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.
Most sensory systems follow the rule “one receptor molecule per receptor cell.” For example, photoreceptors in the fly eye and cones in the human eye each express only one light-sensitive rhodopsin. Rhodopsins are G-coupled protein receptors, a class of ancient signaling molecules that mediate not just vision but also the sense of smell, the inflammatory response, and other physiological processes. However, the mechanisms that regulate mutual exclusion of receptor genes in the visual and olfactory systems are poorly understood. Each ommatidium in the fly eye consists of eight photoreceptors (R1–R8); six of which mediate broad-spectrum motion vision (R1–R6) and two that mediate color vision (R7 and R8). We identified a new class of photoreceptors in the fly retina that violates the one rhodopsin–one receptor rule. This subset of ommatidia, located in the dorsal third of the eye, co-expresses two ultraviolet-sensitive rhodospins (rh3 and rh4) in R7, while maintaining discrimination between green and blue opsins in R8. We took advantage of the genetic tools offered by the fruit fly to show that this co-expression depends on the Iroquois Complex (Iro-C) genes that are both necessary and sufficient to allow the two ultraviolet-sensitive rhosopsins to be expressed in the same R7 cell. These results shed new light on the mechanisms regulating co-expression of rhodopsins in the eye, and may well have implications for regulating co-expression in olfactory receptors and other G-protein coupled systems.
Citation: Mazzoni EO, Celik A, Wernet MF, Vasiliauskas D, Johnston RJ, Cook TA, et al. (2008) Iroquois Complex Genes Induce Co-Expression of rhodopsins in Drosophila. PLoS Biol 6(4): e97. doi:10.1371/journal.pbio.0060097
Academic Editor: Don Ready, Purdue University, United States of America
Received: October 4, 2007; Accepted: March 6, 2008; Published: April 22, 2008
Copyright: © 2008 Mazzoni et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: EOM was supported by an NYU Dean's Dissertation Award. This work was supported by the National Institutes of Health (NIH) grant R01 EY13010 to CD, and investigations were conducted in a facility constructed with support from Research Facilities Improvement #C06 RR-15518–01 from the National Center for Research Resources, NIH.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: PBS, phosphate buffered saline; DRA, dorsal rim area; lGMR , long glass multiple reporter ; p, pale type; Rh, rhodopsin; RT, room temperature; UV, ultraviolet; y, yellow type
The primary role of sensory organs is to probe the environment and to transmit precisely this information to the brain for processing. The visual and olfactory systems are composed of sensory epithelia with thousands of sensory receptor cells, each specifically expressing a single sensory receptor gene out of a much larger repertoire [1–6]. This “one receptor–one neuron” rule allows specific detection of sensory information at the periphery. Together, the architecture of the visual or olfactory organs, the correct specification of the sensory neurons, and the expression of specific sensory receptor molecules are crucial for the acquisition of sensory information. Sensory organs have thus adapted for optimal detection of specific stimuli and often exhibit spatial regionalization within the sensory organ itself. This regionalization also extends into topographic maps in the brain (retinotopy of the visual system, chemotopy in the olfactory system) .
The Drosophila compound eye is composed of approximately 750 simple eyes called ommatidia. Each ommatidium contains eight photoreceptor cells named R1–R8. The light-gathering structures (rhabdomeres) of outer photoreceptors (R1–R6) form an asymmetric trapezoid whose center is occupied by the rhabdomeres of the inner photoreceptors, where the R7 rhabdomere sits on top of that of R8 . The last step in photoreceptor differentiation is the selective expression of one of the photosensitive pigments, the rhodopsins. The expression of a given rhodopsin, along with additional filtering or sensitizing pigments, dictates the color sensitivity of a photoreceptor. Five rhodopsins are expressed in the compound eye. They respect the general rule of “one receptor–one neuron”— R1–R6 cells express Rh1 . The rhodopsins are similar in function to the vertebrate rods in that they are sensitive to a broad range of wavelengths. They are involved in motion detection. Inner photoreceptors (R7 and R8) mediate color vision [9,10], and are thus comparable to vertebrate cones [11,12]. These photoreceptors express the remaining four rhodopsins, which have a restricted spectrum of absorption ranging from ultraviolet (UV) in R7 to blue or green in R8 [1,3,4,13–15].
Although the eye appears to be composed of morphologically identical ommatidia, the main part of the retina consists of a mosaic of two stochastically distributed subtypes of ommatidia: pale type (p) contains a UV-absorbing Rh3 in R7 and a blue-absorbing Rh5 in R8; yellow type (y) contains a different UV-absorbing Rh4 in R7 and green-absorbing Rh6 in R8 [1,14,16]. A filtering pigment, “yellow” sharpens the sensitivity of yR7 and filters out the blue light reaching the green-sensitive underlying yR8 [17,18]. y ommatidia represent ~70% of ommatidia in flies ranging from Musca to Drosophila. These ommatidia can now be defined more accurately based on their Rh content. The Drosophila homolog of the vertebrate dioxin receptor spineless (ss) is responsible for the specification of the retinal mosaic . ss expression in ~70% of R7 cells in pupae commits them to the yR7 fate and to express rh4. The cells that do not express ss become pR7, express rh3, and instruct pR8 to express rh5. By default, the remaining yR8 express rh6 [1,20]. Thus, 30% of ommatidia (p) appear to be more involved in the discrimination of shorter wavelengths, whereas the remaining 70% (y) should be more appropriate for the discrimination of longer wavelengths.
The p and y ommatidia appear to be randomly distributed. The Drosophila eye, like in many insects, has also developed a particularly striking example of sensory system specialization in the dorsal rim area (DRA). DRA ommatidia develop in the dorsal-most row of the eye and have distinct morphological characteristics that enable them to be used to detect the electric vector (e-vector) of light polarization [21,22]. Because polarized light comes from UV-rich sunlight scattered by the atmosphere, this row of ommatidia is limited to the dorsal edge of the eye and must therefore be specified by positional cues [22,23].
