The evolutionary diversity of insect retinal mosaics: common design principles and emerging molecular logic

Abstract

Independent evolution has resulted in a vast diversity of eyes. Despite the lack of a common Bauplan or ancestral structure, similar developmental strategies are used. For instance, different classes of photoreceptor cells (PRs) are distributed stochastically and/or localized in different regions of the retina. Here, we focus on recent progress made towards understanding the molecular principles behind patterning retinal mosaics of insects, one of the most diverse groups of animals adapted to life on land, in the air, under water, or on the water surface. Morphological, physiological, and behavioral studies from many species provide detailed descriptions of the vast variation in retinal design and function. By integrating this knowledge with recent progress in the characterization of insect Rhodopsins as well as insight from the model organism Drosophila melanogaster, we seek to identify the molecular logic behind the adaptation of retinal mosaics to the habitat and way of life of an animal.

Keywords: evolution; insect retina; ommatidia; patterning; regionalization; stochasticity.

Figure 1. Insect retinal mosaics: common design principles, evolutionary and molecular logic

A. General patterning strategies common to many retinas Top: stochastic specification of retinal units within the epithelium creates a retinal mosaic. Middle: Localized specification of marginal units in response to factors emanating from adjacent, non-retinal tissue (grey line), in response to short-range signals (grey arrows). Bottom: The specification of stripes or bands of retinal units can occur at compartment boundaries (light green) or within an otherwise seemingly homogeneous retinal field (purple). In some cases, all retinal units located inside a given compartment show the same, specific specializations (e.g. dark green). B–D. Examples of evolutionary variation between insect ommatidia. B. Top: the fruit fly ommatidium (Drosophila melanogaster) contains eight neuronal photoreceptor cells (PRs), as well as non-neuronal cone cells and pigment cells. Six PRs span the entire thickness of the retina (termed R1–6, green), while R7 (blue) and R8 (red) are situated on top of each other. Inset: Light gathering organelles (rhabdomeres) of all eight PRs are separated from each other (dark green and red circles, respectively), creating an ‘open rhabdom’. (after [120]) Below: electron micrograph showing the open rhabdom structure with ‘inner PRs’ R7 and R8 located in the center (sectioned at the level of R7). C. Top: the ommatidia of a honeybee worker (Apis mellifera) contain nine PRs, 8 of which span the entire retina (R1–8, green), while the shorter cell R9 is always found basally (red). Inset: rhabdomeres of R1–9 are not separated (‘fused rhabdom’, see EM section below). D. Top: the ommatidia of many butterflies such as the swallowtail Papilio contain nine PRs, four of which are located in the distal retina (R1–4, blue), while four are found in the basal half (R5–8, green). The PRs in butterfly ommatidia are therefore tiered. Note the very small cell R9 is always found at the base of the ommatidium (red), with very little contribution to the rhabdom. Insets: the fused rhabdom of Papilio at 3 different levels, illustrating the tiered design. Bottom: transmission electron microscopy of a section through an ommatidium from the butterfly Anthocharis (Pieridae) (from [121]). E,F. Developmental specification of ommatidial cell types in Drosophila. E. Eight cell cluster from wandering third-instar larvae. After initial recruitment from an undifferentiated pool of progenitors, PR cell fates are specified through combinatorial expression of transcription factors, like Spalt (purple) + Senseless (red) in the case of R8, or Spalt + Prospero (blue) for R7. F. Expression of Spalt in ‘inner PRs’ is crucial for their specification into R7 and R8 via Prospero and Senseless, respectively. G. ‘Inner PRs’ R7 and R8 terminate in a deeper level of the brain, with ‘long visual fibers’ (lvf) projecting to the medulla (M) neuropil. ‘Outer PRs’ R1–6 have ‘short visual fibers’ terminating in the lamina (L) neuropil, thereby connecting to distinct post-synaptic partners. H. Based on the molecular (and in some cases: morphological) criteria of inner PRs, Drosophila ommatidia can be subdivided into five subtypes, which will be discussed below.

Figure 2. Common features and differences between ommatidial mosaics in different insects

