end result of an activated rhodopsin is the same—a neural signal from the photoreceptor axon (invertebrates) or downstream neurons (retinal ganglion cells in vertebrates).

The fruit fly Drosophila melanogaster is an excellent model for studying color vision because it has a sophisticated “hardware” to detect colors (including four different color-sensitive opsins) and can functionally and behaviorally respond to colors [11, 12, S. Yamaguchi and C. Desplan, unpublished results]. Although distantly related, flies and vertebrate opsins share the same biophysical properties, and Drosophila’s genetic malleability has supported fundamental discoveries in eye development, photoreceptor differentiation, visual transduction, and visual information processing.

ANATOMY AND MOLECULAR ASPECTS OF COLOR-SENSITIVE OPSINS IN THE DROSOPHILA EYE

Structure of the Drosophila Eye: Ommatidia, Photoreceptors, and Rhodopsins

The Drosophila eye is organized into a latticed array of about 800 unit eyes, or ommatidia. Each ommatidium contains about 21 cells: 8 photoreceptors, 4 cone cells, a bristle cell, and about 8 accessory cells that help form the lens and pigment cells that shield photoreceptors (PRs) from light coming from other ommatidia [13]. The photoreceptors collect light in large, apical extensions of the membrane called rhabdomeres that contain densely packed rhodopsin molecules. The detection of light by rhodopsin in a photoreceptor initiates the signal phototransduction cascade that converts photon energy into an electrical impulse sent to higher-order neurons for visual information processing.

According to their morphology, axonal projections, and opsin expression, the eight adult photoreceptors can be grouped into two main functional categories (Fig. 1):

1.The “outer” photoreceptors R1–R6 are specialized in dim-light vision and motion detection. Outer photoreceptors capture photons with high efficiency due to their broad-spectrum rhodopsin Rh1 as well as the large diameter and length of their

Fig. 1. Organization of the Drosophila eye. A Whole-eye view of a retina. Note the precise lattice of ommatidia. B Electron micrograph of a cross section through an ommatidium. The outer photoreceptors (R1–R6) are arranged in a chiral trapezoid, the center of which is occupied by the inner photoreceptors (R7 and R8), with R7 positioned on top of R8. C Longitudinal view of a single ommatidium. Light enters through the lens, at the top, and passes through the eight photoreceptor rhabdomeres.

rhabdomeres, which extend from the basal to the apical side of the retina [14]. R1–R6 axons project to and terminate in the first optic ganglion, the lamina. They can be considered functionally analogous to vertebrate rods. These six outer photoreceptors are organized in a chiral trapezoid, which has a center that is occupied by the two “inner” photoreceptors, R7 and R8.

2.The “inner” photoreceptors R7 and R8 form the color detection system in the fly retina. The rhabdomeres of inner photoreceptors are in the same optical path, one beneath the other. R7 is located apically, closer to the lens, and expresses ultraviolet (UV)-sensitive Rh3 or Rh4. R8 is directly underneath R7 and expresses either blue-sensitive Rh5 or green-sensitive Rh6. R7 and R8 have thinner and shorter rhabdomeres than R1–R6, and their axons terminate in the second optic ganglion, the medulla [15]. Inner photoreceptors are similar to vertebrate cones in their role for color vision [16].

An additional layer of organization is imposed on the inner photoreceptors through the specific pairing of rhodopsins expressed in R7 and R8 (Fig. 2). R7 cells that express UV-sensitive Rh3 are always found in ommatidia with R8 cells that express the bluesensitive Rh5 [17, 18]. This Rh3/Rh5 rhodopsin expression in R7 and R8 photoreceptors defines the “pale” (p) ommatidial subtype. Conversely,R7 cells expressing a different UV-sensitive Rh4 are always found together with an R8 that expresses the green-sensitive Rh6, defining the “yellow” (y) ommatidial subtype [19, 20]. These two ommatidial subtypes are interspersed randomly within the fly retina [21], with 30% of the pale subtype and the remaining 70% of the yellow subtype. It is believed that the differences in rhodopsin between each subtype play a crucial role for the fly’s ability to discriminate between colors, with the p ommatidia discriminating among shorter wavelengths (UV to blue), and the y ommatidia discriminating longer wavelengths extending to the green. Thus, comparisons between wavelengths detected by R7 and R8 from the same ommatidium and comparisons among neighboring p and y R7/R8 subtypes are likely the first two steps in color vision processing.

A third class of photoreceptors is located in the dorsal-most row of ommatidia (dorsal rim area, DRA) near the head cuticle. In these ommatidia, R7 and R8 both express Rh3 [22], which makes them inappropriate for color vision. Instead, the DRA inner photoreceptors detect the vector of oscillation plane of polarized light for spatial navigation [23, 24]. A fourth class of ommatidia, which does not exist in Drosophila, is found in the male Musca domestica in a large region of the eye where R7 expresses a Rh1-like molecule and projects to the lamina part of the optic lobe, thus leading to ommatidia that have seven motion detection photoreceptors instead of six and no color vision in this part of the retina [20]. This was entitled the “love spot” because it is used by males to track females during in-flight mating behavior. Because the DRA and Musca love spot are not involved in color vision, they are not discussed further.

Molecular Genetics and Evolution of Rh5 and Rh6

The cloning of the gene encoding Rh5 [17, 18] provided the first opportunity to examine R8 opsin expression and to investigate color sensitivity in Drosophila. Introduction of either Rh5 or Rh6 into mutant strains that lack Rh1 expression is capable of restoring light response, indicating that these genes encode functional rhodopsins [25]. Flies expressing Rh5

Fig. 2. Retinal mosaic in Drosophila. A Two main ommatidial subtypes can be distinguished: “pale” (p) and “yellow” (y). The model for p/y specification includes two steps: First, R7 cells choose between p and y fates by the stochastic expression of a transcription factor. Consequently, R7 cells impose their subset choice onto the underlying R8 cells (instruction). B Dorsal rim area ommatidia are always found in one or two rows at the dorsal periphery of the adult retina, whereas p (Rh3, Rh5) and y (Rh4, Rh6) ommatidia are distributed randomly through the retina in a 30:70 ratio. eq equator.

show peak sensitivity to wavelengths of 437 nm, corresponding to blue light, whereas Rh6 flies have a prominent peak at 508 nm, or green light (Table 1 [25]).

The Rh5 protein shares several structural features with other insect opsins, such as the seven-transmembrane domains, an N-glycosylation site required for protein maturation [26], two cysteine residues that form a disulfide bond, and serine and threonine phosphorylation sites in the cytoplasmic C-terminal domain. In addition, Rh5 contains a tyrosine that is thought to serve as a counterion for the protonated Schiff base in the retinal chromophore and may affect light sensitivity.