Regionalization of tissues often starts very early during organogenesis and often involves conserved molecular mechanisms that are important for patterning tissues as different as Drosophila sensory systems or vertebrate limb buds. In the Drosophila eye imaginal disc, dorso-ventral compartmentalization involves the differential expression of genes of the Iroquois Complex (Iro-C). Iro-C genes encode conserved homeodomain transcription factors from the TALE class —araucan (ara), caupolican (caup), and mirror (mirr)—and their genomic organization as a cluster of three genes is conserved from flies to mammals [25,26]. In Drosophila, ara and caup have almost identical patterns of expression , whereas mirr is more divergent. Among other functions, these three genes have been implicated in very early stages of eye-antennal disc development as “dorsal selectors” that are required for the correct specification of dorsal head structures and for the formation of the dorsal compartment of the eye [28–30]. During larval development, the Iro-C genes are expressed in dorsal nondifferentiated cells of the eye imaginal disc and are then down-regulated once neurogenesis has begun. This expression distinguishes different cell fates on either side of the dorso-ventral boundary and is necessary to establish the organizer center at the equator (reviewed in ). Although expression of Iro-C genes fades away after the morphogenetic furrow, their expression reappears in the adult. Iro-C genes are necessary to specify the DRA: ommatidia near the edge of the disc are exposed to wingless signaling and become DRA ommatidia only when they are located dorsally [22,23].
Here we describe a new function for Iro-C genes in photoreceptor development: they define a subtype of ommatidia that is restricted to the dorsal region of the eye in which the “one receptor–one neuron” rule is broken. These ommatidia are positioned in the dorsal part of the retina and co-express the two genes encoding UV-absorbing Rhs—rh3 and rh4—in R7 cells. This co-expression results from the induction of rh3 in yR7 cells while pR7 are normal. Therefore, the mutual exclusion pathway that prevents co-expression of sensory receptors appears to be disabled by the activity of the Iro-C genes, allowing the expression of two sensory receptors in a single cell.
A Novel Class of Dorsal Ommatidia That Co-Expresses rh3 and rh4 in R7
It is widely accepted that individual Drosophila photoreceptors express a single rhodopsin gene: rh1 in R1–R6, rh3 or rh4 in R7 [15,31] (Figure 1A), and rh5 or rh6 in R8 [1,3]. However, careful examination of antibody stainings on whole-mounted retinas revealed a surprising exception to this rule: a fraction of R7 cells co-expresses both rh3 and rh4 (Figure 1A and 1B) in a region that starts near the dorsal edge of the eye, outside the DRA, and extends toward the equator, spanning approximately one-third of the eye at its maximum point (Figure 1A). This phenomenon is also clearly observed in cross-sections of the eye (Figure 1C). In the ventral region of the eye, Rh3 and Rh4 proteins are present at a high level in R7 cells and are never found in the same cell (Figure 1C, “V”). In contrast, all R7 cells located in the dorsal eye contain Rh3, either alone or in combination with Rh4 (p and y subtypes, respectively, see below) (Figure 1C, “D”). In R7 cells co-expressing rh3 and rh4, the level of Rh3 protein is lower than in non–co-expressing cells (Figure 1A and 1B). Together, these data suggest that a subset of dorsal ommatidia induce rh3 expression in rh4-expressing yR7 cells (Figure 1B). Rh3 and Rh4 colocalization was observed using different combinations of primary antibodies, indicating that this is not an artifact of a particular pair of antibodies (unpublished data), and co-expression is present in all wild-type backgrounds tested to date (yw and all other Gal4 and upstream activating sequence (UAS) lines used in this study), suggesting that this is a conserved feature of the Drosophila eye.
(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.
In our previous studies, we had detected expression of an rh3 promoter fusion to a green fluorescent protein (GFP) reporter  in most ommatidia located in the dorsal eye. To distinguish whether the mutual exclusion or co-expression of rhodopsins in one cell results from transcriptional or post-transcriptional regulation, we performed double in situ hybridization to visualize rh3 and rh4 mRNA. In the ventral and central regions of the eye, rh3 and rh4 mRNA are present at high levels in a mutually exclusive manner (Figure 1D, “V”). However, in the dorsal eye, all R7 cells contain rh3 mRNA, either alone or in combination with rh4 mRNA (Figure 1D, “D”). Moreover, staining of rh3-lacZ reporter constructs consistently reveals expanded, weak rh3 transcription in all ommatidia in the dorsal eye, whereas restricted expression to p ommatidia is observed in the remaining part of the retina (unpublished data and ). Together, these data indicate that there is localized transcriptional control of rh3 and rh4 that allows their co-expression in the dorsal retina.
We quantified the frequency of R7 cells co-expressing UV-opsins. In line with previous observations, antibody stainings on dissociated ommatidia identified the three previously described subtypes of ommatidia [1,3]: DRA ommatidia that contain Rh3 in both R7 and R8 (Figure 2A), p ommatidia that contain Rh3 and Rh5 (Figure 2B), and y ommatidia that contain Rh4 and Rh6 (Figure 2C) . In addition, a small proportion (5.7%; 6/106) of all ommatidia (dorsal or ventral) express Rh3 in R7 (without Rh4) associated with Rh6 in R8. These likely correspond to the previously described rare Rh3/Rh6 “odd coupled” ommatidia where the signal from pR7 fails to induce rh5 in R8 (unpublished data and [19,20,22]). However, we also identified a fourth subtype of R7 cells that contain both Rh3 and Rh4 in R7 cells (Figure 2D). These represent ~10% of all ommatidia and are always coupled with Rh6-expressing R8 cells (Figure 2D). Stainings with anti-Rh4, anti-Rh5, and anti-Rh6 antibodies never revealed expression of Rh4 and Rh5 in the same ommatidium (0/200). yR7 cells contain a pigment that gave rise to their name (“yellow”) that is visible under the confocal microscope after neutralization of the cornea . To further confirm that these co-expressing cells are yR7, we imaged the eyes of flies by confocal microscopy to visualize the “yellow” pigment as well as red fluorescent protein (RFP) controlled by the rh3 promoter (rh3>RFP). As expected, “yellow” and rh3>RFP do not overlap in the ventral eye, because “yellow” marks yR7 cells and rh3>RFP labels pR7 cells. However, in the dorsal eye, “yellow” overlaps with rh3>RFP (Figure 2E).
