Iroquois complex genes induce co-expression of rhodopsins in Drosophila

Abstract

The Drosophila eye is a mosaic that results from the stochastic distribution of two ommatidial subtypes. Pale and yellow ommatidia can be distinguished by the expression of distinct rhodopsins and other pigments in their inner photoreceptors (R7 and R8), which are implicated in color vision. The pale subtype contains ultraviolet (UV)-absorbing Rh3 in R7 and blue-absorbing Rh5 in R8. The yellow subtype contains UV-absorbing Rh4 in R7 and green-absorbing Rh6 in R8. The exclusive expression of one rhodopsin per photoreceptor is a widespread phenomenon, although exceptions exist. The mechanisms leading to the exclusive expression or to co-expression of sensory receptors are currently not known. We describe a new class of ommatidia that co-express rh3 and rh4 in R7, but maintain normal exclusion between rh5 and rh6 in R8. These ommatidia, which are localized in the dorsal eye, result from the expansion of rh3 into the yellow-R7 subtype. Genes from the Iroquois Complex (Iro-C) are necessary and sufficient to induce co-expression in yR7. Iro-C genes allow photoreceptors to break the "one receptor-one neuron" rule, leading to a novel subtype of broad-spectrum UV- and green-sensitive ommatidia.

Figure 1. R7 Cells in the Dorsal Eye Co-Express rh3 and rh4

(A and B) Optical sections through a control eye stained for Rh3 (cyan) and Rh4 (red). Dorsal to the top. (A) R7 level: In ventral regions, R7 contain only Rh3 or Rh4. Dorsal R7 cells contain only Rh3, or Rh3 plus Rh4 (marked by dotted line). (B) Detailed view of the mid-dorsal region. The most dorsal yR7 cells co-express Rh3 and Rh4, while more ventral yR7 cells only express Rh4. (C) Transverse cryosection of a control eye stained for Rh3 (cyan) and Rh4 (red). Dorsal ommatidia clearly co-express Rh3 and Rh4. DRA ommatidia express Rh3 in both R7 and R8. D=dorsal, V=ventral. (D) In situ hybridization on a transverse section of a control eye with fluorescent probes for rh3 (green) and rh4 (red) mRNA. Co-expression of rh3 and rh4 mRNA is observed is observed in the dorsal retina. D = dorsal, V = ventral.

Figure 2. rh3 and rh4 Are Co-Expressed in yR7 Cells

(A–D) Antibody staining of wild-type dissociated ommatidia. (A) DRA subtype: Rh3 (blue) is present in both R7 and R8. (B) p subtype: Pairing between Rh3 (blue) in R7 and Rh5 (yellow) in R8 is observed. (C) y subtype: Pairing between Rh4 (red) in R7 and Rh6 (green) in R8 is observed. (D) In the “dorsal” y subtype, Rh3 (blue) and Rh4 (red) are present in the same (pink) R7 and they are coupled with Rh6 (green) in R8. (E) Visualization of the “yellow” pigment (green) and rh3>RFP (red) using cornea neutralization technique of dorsal and ventral regions of the eye. The “yellow” pigment can be visualized due to its inherent fluorescence in the green channel. Arrowheads point to yR7 rhabdomeres. Note that, contrary to “yellow,” RFP is not restricted to the rhabdomere, allowing the whole cell to be visualized. Residual signal in pigment cells surrounding each ommatidium is also observed in the green channel.

Figure 3. Iro-C Is Expressed in R7 Cells That Co-Express Rh3 and Rh4

(A) Larval eye imaginal disc stained for Iro-C>GFP (green). Iro-C is expressed in the dorsal half of the eye disc (top), whereas photoreceptor development is indicated by the neural marker ElaV (red). (B) Pupal eye (∼24 h APF): Iro-C>GFP (green) is expressed as a gradient in the dorsal (top) eye. The neural marker ElaV (blue) and the R7 marker Pros (red) are also shown. (C) Close up of the region of the eye where Iro-C>GFP expression (green) fades and can only be seen in R7 (marked by Pros (red)). (D) Iro-C-nuZ (green) expression in the adult eye. Expression in the dorsal eye (top) co-localizes with ElaV (blue) and Pros (red) in R7. Arrowheads indicate some R7 cell nuclei. (E) Iro-C-nuZ (green) is expressed in the adult eye in the dorsal region (left) where Rh3 (blue) and Rh4 (red) are co-expressed in R7 (arrowheads). (F) Iro-C-nuZ (green) expression with R8 specific markers Rh6 (red) and Rh5 (blue). (G) Iro-C mutant clones (marked by the absence of β-Gal; green) in the dorsal region of the eye stained with Rh3 (blue) and Rh4 (red). Mutant R7 do not co-express Rh3 and Rh4. Outside of the clone, all R7 cells that express Rh4 also express Rh3. White arrows mark Iro-C mutant R7 (β-Gal negative) that express only Rh4. Dorsal to the top.

Figure 4. Over-Expression of caup Induces rh3 and rh4 Co-Expression

(A) Optical section through the center (close to the equator, where there is no co-expression in control eyes, see Figure1) of eyes expressing caup in all photoreceptors under the lGMR promoter (lGMR>caup) and stained for Rh3 (cyan) and Rh4 (red). All yR7 cells containing Rh4 also contain Rh3 (white). pR7 cells only express Rh3 (cyan). (B) The same genotype stained for Rh5 (blue) and Rh6 (green) shows few R8 cells that contain Rh5; those are more frequent in ventral regions of the eye. (C) Similar staining as in (A) but caup is expressed in all R7 cells late during pupation (PanR7>caup). As in (A), most R7 cells that contain Rh4 also contain Rh3. pR7 only contain Rh3. (D) In the R8 layer, Rh5 (blue) and Rh6 (green) show a normal ratio. (E) Optical section through the center (equatorial) of the retina that expresses caup late during pupation only in yR7 (rh4>caup). Most yR7 cells contain both Rh3 and Rh4. Arrows indicate nontransformed ommatidia close to the equator. (F) In the R8 layer, Rh5 (blue) and Rh6 (green) show a normal ratio.

Figure 5. Generation of Retinal Subtypes

(A) During development, the first cell-fate decision divides photoreceptors into two different subtypes, inner (R7 and R8) and outer (R1–R6). This separation is mediated by the spalt (sal) genes in inner photoreceptors. After the acquisition of a generic ‘inner' fate, prospero (pros) is expressed in R7 and directs cells towards the R7 fate. senseless (sens) plays a parallel role in R8 cells and directs cells towards the R8 fate. The dorsal most row of ommatidia is then specified as DRA by the expression of homothorax (hth) and becomes a polarized light detector. The main part of the retina is then patterned into the y and p subtypes. orthodenticle (otd) is expressed in all photoreceptors and is required for the direct activation of rh3 and rh5 in the p subtype. Expression of spineless (ss) in a subset of R7 cells then generates the y subtype. Iro-C genes ara and caup then act in yR7 cells and allow the co-expression of rh3 and rh4. (B) Distribution of ommatidial subtypes along the dorso-ventral axis of the retina. DRA photoreceptors are located most dorsally. The dorsal eye contains either pure p or dorsal y ommatidia generated in response to Iro-C that extends through the dorsal eye. The central and ventral regions of the eye contain true p and y ommatidia.