Stochastic spineless expression creates the retinal mosaic for colour vision

Abstract

Drosophila colour vision is achieved by R7 and R8 photoreceptor cells present in every ommatidium. The fly retina contains two types of ommatidia, called 'pale' and 'yellow', defined by different rhodopsin pairs expressed in R7 and R8 cells. Similar to the human cone photoreceptors, these ommatidial subtypes are distributed stochastically in the retina. The choice between pale versus yellow ommatidia is made in R7 cells, which then impose their fate onto R8. Here we report that the Drosophila dioxin receptor Spineless is both necessary and sufficient for the formation of the ommatidial mosaic. A short burst of spineless expression at mid-pupation in a large subset of R7 cells precedes rhodopsin expression. In spineless mutants, all R7 and most R8 cells adopt the pale fate, whereas overexpression of spineless is sufficient to induce the yellow R7 fate. Therefore, this study suggests that the entire retinal mosaic required for colour vision is defined by the stochastic expression of a single transcription factor, Spineless.

Figure 1. The yR7 subtype is lost in spineless mutants

a, Three subtypes can be identified on the basis of molecular markers: ‘pale’ (blue), ‘yellow’ (yellow) and DRA (pink) ommatidia together form the wild-type retinal mosaic (schematic representation;dorsal is to the top). b, Schematic representation of the ss phenotype in R7 cells. c, Transverse section through a wild-type (WT) adult eye (left panel; dorsal is to the left). The arrow denotes the DRA. Ratio of R7 opsins in a wild-type whole-mount adult retina (right panel; dorsal is to the top) stained for Rh3 (red) and Rh4 (cyan). d, Transverse section through a ss^D115.7 whole-mutant adult eye (left panel). Rh3 (red) is expanded and Rh4 (cyan) is completely lost. Opsin expression in a mutant whole-mount adult retina is also shown (right panel). e, Whole-mount retina with mitotic clones lacking Spineless (marked by the absence of expression of the armadillo-lacZ (Arm-Z) construct, blue). Rh4 (green) is always absent from mutant clones. Rh3 expression is shown in red. f, Whole-mount retina with MARCM clones lacking Spineless (marked by the presence of GFP, green). Every GFP-positive cell expresses Rh3 (red), whereas Rh4 (blue) is always absent from mutant cells.

Figure 2. R8 phenotype of spineless mutant eyes

a, Whole-mount retina from a ss mutant fly. The pR8 subtype (Rh5, blue) is expanded to almost all R8 cells (Rh6, green). b, Unusual mis-coupling of Rh3 (red) in R7 and Rh6 (green) in R8 of the same ommatidium is frequently observed in ss mutant retinas. Rh5 expression is shown in blue. c–f, Transverse sections stained for Rh5 (blue) and Rh6 (green): an adult wild-type eye (c), a ss mutant eye (d), a sev mutant eye (e), and a double-mutant (sev; ss) eye (f), which manifests the same R8 phenotype as sev mutants (expansion of yR8 cells and loss of pR8 cells).

Figure 3. spineless is sufficient to induce the yR7 fate

a, Summary of the ss gain-of-function phenotype. All R7 cells adopt the yR7 fate (yellow); the fate of DRA ommatidia is unclear (grey). b, Water immersion microscopy on living wild-type flies expressing the yR7-specific reporter Rh4-GFP. Expression is restricted to one inner PR in a large subset of ommatidia. c, Rh4-driven GFP expression is dramatically expanded in LGMR>ss flies, as visualized by water immersion. d, In sGMR>ss flies, Rh4 (cyan) is expanded through the whole ss-overexpressing retina (compare with Fig. 1c). e, Rh4 (cyan) is completely lost in sev mutants, although Rh3 (red) is present in DRA R8 cells (arrow). f, Overexpression of ss (sGMR>ss) leads to ectopic Rh4 (cyan) in sev mutants. Rh3 (red) is restricted to DRA R8 cells (arrow). g, In whole-mount retinas from sGMR>ss flies, variegated expression of ss leads to the expansion of Rh4 (cyan) into a variable number of PRs per ommatidium; coexpression with Rh3 (red) is never observed (arrows). h, In wild-type eyes, the outer-PR opsin Rh1 (red) and Rh4 (cyan) never co-localize in the same PR cell. i, Ectopic expression of ss in sGMR>ss flies leads to massive expansion of Rh4 (cyan), with some co-localization (arrows) with the outer-PR opsin Rh1 (red).

Figure 4. Control of PR cell fates by spineless

a, In whole-mount retinas from PanR7>ss flies, all R7 cells express Rh4 (cyan), whereas Rh3 (red) is absent (left panel). Late expression of ss in R7 has no effect on the ratio between Rh5 (blue) and Rh6 (green) expression (right panel). b, Section of a PanR7>ss eye. As the fate of R8 is not affected by late ss expression in R7 cells, there is a great number of ‘odd-coupled’ Rh4 (cyan)/Rh5 (blue) ommatidia. The arrow indicates the DRA. c, In whole-mount retinas from PanR7>ss flies on a ss mutant background, Rh3 (red) is not expressed, while every R7 cell expresses Rh4 (cyan) (left panel). Late overexpression of ss in all R7 cells on a ss mutant background does not lead to a reprogramming of R8 cells as most R8 cells express Rh5 (blue) and few dorsal R8 express Rh6 (green) (right panel).

Figure 5. Mosaic expression of spineless in the developing retina

a, In situ hybridization of a whole-mount pupal retina with an antisense ss probe (green) and an ELAV antibody (blue). ss expression can be observed in all bristle cells (asterisks), as well as in one PR (circles) per ommatidium in only 60–80% of all ommatidia. Note that ss levels vary from cell to cell. b, The PR cell positive for ss (green) is identified as an R7 cell by co-staining with the R7-specific marker Prospero (Pros, red). Neurons are marked with ELAV (blue). c, Nuclear β-gal (nβ-Gal, red) driven under the control of ss_eye-Gal4 reveals mosaic expression of ss in one cell per cluster. Neurons are marked with ELAV (blue). Dorsal is to the left in all panels.

Figure 6. Stochasticity of ss expression and a revised model for retinal patterning in Drosophila

a, b, The effect of a 30-min pulse of ss expression at ~50% pupation leads to an almost 100% transformation of R7 to the y fate (a), or almost every PR (b). c, Top: transient expression of ss_eye-Gal4 (red) during pupation before the onset of opsin expression (blue). Bottom: variable expression of ss (different tones of red) in R7 cells. d, Left: the ss data suggest that ~70% of the R7 cells get promoted into the yR7 fate (Rh4, yellow) by expressing ss. The pR7 subtype (Rh3, red) therefore represents the R7 ‘default state’. Right: in ss-positive yR7 cells, the ability to communicate with the underlying R8 is abolished, resulting in y ommatidia as the R8 default state is expression of Rh6 (green). Only pR7 retain the competence to instruct the pR8 fate (Rh5, blue).