(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.
We have thus identified a class of dorsal ommatidia that express both rh3 and rh4 in R7, and rh6 in R8. These ommatidia represent a subset of y ommatidia that also express rh3 in addition to the endogenous rh4. Ommatidia containing Rh3/Rh5 make up ~30% of all ommatidia as evaluated by quantification of dissociated ommatidia (there is no Rh4/Rh5 coupling, and Rh5-positive ommatidia represent ~30% [178/636] of all ommatidia). The remaining ~70% of ommatidia express rh6 (458/636). Stainings with anti-Rh3 and anti-Rh4 antibodies revealed that ~30% of R7 express only rh3 (85/273), ~60% express only rh4 (158/273), and ~10% co-express rh3 and rh4 (30/273). Thus, the ~70% rh6-expressing ommatidia can be divided into two subtypes: ~60% of all ommatidia express rh4/rh6 and ~10% express (rh3 + rh4)/rh6, representing y ommatidia in the dorsal region of the eye.
Genes of the Iro-C Complex Are Expressed Dorsally in the Retina
Iro-C genes control dorsal identity during early eye development; therefore, we analyzed their expression in an effort to identify the determinants of this “dorsal” identity [28,30]. As mentioned earlier, these genes are expressed transiently during early larval stages of eye disc development (Figure 3A). ara and caup (but not mirr) are re-expressed in the adult in the dorsal retina. To perform a more detailed analysis of the Iro-C gene expression pattern, we used reporter lines (Iro-C-nuZ or Iro-C-Gal4), that are insertions in the Iro-C complex and are believed to reflect the expression of both ara and caup [21,27]. At 24 h after puparium formation (APF), the Iro-C-Gal4 reporter is highly expressed in all photoreceptors in the dorsal eye (Figure 3B). The level of expression gradually decreases toward the equator due to fewer and fewer cells per cluster that express the reporter. Ultimately, only R7 cells, identified with the R7-specific marker Prospero (Pros), express the reporter (Figure 3C). In the adult, the expression of the reporter persists in outer photoreceptors, as well as in R7 and R8 as previously shown . This expression pattern correlates with the distribution of y ommatidia that co-express rh3 and rh4 in R7 and express rh6 in R8 (Figure 3E and 3F) (see below for discussion). Thus, in the adult retina, the Iro-C genes ara and caup are specifically expressed in the region of the eye where there is co-expression of rhodopsins.
(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.
Iro-C Genes Are Necessary for Co-Expression of rh3 and rh4 in R7
The similarity between the expression profile of the Iro-C genes ara and caup in the region of the eye where rh3 and rh4 are co-expressed suggested that these transcription factors regulate this newly defined subset of ommatidia. To test this hypothesis, we induced clones of cells that were mutant for Iro-C by using a deficiency that covers ara and caup and deletes most of the regulatory sequences of mirr [27,34]. Ventral clones are easily recovered but, as expected, they do not have a visible phenotype. While small dorsal clones do not produce a strong morphological phenotype, large clones often lead to the formation of ectopic eye tissue near the dorsal head cuticle, presumably because they create a new organizer between Iro-C + and Iro-C – cells (unpublished data)[28,30]. In the few dorsal mutant clones recovered, R7 cells co-express rh3 and rh4 in the surrounding heterozygous tissue, whereas in mutant tissue, R7 cells contain only Rh3 or Rh4 (Figure 3G). Thus, similar to the adult ventral eye where Iro-C is not expressed, dorsal Iro-C mutant R7 cells exclusively contain either Rh3 or Rh4. Therefore, Iro-C expression in the dorsal eye appears to be required for rhodopsin co-expression in R7 cells of dorsal y ommatidia.
To study whether the ara and caup genes are sufficient to induce rhodopsin co-expression, we performed a series of mis-expression experiments. We observed essentially the same phenotype when over-expressing ara and/or caup, with the only difference being that the over-expression of both genes produces a more severe morphological phenotype than the expression of either one of them alone. We only show experiments using UAS-caup, but the same set of data is presented for ara in Figure S1. Because mirr is not expressed at this stage, we did not investigate its mis-expression phenotype. To mis-express ara and caup genes, we used the long glass multiple reporter-Gal4 (lGMR-Gal4) driver whose expression is restricted to all photoreceptors. lGMR expression starts during larval stages, after photoreceptors are specified at the morphogenetic furrow and is maintained throughout photoreceptor development and adulthood [19,35]. Over-expression of caup or ara at 25 °C leads to strong morphological defects in the eye, likely due to the prolonged expression of Iro-C genes when they are normally down-regulated during photoreceptor development. However, lowering Gal4 activity by raising flies at 18 °C induces robust lGMR>caup–dependent co-expression of rh3 and rh4 specifically in all yR7 cells (Figure 4A), whether ventral or dorsal. Importantly, caup-induced expansion of rh3 in yR7 cells does not repress rh4 expression. lGMR>caup over-expression does not induce ectopic expression of rh3 in outer (R1–R6) or in R8 photoreceptors, and co-expression of rh5 and rh6 is not observed. However, lGMR>caup does increase to various degrees the proportion of rh6-expressing R8 cells with a corresponding decrease in rh5-expressing cells (Figure 4B). This expansion of Rh6 in R8 cells produces mis-coupling between R7 and R8 cells, resulting in an increase in ommatidia containing Rh3 in R7 and Rh6 in R8. Our interpretation is that, because lGMR>Iro-C produces morphological defects in the eye, the communication between R7 and R8 might be disrupted. In the absence of a signal from R7 to R8, most R8 cells express the default rh6 (as in sevenless mutant eyes) [1,20].
