for the cross-section in B, dorsal is upwards. (A,B) L3 eye disc of a hs-Gal4; UAS-tsh larvae, in which tsh was transiently induced during L2 through a heat-pulse, shows clusters of 22C10 (red) photoreceptors on the PE (marked by Hth nuclei, blue). (A) Focal plane through the PE showing the axons (white arrowhead) and cell bodies (white arrow); the ME photoreceptor cell bodies are also seen in this plane (orange arrow). (B) Confocal z-section through the same disc where the normal (orange) and the tsh-induced PE (white) photoreceptors (arrows) plus associated axons (arrowheads) can be distinctly seen. (C,E) Expression patterns of MD705-GAL4 (705>GFP) and MS-1096-GAL4 (1096>GFP) in late L3 eye discs. (C) MD705-GAL4 drives expression along the dorsoanterior margin and PE. (E) MS1096-GAL4 in both dorsal and ventral PE and margin of the eye disc, although more strongly ventrally. Fate maps (Haynie and Bryant, 1986) indicate that the dorsoanterior disc margin and PE give rise to dorsal anterior head capsule (including the frontal, vertical and post-vertical bristles) while anteroventral margin and PE develops into anteroventral head capsule (including the vibrissae, post-gena and lower post-occipital bristles). (D) MD705- and (F) MS1096-driven tsh expression results in PE photoreceptor differentiation (arrow). In 1096>Tsh discs, the region where these photoreceptors develop loses tsh and hth expression (close-up in Fi and Fii). (G) MS1096-driven tsh expression results in ventral ectopic eyes (arrow) that replace ventral head structures (loss of postgenal structures) and in small dorsal patches of photoreceptors normally accompanied by a bristle (arrowhead). (H) MS1096-driven expression of ey does not induce ectopic eyes in equivalent positions (arrow) in the head, although it does so in the wing hinge (J), where tsh is present (Bessa et al., 2002). (I) Expression of MS1096 detected by X-Gal histochemistry in MS1096-GAL4/UAS-lacZ adult heads. Signal is detected surrounding the eye in the dorsoanterior (arrowhead) and ventroanterior head capsule (arrow), in agreement with the fate map of the corresponding PE domains in eye discs. Bolwig's organ nerve (bn) runs along the peripodial layer of the eye disc and is detected by 22C10.

View larger version (90K): [in a new window]   Fig. 6. Reduction of tsh function results in small eyes. (A,B) Lateral views of (A) wild-type and (B) ey-GAL4; arm-GAL4/UAS-tshRNAi adult heads. (C-F) Clones expressing tshRNAi, positively marked with lacZ (blue) and Tsh (green). (C,D) Late L3 disc showing autonomous loss of Tsh-immunoreactivity (arrows). (E,F) Close up of a dorsal clone in which cells have different levels of Tsh immunoreactivity: none (arrow) or normal (arrowhead).