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. 2013 Sep 15;381(2):482-90.
doi: 10.1016/j.ydbio.2013.07.001. Epub 2013 Jul 11.

The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes

Hui-Yi Hsiao  1 , David Jukam, Robert Johnston, Claude Desplan
Affiliations

The neuronal transcription factor erect wing regulates specification and maintenance of Drosophila R8 photoreceptor subtypes

Hui-Yi Hsiao et al. Dev Biol. .

Abstract

Signaling pathways are often re-used during development in surprisingly different ways. The Hippo tumor suppressor pathway is best understood for its role in the control of growth. The pathway is also used in a very different context, in the Drosophila eye for the robust specification of R8 photoreceptor neuron subtypes, which complete their terminal differentiation by expressing light-sensing Rhodopsin (Rh) proteins. A double negative feedback loop between the Warts kinase of the Hippo pathway and the PH-domain growth regulator Melted regulates the choice between 'pale' R8 (pR8) fate defined by Rh5 expression and 'yellow' R8 (yR8) fate characterized by Rh6 expression. Here, we show that the gene encoding the homolog of human Nuclear respiratory factor 1, erect wing (ewg), is autonomously required to inhibit warts expression and to promote melted expression to specify pR8 subtype fate and induce Rh5. ewg mutants express Rh6 in most R8s due to ectopic warts expression. Further, ewg is continuously required to maintain repression of Rh6 in pR8s in aging flies. Our work shows that Ewg is a critical factor for the stable down-regulation of Hippo pathway activity to determine neuronal subtype fates. Neural-enriched factors, such as Ewg, may generally contribute to the contextual re-use of signaling pathways in post-mitotic neurons.

Keywords: Drosophila; Ewg; Hippo pathway; Photoreceptors; R8; Retina; Rhodopsin.