(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.
We have previously shown that the decision between p and y fates is made during early pupation, when ss is activated in yR7 precursors, after lGMR-Gal4 starts to be expressed and long before rhodopsins are expressed . To test whether Iro-C genes can cell-autonomously induce rhodopsin co-expression after the p versus y decision is made, ara and caup genes were expressed using a promoter that is expressed at late stages in development. PanR7-Gal4 is a combination of rh3 and rh4 promoters that is expressed in every R7 cell and in DRA R8 cells , starting at late pupal stages when rhodopsin expression starts . Over-expression of caup using this late driver induces co-expression of rh3 and rh4 in the majority of R7 cells (Figure 4C), which are likely yR7 cells. To test whether a very late signal can induce co-expression in yR7, we expressed Iro-C genes using a rh4-Gal4 driver, which is only expressed in yR7 cells. This should allow R7 to be normally specified as yR7 and turn on rh4, which would then supply the Iro-C signal. Again, mis-expression of caup using this driver induces expression of rh3 in most rh4-expressing cells (Figure 4E). The phenotype is stronger in the central or more-dorsal areas than in ventral regions where this driver is not able to transform all yR7 cells, because it might lack the strength of the PanR7 driver. Together, these results suggest that there is an endogenous sub-threshold level of Iro-C in the dorsal eye close to the equator that is not sufficient to induce co-expression of rh3 and rh4 in a wild-type situation. The PanR7- and rh4-Gal4 drivers must only add limited amount of ara or caup, or provide it late, such that not all yR7 cells co-express. Neither PanR7- nor rh4-Gal4 drivers induce phenotypes in R8 cells (31.7% and 33% of rh5 expression, respectively) (Figure 4D and 4F), suggesting that, as expected, the early decision between p and y fates is not affected. In addition, the expression of caup only in R8 with rh5- and rh6-Gal4 drivers does not produce a visible phenotype (Figure S1G). Therefore, the presence of the Caup or Ara transcription factors in yR7 cells, even very late in development, instructs them to co-express rh3 and rh4.
The Drosophila retina presents a stochastic distribution of ommatidial subclasses. As described before, in ~30% of ommatidia, R7 express rh3 and R8 express rh5, whereas in the remaining ~70%, R7 express rh4 and R8 express rh6 [1,16,37]. These numbers are correct if we consider only R8 rhodopsin expression. However, the data presented here indicate that, if one considers the distribution of R7 rhodopsins, the y ommatidial subtype should be divided into two subpopulations: the “classical y” subtype that expresses only rh4 in R7 and rh6 in R8; and the “dorsal y” subtype that expresses both rh3 and rh4 in R7 and rh6 in R8. Thus, Drosophila contain four (or even five, if we consider the “odd coupled” ommatidia) rather than the three classes that were previously described: DRA, p, y, and “dorsal y” ommatidia.
The expression of ara and caup allows co-expression of rhodopsins in R7 cells by inducing the expression of rh3 in rh4-expressing cells. Although Iro-C gene products could activate the rh3 promoter directly, it should be noted that they are expressed in all photoreceptors in the dorsal eye, but rh3 is only induced in rh4-expressing R7. In addition, over-expression of Iro-C in all photoreceptors using lGMR-Gal4 only induces rh3 in R7, and not in other photoreceptors (Figure 3A).
During development, photoreceptors are subdivided first into two different subtypes, inner (R7 and R8) and outer (R1–R6) by the expression of the two transcription factors encoded by the spalt (sal) complex in inner photoreceptors (Figure 5A). After photoreceptors acquire a generic “inner” fate, prospero is expressed in R7 and directs it away from the R8 fate and toward an R7 fate. Similarly, senseless plays a parallel role in R8 cells to prevent R7 differentiation . At this stage, R7 and R8 are specified as photoreceptors, but they are not patterned in terms of rhodopsin expression. The dorsal-most row of ommatidia is then specified as DRA by the expression of homothorax (hth), which differentiates them from the rest of the retina to become polarized light detectors (Figure 5A and 5B). The main part of the retina is then patterned into the y and p subtypes by the expression of ss during pupation in a subset of R7 cells (Figure 4A). R7 cells that do not express ss become pR7 cells by default . rh3 is activated by orthodenticle (otd), which is present in all photoreceptors, and thus its activity must be actively repressed in yR7 cells. ara and caup might be the signal in yR7 cells that allows the expression of the default state rh3 and breaks the mutual exclusion pathway between rhodopsins (Figure 4A and 4B). R8 cells that are located below dorsal yR7 cells cannot co-express rhodopsins because a bi-stable loop between warts and melted does not allow an ambiguous choice between the rh5 and rh6 fates after the decision is made .
(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.
Functional Consequences of Sensory Receptor Co-Expression
The co-expression of rhodopsins in R7 is restricted to the dorsal eye, which faces the sky. The biological significance of these particular ommatidia in Drosophila is not known. The Drosophila “dorsal y” ommatidia that contain both UV-Rh3 and UV-Rh4 in R7 and green-absorbing Rh6 in R8 provide a unique configuration to measure the ratio between UV and long wavelengths: They contain two UV opsins in R7, providing broad UV sensitivity that is expanded toward shorter wavelengths by Rh3, along with a blue-filtering pigment that prevents short wavelengths to penetrate the R8 layer containing the green-absorbing Rh6 . These ommatidia might be used to discriminate between the “solar” and “antisolar” halves of the sky, necessary to navigate in the correct direction .
Although the exclusion of sensory receptors is a general rule, co-expression to achieve a novel sensitivity might be used in special cases when the expression of a single receptor is not sufficient to confer high enough sensitivity. Although the mouse retina is dominated by rods, it also contains cone cells. The majority of these cone cells co-express both S (blue) and M (green) opsins . Presumably, mice live in a dark environment and are mostly color blind; the co-expression might be useful for optimal utilization of cones. The eye of butterflies also displays co-expression of two rhodopsins in several of their photoreceptors, perhaps to expand the spectrum of sensitivity of photoreceptors in species that do not have a rhodopsin with broad absorption spectrum such as Rh1, which is unique to Diptera [42–44].
Vertebrate olfactory neurons also express only one olfactory receptor gene per olfactory receptor neuron, and a direct feedback from the expressed receptor molecule has been proposed to ensure that this rule is stringently applied [45–47]. However, it cannot be excluded that two olfactory receptor genes are co-expressed, because their large number prevents comprehensive expression studies. Indeed, in Drosophila, a striking example of co-expression of two chemosensory receptors that mediate sensitivity to CO2 was recently described for the olfactory system . The expression of each receptor is not sufficient to confer olfactory CO2-chemosensation on its own, but their combined expression does. Therefore, the addition of multiple receptors might not only increase the receptive spectrum of cells, but might also confer sensitivity to new stimuli. In the CO2 sensitivity case, the co-expression is crucial for the fly to detect a repellent smell that indicates danger. Thus, precise regulation of receptor co-expression must be achieved.
Stochastic Versus Regionalized Specification in the Retinal Mosaic
The spatial specialization induced by Iro-C genes in the fly retina is not the only example where regionalized specification occurs within sensory systems. For example, in the “love spot” of the housefly Musca, the antero-dorsal region of the male eye has presumably lost color vision, because R7 cells are transformed into motion detecting outer photoreceptors that express Rh1 . The human eye also has geographic specialization: the center of the eye (fovea) contains exclusively cones that are involved both in acute and color vision in bright light. The periphery of the eye is mostly composed of rods and is involved in dim light vision (reviewed in ). The mouse olfactory system also exhibits specialization where the main olfactory epithelium that is responsible for detection of general odorants is separated from the vomeronasal organ that is involved in pheromone detection . Drosophila also has two olfactory organs, the antenna and the maxillary palps, which express different sets of olfactory receptors and are likely involved in the detection of different types of odors .
Conserved Role for Iro-C in Sensory Receptor Co-Expression?
Iro-C genes may not only be responsible for relieving the “one receptor–one neuron” constraint in the Drosophila eye, but may also allow receptor co-expression elsewhere. For instance, members of the orthologous family, the Irx genes, are expressed in mouse photoreceptors where opsin co-expression is observed [53–55]. Although the terminal differentiation of bipolar cells is affected in mice with mutant Irx5 , it will be of interest to study cone opsin expression in this and other Irx mutants to test whether these genes are also involved in the co-expression of opsins.
Mouse olfactory neurons do not express Irx5 or Irx6 , and they do not express more than one olfactory receptor gene . In contrast, recent comprehensive studies in the Drosophila antenna and maxillary palp have identified a subgroup of olfactory receptor neurons that co-express two divergent receptors [57,58]. Interestingly, cells that co-express different olfactory receptor genes are the only neurons that express Iro-C genes in the maxillary palp (EOM, AC, and CD; unpublished observations). Unfortunately, the loss of Iro-C function in this tissue leads to re-specification of these neurons toward other non-neuronal fates (EOM, AC, and CD; unpublished observations), preventing us from further testing the involvement of Iro-C genes in the lack of exclusion.
Genes directly controlled by Iro-C transcription factors are still elusive. Binding sites for Mirr that presumably mediate repression of fringe in the dorsal eye disc were recently described . The identification of target genes of the Iro/Irx family might shed some light on the regulation of the pathway that maintains mutual exclusion of sensory receptors.
Materials and Methods
Drosophila strains and crosses.
Flies were raised on standard corn meal–molasses–agar medium and grown at room temperature (24 ± 1 °C) unless otherwise noted. y1w67 flies were used as control for Rhodopsin expression. As the red color of adult eyes interferes with fluorescent immunostainings, the eyes were rendered white by using an RNAi construct against the white gene  when a white marker gene was introduced in the genetic background by P-element transgenes. lGMR-Gal4 was produced by a pentamerized Glass binding site , UAS-ara and caup were gifts from J. Modolell. irorF209-PZ and Df(3L)iroDFM3 were obtained from the Bloomington Stock Center. Iro-C-Gal4 was created by replacing the P element in irorF209-PZ with one containing Gal4. rh3-, rh4, and PanR7-Gal4 drivers were described in . To visualize “yellow” and rh3 expression with a reporter, we used flies containing rh3-LexA and lexAop-RFP. Clones were generated using the standard FLP/FRT technique.
Antibodies and dilutions used were as follows: mouse anti-Rh3 and anti-Rh4 (1:100) and mouse anti-Rh5 (1:50) (gift from S Britt, University of Colorado); rabbit anti-Rh4 (1:400) (gift from C. Zuker, University of California San Diego); rabbit anti-Rh6 (1:5000); rabbit anti-βGal (1:5000) (Cappel); mouse anti-βGal (1:500) (Promega); mouse anti-pros (1:50) (Developmental Studies Hybridoma Bank); rat anti-ElaV (1:10) (DSHB); and rabbit anti-GFP (1:800) (Biogenesis). Chicken anti-Rh3 was from . All secondary antibodies were Alexa-conjugated (1:800) (Molecular Probes). Throughout the paper, Rh3 and Rh4 were stained using antibodies generated in mouse and rabbit, respectively, because they are significantly better that the other two.
Antibody stainings for larval and pupal retinas.