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Figures

Fig. 1
Fig. 1. Rh5 is expressed in fewer R8 cells in ewg mutants
(A) Two subtypes of ommatidia: pale ommatidia have Rh3 in R7 paired with Rh5 in R8 whereas yellow ommatidia contain Rh4 in R7 and Rh6 in R8. Outer photoreceptors (R1–R6) all express Rh1. (B) Model showing how R7 is specified into pale and yellow R7s, and how the Hippo pathway and Melted regulate R8 subtype specification. Model modified from (Jukam and Desplan, 2011). (C–G) Confocal images of adult retina showing antibody staining of Rh5 (blue) and Rh6 (red) to mark the two R8 subtypes: (C) y1w67 retinas are used as wild-type controls with a ratio of Rh5 to Rh6 of 35:65. (D) ewgl1 mutant retina showing a lower number of Rh5 expressing R8. The ratio of Rh5:Rh6 is 10:90. (E) Using elav-Gal4 to drive expression of the ewg-cDNA in ewgl1 mutant restores the ratio of Rh5:Rh6 to wild type. (F) Overexpression of Ewg using senseless-Gal4 shows no significant changes in the Rh5:Rh6 ratio. (G) panR8>ewg-cDNA retinas have a wild-type Rh5:Rh6 ratio. (H) Quantification of Rh5 and Rh6 in ewg mutants. The graph shows the percentage of Rh5 (blue) and Rh6 (red) in R8. An unpaired t-test was performed to calculate the difference in mean % Rh5. Wild-type: n=10 retinas, N=3675 ommatidia; ewgl1 mutant: n=11 retinas, N=3214 ommatidia; ewgl1, elav>ewg-cDNA: n=11, N=3321 ommatidia; all other genotypes throughout paper: n≥4, N≥ 800 ommatidia. *** p<0.001, error bars are mean ± one standard deviation (s.d.).
Fig. 2
Fig. 2. ewg is expressed in all photoreceptors and functions autonomously to specify R8 subtypes
(A–E) Ewg (red) is co-expressed with Elav (green), a neuronal marker, in all photoreceptors starting from 3rd instar larval stage and remains in pupal and adult retinas. (A'–E') Ewg (red) channel only. (A) y1w67eye imaginal disc at the 3rd instar stage. Ewg is co-expressed with Elav only in differentiated photoreceptors. Expression of Ewg remains at 0% (B), 50% (C), 75% pupation (D) and throughout adult stages (E). Ewg is visible both in nuclei of outer and R7 photoreceptors (outer layer) as well as in R8 in the lower nuclear layer. (F) Most ewg R8 mutant cells (arrow) in clones marked by GFP (green) express Rh6 (red). (F') Red (Rh6) and blue (Rh5) channel only. Among 60 mutant R8s, 56 expressed Rh6. (G) One ewg mutant R8 (arrow) marked by GFP (green) expresses Rh5 (blue). (G') Red (Rh6) and blue (Rh5) channel only.
Fig. 3
Fig. 3. The Ewg activation domain is required for R8 subtype specification
(A) Schematic of ewg genomic organization and four isoforms of Ewg. Exons are indicated in blue. Modified from (Haussmann and Soller, 2010). (B) Expression of the ΔDJ isoform fails to restore the normal ratio of Rh5 (blue) to Rh6 (red). (C) Expression of the ΔD isoform restores a wild type Rh5:Rh6 ratio. (D) Expression of the ΔJ isoform is not able to rescue the ewg mutant phenotype. (E) Quantification of Rh5 and Rh6 rescue with different Ewg isoforms. An unpaired t-test was performed to calculate the difference in mean % Rh5. ewgl1; elav-ewg-cDNA, n=11 retinas, N=3321 ommatidia; ewgl1; elav-ΔDJ, n=5 retinas, N=1656 ommatidia; ewgl1; elav-ΔD, n=6 retinas, N=2096 ommatidia; ewgl1; elav-ΔJ, n=6 retinas, N= 2103 ommatidia. *** p<0.001, error bars are mean ± one standard deviation (s.d.)
Fig. 4
Fig. 4. ewg functions in R8 subtype specification and is required to maintain repression of Rh6 in pR8 photoreceptors
(A–C) Confocal images of 4 weeks old adult retina stained with antibodies against Rh5 (blue) and Rh6 (red) with zoomed-in images. (A) ewgl1 mutant retina shows expanded expression of Rh6 in all R8. Arrows point R8 cells co-expressing Rh5 and Rh6. (A') Rh6 channel only. (A”) Rh5 channel only. Note the co-expression of Rh5 and Rh6 in the bottom panels of A and A'. (B) A four week old wild-type retina shows no expansion of Rh6 expression, and maintains mutually exclusive Rh5 and Rh6 expression. (B') Rh6 channel only. (B”) Rh5 channel only. (C) ewgl1 mutant rescued with ewg cDNA shows no expansion of Rh6 in 4 weeks old flies. (C') Rh6 channel only. (C”) Rh5 channel only.
Fig. 5
Fig. 5. ewg is required to specify the pR8 subtype fate
In controls (A) warts-lacZ (antibody to β–galactosidase, blue) is expressed only in yR8s, marked by Rh6 (red) while pR8s are marked by Rh5 (green) while (B) melted-lacZ expression (blue) in pR8s (labeled with Rh5) is mutually exclusive with warts-lacZ expression in yR8s (marked by Rh6). (C–F) ewgl1 mutant retinas. (C) R8s co-express Rh6 and warts-lacZ. (C') warts-lacZ channel only. (D) Not all R8s expressing Rh5 contain melted-lacZ. The white arrow marks a photoreceptor expressing Rh5 that lacks melted-lacZ expression. (D') melted-lacZ channel only. (E–F) Two weeks old ewgl1 mutant flies. (E) Small amounts of Rh6 are co-expressed in R8s that express Rh5. In those R8s (white arrow), melted-lacZ expression is lost (E') melted-lacZ channel only. (F) warts-lacZ expands in R8s that are co-expressed with Rh5 and Rh6 (arrows). (F') warts-lacZ channel only.
Fig. 6
Fig. 6. ewg acts genetically upstream of warts and melted
(A–H) Confocal images of adult retinas stained with antibodies for Rh5 (blue) and Rh6 (red). (A) Over-expression of melted driven by IGMR-Gal4 results in Rh5 expressed in all R8s. (B) In ewgl1; IGMR>melted flies, overexpression of melted suppresses the ewgl1 phenotype, leading to Rh5 expression in all R8s. (C) In a warts mutant, all R8s are converted to pR8 expressing Rh5. (D) ewgl1; warts− double mutants show Rh5 expression in all R8s. (E) Overexpression of a dominant negative form of Merlin (merDN) in all PRs leads to expression of Rh5 in all R8s. (F) A merDN;ewg double mutants show Rh5 expression in all R8s. (G) Over-expression of yki driven by GMR causes Rh5 expression in all R8s. (H) Over-expression of yki in ewgl1mutants suppresses the ewg phenotype and leads to Rh5 expression in all R8s. (I) Model of ewg interaction with the Warts-Melted feedback loop. ewg acts upstream of melted to promote its expression, allowing expression of Rh5 in pR8s. ewg might also be required to repress the Hippo pathway, leading to Rh5 expression.
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