Antibody stainings for larval and pupal retinas were essentially the same except for the collection of tissue. The protocols merge after the fixation step. Cerebral complexes of late third instar larvae were dissected in phosphate buffered saline (PBS) (1×) and fixed in PBS + 4% paraformaldehyde for 20 min at room temperature (RT). Pupal cases were collected at 24 h after puparium formation at 25 °C and the head was dissected in ice cold PBS (1x). Several eye-brain complexes were extracted by gentle pipetting and collected in PBS (1×) on ice. After 20 min fixation using PBS (1×) + 4% formaldehyde at RT, the samples were washed four times with PBS + 0.1% Triton-X-100 (PBT). The first antibody was added overnight at 4 °C. After four washes with PBT, the secondary antibody was added for at least 2 h at RT. After another four washes in PBT, each retina was separated from the brain by using two tungsten needles and then mounted flat in Vectashield (Vector Laboratories).
Antibody stainings for frozen fly head sections.
10-μm horizontal eye sections were produced using a cryostat (Zeiss) and deposited on Superfrost PLUS slides (Fisher). The slides were then fixed 15 min in PBS (1×) + 4% formaldehyde. After four washes with PBT, the first antibody was added overnight at 4 °C. After four washes with PBT, the secondary antibody was added for at least 2 h at RT. After four washes with PBT, the slides were mounted in Aquamount.
Antibody stainings for adult whole-mounted retinas.
Adult retinas were dissected out and after a rinse with PBS (1×), they were fixed for 15 min with 4% formaldehyde at RT. After three washes in PBT, the retinas were incubated with the primary antibodies diluted in BNT (PBS, 0.1% BSA, 0.1% Tween-20, 250 mM NaCl) overnight at 4 °C. After two rinses and a 30 min wash with PBT, the retinas were incubated with secondary antibodies for 2–4 hours at RT. Two quick rinses with PBT were followed by an overnight wash at 4 °C. Retinas were cleaned of any remaining cuticle and mounted in Vectashield.
Antibody stainings in dissociated ommatidia.
The retina of 3–5 dissected eyes were removed from the cornea and dissociated on a slide using dissection needles in a drop of PBS. After the samples dried at RT, they were fixed with 4% formaldehyde and staining was carried out as for frozen sections.
In situ hybridization for cryosectioned adult retinas.
Adult retinas were dissected as described for antibody stainings. Dissected retinas were mounted on Superfrost PLUS slides (Fisher) and dried for 2 h at 65 °C. After fixation for 15 min with 4% paraformaldehyde, the slides were washed in PBS, treated with Proteinase K for 5 min at 37 °C and refixed for 10 min. Following a short PBS wash, the slides were treated with 0.2 M HCl for 10 min, washed in PBS, and acetylated with 0.1 M triethanolamine. Retinas were hybridized overnight at 65 °C with 100 μl hybridization buffer (50% formamide, 5× SSC, 5× Denhardt's, 250 μg/ml yeast tRNA, 500 μg/ml herring sperm DNA, 50 μg/ml heparin, 2.5 mM EDTA, 0.1% Tween-20, 0.25% CHAPS) containing a digoxygenin-labeled rh3 probe and a fluorescein-labeled rh4 probe. After a series of washes in 5× SSC; 50% formamide, 2× SSC; 2× SSC; 0.2× SSC, and 0.1× SSC (5, 30, 20, 20, and 20 min, respectively), the rh3 probe was detected using HNPP/Fast Red (Roche) and the rh4 probe was detected using the TSA Biotin System (Perkin Elmer) and streptavidin-Alexa488 according to the manufacturers suggestions.
Neutralization of the cornea.
Anesthetized flies were fixed to a Petri dish using nail polish. Then, flies were submerged in water and visualized using a 20× water immersion lens. To visualize “yellow”, FITC settings were used .
Figure S1. Over-Expression of ara in R7 and caup in R8
(A) Optical section through the center (equatorial) of eyes expressing ara in all photoreceptors under the lGMR promoter (lGMR>ara) and stained for Rh3 (cyan) and Rh4 (red). Almost all yR7 cells containing Rh4 also contain Rh3 (white). pR7 cells only contain Rh3.
(B) Fly eyes of the same genotype stained for Rh5 (blue) and Rh6 (green) show few R8 cells containing Rh5; those are more frequent in ventral regions of the eye.
(C) Similar staining as in (A), but ara is expressed in all R7 cells late during pupation (PanR7>ara). As in (C), 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 (equator) of the retina that expresses ara late during pupation only in yR7 (rh4>ara). 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.
(G) Over-expression of caup using R8 specific drivers (rh5+rh6>caup) does not induce any phenotype.
(6.05 MB PDF)
We would like to thank S. Britt for antibodies against Rh3, Rh4, and Rh5; Charles Zuker for antibodies against Rh4; the Bloomington Stock Center for mutant stocks and Gal4 drivers; and John Carlson and Anand Ray for discussions about olfactory neurons. We are also grateful to members of the Desplan lab and FlyNet at New York University Department of Biology for their advice. Hynek Wichterle provided helpful comments on the manuscript.
EOM, AC, MFW, and CD conceived and designed the experiments. EOM, AC, MFW, and DV performed and analyzed the experiments. RJJ and TAC contributed reagents. TAC and FP made the initial observations. EOM, AC, and CD wrote the paper.
- 1. Papatsenko D, Sheng G, Desplan C (1997) A new rhodopsin in R8 photoreceptors of Drosophila: evidence for coordinate expression with Rh3 in R7 cells. Development 124: 1665–1673.
- 2. O'Tousa JE, Baehr W, Martin RL, Hirsh J, Pak WL, et al. (1985) The Drosophila ninaE gene encodes an opsin. Cell 40: 839–850.
- 3. Chou WH, Hall KJ, Wilson DB, Wideman CL, Townson SM, et al. (1996) Identification of a novel Drosophila opsin reveals specific patterning of the R7 and R8 photoreceptor cells. Neuron 17: 1101–1115.
- 4. Zuker CS, Montell C, Jones K, Laverty T, Rubin GM (1987) A rhodopsin gene expressed in photoreceptor cell R7 of the Drosophila eye: homologies with other signal-transducing molecules. J Neurosci 7: 1550–1557.
- 5. Pollock J, Benzer S (1988) Transcript localization of four opsin genes in the three visual organs of Drosophila: RH2 is ocellus specific. Nature 333: 779–782.
- 6. Vosshall LB, Amrein H, Morozov PS, Rzhetsky A, Axel R (1999) A spatial map of olfactory receptor expression in the Drosophila antenna. Cell 96: 725–736.
- 7. Luo L, Flanagan JG (2007) Development of continuous and discrete neural maps. Neuron 56: 284–300.
- 8. Hardie R (1985) Functional organization of the fly retina. In: Ottoson D, editor. Progress in sensory physiology. Berlin: Springer. pp. 1–79.
- 9. Yamaguchi S, Wolf R, Desplan C, Heisenberg M (2008) Motion vision is independent of color in Drosophila. Proc Natl Acad Sci U S A. E-pub ahead of print.
- 10. Tang S, Guo A (2001) Choice behavior of Drosophila facing contradictory visual cues. Science 294: 1543–1547.
- 11. Nathans J (1999) The evolution and physiology of human color vision: insights from molecular genetic studies of visual pigments. Neuron 24: 299–312.
- 12. Miller G, Hansen K, Stark W (1981) Phototaxis in Drosophila: R1-R6 input and interaction among ocellar and compound eye receptors. J Insect Physiol 27: 813–819.
- 13. Fryxell KJ, Meyerowitz EM (1987) An opsin gene that is expressed only in the R7 photoreceptor cell of Drosophila. EMBO J 6: 443–451.
- 14. Huber A, Schulz S, Bentrop J, Groell C, Wolfrum U, et al. (1997) Molecular cloning of Drosophila Rh6 rhodopsin: the visual pigment of a subset of R8 photoreceptor cells. FEBS Lett 406: 6–10.
- 15. Montell C, Jones K, Zuker C, Rubin G (1987) A second opsin gene expressed in the ultraviolet-sensitive R7 photoreceptor cells of Drosophila melanogaster. J Neurosci 7: 1558–1566.
- 16. Chou W, Huber A, Bentrop J, Schulz S, Schwab K, et al. (1999) Patterning of the R7 and R8 cells of Drosophila: evidence for induced and default cell-fate specification. Development 126: 607–616.
- 17. Franceschini N, Kirschfeld K, Minke B (1981) Fluorescence of photoreceptor cells observed in vivo. Science 213: 1264–1267.
- 18. Stark WS, Thomas CF (2004) Microscopy of multiple visual receptor types in Drosophila. Mol Vis 10: 943–955.
- 19. Wernet M, Mazzoni EO, Celik A, Duncan A, Duncan I, et al. (2006) Stochastic spineless expression creates the retinal mosaic for color vision. Nature 440: 174–180.
- 20. Chou WH, Huber A, Bentrop J, Schulz S, Schwab K, et al. (1999) Patterning of the R7 and R8 photoreceptor cells of Drosophila: evidence for induced and default cell-fate specification. Development 126: 607–616.
- 21. Labhart T, Meyer EP (1999) Detectors for polarized skylight in insects: a survey of ommatidial specializations in the dorsal rim area of the compound eye. Microsc Res Tech 47: 368–379.
- 22. Wernet M, Labhart T, Baumann F, Mazzoni EO, Pichaud F, et al. (2003) Homothorax switches function of Drosophila photoreceptors from color to polarized light sensors. Cell 115: 267–279.
- 23. Tomlinson A (2003) Patterning the peripheral retina of the fly: decoding a gradient. Dev Cell 5: 799–809.
- 24. Burglin TR (1997) Analysis of TALE superclass homeobox genes (MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domain conserved between plants and animals. Nucleic Acids Res 25: 4173–4180.
- 25. Gomez-Skarmeta JL, Modolell J (2002) Iroquois genes: genomic organization and function in vertebrate neural development. Curr Opin Genet Dev 12: 403–408.
- 26. Cavodeassi F, Modolell J, Gomez-Skarmeta JL (2001) The Iroquois family of genes: from body building to neural patterning. Development 128: 2847–2855.
- 27. Gomez-Skarmeta JL, del Corral RD, de la Calle-Mustienes E, Ferre-Marco D, Modolell J (1996) Araucan and caupolican, two members of the novel iroquois complex, encode homeoproteins that control proneural and vein-forming genes. Cell 85: 95–105.
- 28. Cavodeassi F, Diez del Corral R, Campuzano S, Dominguez M (1999) Compartments and organising boundaries in the Drosophila eye: the role of the homeodomain Iroquois proteins. Development 126: 4933–4942.
- 29. Dominguez M, de Celis JF (1998) A dorsal/ventral boundary established by Notch controls growth and polarity in the Drosophila eye. Nature 396: 276–278.
- 30. Pichaud F, Casares F (2000) homothorax and iroquois-C genes are required for the establishment of territories within the developing eye disc. Mech Dev 96: 15–25.
- 31. Fortini ME, Rubin GM (1990) Analysis of cis-acting requirements of the Rh3 and Rh4 genes reveals a bipartite organization to rhodopsin promoters in Drosophila melanogaster. Genes Dev 4: 444–463.
- 32. Pichaud F, Desplan C (2001) A new visualization approach for identifying mutations that affect differentiation and organization of the Drosophila ommatidia. Development 128: 815–826.
- 33. Tahayato A, Sonneville R, Pichaud F, Wernet MF, Papatsenko D, et al. (2003) Otd/Crx, a dual regulator for the specification of ommatidia subtypes in the Drosophila retina. Dev Cell 5: 391–402.
- 34. Diez del Corral R, Aroca P, Gomez-Skarmeta JL, Cavodeassi F, Modolell J (1999) The Iroquois homeodomain proteins are required to specify body wall identity in Drosophila. Genes Dev 13: 1754–1761.
- 35. Moses K, Rubin GM (1991) Glass encodes a site-specific DNA-binding protein that is regulated in response to positional signals in the developing Drosophila eye. Genes Dev 5: 583–593.
- 36. Earl JB, Britt SG (2006) Expression of Drosophila rhodopsins during photoreceptor cell differentiation: insights into R7 and R8 cell subtype commitment. Gene Expr Patterns 6: 687–694.
- 37. Fortini M, Rubin G (1990) Analysis of cis-acting requirements of the Rh3 and Rh4 genes reveals a bipartite organization to rhodopsin promoters in Drosophila melanogaster. Genes & Development 4: 444–463.
- 38. Xie B, Charlton-Perkins M, McDonald E, Gebelein B, Cook T (2007) Senseless functions as a molecular switch for color photoreceptor differentiation in Drosophila. Development 34: 4243–4253.
- 39. Mikeladze-Dvali T, Wernet MF, Pistillo D, Mazzoni EO, Teleman AA, et al. (2005) The growth regulators warts/lats and melted interact in a bistable loop to specify opposite fates in drosophila R8 photoreceptors. Cell 122: 775–787.
- 40. Rossel S, Wehner R (1984) Celestial orientation in bees: the use of spectral cues. J Comp Physiol 155: 605–613.
- 41. Applebury ML, Antoch MP, Baxter LC, Chun LLY, Falk JD, et al. (2000) The murine cone photoreceptor: a single cone type expresses both S and M opsins with retinal spatial patterning. Neuron 27: 513–523.
- 42. Kitamoto J, Ozaki K, Arikawa K (2000) Ultraviolet and violet receptors express identical mRNA encoding an ultraviolet-absorbing opsin: identification and histological localization of two mRNAs encoding short-wavelength-absorbing opsins in the retina of the butterfly Papilio xuthus. J Exp Biol 203: 2887–2894.
- 43. Arikawa K, Mizuno S, Kinoshita M, Stavenga DG (2003) Coexpression of two visual pigments in a photoreceptor causes an abnormally broad spectral sensitivity in the eye of the butterfly Papilio xuthus. J Neurosci 23: 4527–4532.
- 44. Sison-Mangus MP, Bernard GD, Lampel J, Briscoe AD (2006) Beauty in the eye of the beholder: the two blue opsins of lycaenid butterflies and the opsin gene-driven evolution of sexually dimorphic eyes. J Exp Biol 209: 3079–3090.
- 45. Serizawa S, Miyamichi K, Nakatani H, Suzuki M, Saito M, et al. (2003) Negative feedback regulation ensures the one receptor-one olfactory neuron rule in mouse. Science 302: 2088–2094.
- 46. Shykind BM, Rohani SC, O'Donnell S, Nemes A, Mendelsohn M, et al. (2004) Gene switching and the stability of odorant receptor gene choice. Cell 117: 801–815.
- 47. Feinstein P, Bozza T, Rodriguez I, Vassalli A, Mombaerts P (2004) Axon guidance of mouse olfactory sensory neurons by odorant receptors and the beta2 adrenergic receptor. Cell 117: 833–846.
- 48. Jones WD, Cayirlioglu P, Kadow IG, Vosshall LB (2007) Two chemosensory receptors together mediate carbon dioxide detection in Drosophila. Nature 445: 86–90.
- 49. Hardie RC (1983) Projection and connectivity of sex-specific photoreceptors in the compound eye of the male housefly (Musca domestica). Cell Tissue Res 233: 1–21.
- 50. Ahnelt PK, Kolb H (2000) The mammalian photoreceptor mosaic-adaptive design. Prog Retin Eye Res 19: 711–777.
- 51. Mombaerts P (2004) Genes and ligands for odorant, vomeronasal and taste receptors. Nat Rev Neurosci 5: 263–278.
- 52. Stocker RF (2001) Drosophila as a focus in olfactory research: mapping of olfactory sensilla by fine structure, odor specificity, odorant receptor expression, and central connectivity. Microsc Res Tech 55: 284–296.
- 53. Cohen DR, Cheng CW, Cheng SH, Hui CC (2000) Expression of two novel mouse Iroquois homeobox genes during neurogenesis. Mech Dev 91: 317–321.
- 54. Cheng CW, Chow RL, Lebel M, Sakuma R, Cheung HO, et al. (2005) The Iroquois homeobox gene, Irx5, is required for retinal cone bipolar cell development. Dev Biol 287: 48–60.
- 55. Houweling AC, Dildrop R, Peters T, Mummenhoff J, Moorman AF, et al. (2001) Gene and cluster-specific expression of the Iroquois family members during mouse development. Mech Dev 107: 169–174.
- 56. Rawson NE, Eberwine J, Dotson R, Jackson J, Ulrich P, et al. (2000) Expression of mRNAs encoding for two different olfactory receptors in a subset of olfactory receptor neurons. J Neurochem 75: 185–195.
- 57. Fishilevich E, Vosshall LB (2005) Genetic and functional subdivision of the Drosophila antennal lobe. Curr Biol 15: 1548–1553.
- 58. Couto A, Alenius M, Dickson BJ (2005) Molecular, anatomical, and functional organization of the Drosophila olfactory system. Curr Biol 15: 1535–1547.
- 59. Bilioni A, Craig G, Hill C, McNeill H (2005) Iroquois transcription factors recognize a unique motif to mediate transcriptional repression in vivo. Proc Natl Acad Sci U S A 102: 14671–14676.
- 60. Lee YS, Carthew RW (2003) Making a better RNAi vector for Drosophila: use of intron spacers. Methods 30: 322–329.
- 61. Cook T, Pichaud F, Sonneville R, Papatsenko D, Desplan C (2003) Distinction between color photoreceptor cell fates is controlled by Prospero in Drosophila. Dev Cell 4: 853–864.