A. The Drosophila retina contains two ommmatidial subtypes named ‘yellow’ and ‘pale’, which are distributed stochastically, at an un-even ratio (65/35). They differ in the Rhodopsins expressed in R7 (choice between UV opsins Rh3 and Rh4) and R8 (Blue opsin Rh5, or Green opsin Rh6), thereby creating a mosaic of chromatic sensitivities. The subtypes are first defined in R7 cells by stochastic expression of the transcription factor Spineless in the ‘yellow’ R7 subtype, where it represses the ‘pale’ fate. Only ‘pale’ R7 cells instruct underlying R8 cells to acquire the same subtype fate, while ‘yellow’ R8 cells choose their fate by default. A cellular signal transduction pathway maintains the chosen subtype fate in R8 cells by mutual repression. BB = broad band Rhodopsin associated with a UV-sensitizing pigment. B. Stochastic distribution of ‘pale’/’yellow’ rhodopsins Rh3 (red) and Rh4 (blue) in the R7 layer of the adult Drosophila retina. C. Very similar stochastic distribution of Rh5 (purple) and Rh6 (green) in the R8 layer. D. Molecular mechanism driving stochastic expression of spineless in R7 cells. Activating (green) and repressing (red) cis-regulatory elements determine on-off expression. Furthermore, interchromosomal long-range communication (via ‘intercom’, blue) modulates the frequency of expression, and coordinates expression state between alleles. As a result, both alleles are expressed in the same random subsets of R7 cells. Spineless encodes a PAS-bHLH transcription factor which then activates ‘yellow’-specific downstream genes. E. The retinal mosaic of the honeybee worker (Apis mellifera) contains three stochastically distributed subtypes, named I (44%), II (46%), and III (10%). Similarly to Drosophila the mosaic is defined by differences in Rhodopsin expression in two PRs with ‘long visual fibers’, R1 (choice between UV or B/blue) and R5 (UV or B/blue). The identity of R9 remains obscure due to its small size. Similar to Drosophila all PRs with ‘short visual fibers’ (Svf) express the same long-wavelength Rhodopsin (G/green). (Long visual fiber PRs are shown in the center of the ommatidial schematic, following the nomenclature of Friedrich et al., 2011). F. Stochastic expression of honeybee rhodopsin genes in the adult retina visualized by in-situ hybridization against UV (left) and blue opsins (right). G. The ventral retina of the swallowtail butterfly Papilio also contains three stochastically distributed ommatidial subtypes named I (~50%), II (~25%), and III (~25%). Like in the honeybee, distal PRs with long visual fibers (R1 and R2) choose between expression of UV and blue Rhodopsins. The basal PR always co-expresses two long-wavelength Rhodopins (LW1 and LW2). Unlike in Drosophila and honeybees, the mosaic is not uneven and extends to PRs with Svf: two cells always co-express the same two long-wavelength Rhodopsins as PR R9, while the remaining four cells manifest different combinations of long wavelength Rhodopsins. The spectral sensitivity of most of these cell types is further modulated by the presence of additional pigments (V* = violet; R = red-sensitive; BB = broad band; DG = double-peaked green). H. Schematic of Papilio type I–III ommatidia depicting the additional pigments: while all three subtypes contain purple pigment (PP) granules distally in R1 and R2, they differ in perirhabdomeral pigment content: red (type I and II) or yellow (type III), resulting in different coloring when back-illuminated (inset, top right). Finally, only type II ommatidia containtain 3-hydroxyretinol, visible by UV-induced fluorescence (inset, top center). This pigment shifts the sensitivity of type II R1 and R2 UV PRs towards violet light (see spectral sensitivities at bottom).

Figure 3. Localized specification of dorsal rim ommatidia of flies, crickets, and desert locusts

A. Scanning EM with the approximate location of 1–2 rows of specialized ommatidia in the dorsal rim area (DRA) in Drosophila. Inset: only DRA ommatidia express the UV Rhodopsin Rh3 in both R7 andR8. B. In the DRA, the diameter of R7 and R8 PR rhabdomeres is enlarged (compare with Figure 1B). Furthermore, rhabdomeric microvilli are untwisted and oriented at an angle of 90 degrees (symbolized by the double-headed arrows). C. During development, the DRA fate is induced by combining positional information provided by the dorsal selector genes of the Iroquois complex (Iro-C, blue) and high levels of Wingless signaling (Wg, green), which induce an unknown diffusible signal emanating from the adjacent head tissue all around the eye. D. The homeodomain transcription factor Homothorax specifically marks R7 and R8 nuclei of DRA ommatidia (Hth shown in green, co-labeled with thee pan-neuronal marker Elav in blue), where it is both necessary and sufficient to induce the DRA fate. E. Summary of Rhodopsin expression in cricket (Gryllus bimaculatus) DRA ommatidia. Like in Drosophila, cricket ommatidia contain eight PRs, at least one of which has long visual fibers (identity of the proximal cell remains unclear). Five PRs in the DRA (Lvf + 4 Svf) express the Blue Rhodopsin. The proximal cell expresses the UV Rhodopsin, while 2 cells do not contribute to the Rhabdom (shown in white; Rhodopsin expression unknown). F. In-situ hybridizations visualizing the expression of cricket Rhodopsins in the adult retina. Note expression of the blue opsin outside the DRA, only in a previously undescribed ventral band of ommatidia. Inset, top: summary and scanning EM of DRA morphology where two groups of PRs forming orthogonally oriented, untwisted rhabdomeric microvilli: R7 versus R1,2,5,6 (white ‘T’ indicates orientation of the ommatidium). G. Summary of Rhodopsin expression in cricket central ommatidia: the Lvf cell most likely expresses a UV Rhodopsin, while Rhodopsin expression in the proximal cell remains unknown. The six remaining Svf PRs most likely express the same green Rhodopsin (exact PR identities could not be determined: question mark). H–J. Summary of Rhodopsin expression in ommatidial subtypes in the retina of the desert locust (Schistocerca gregaria). H. DRA ommatidia are dramatically specialized, with all PRs expressing only the blue (B) Rhodopsin. I. Specialized morphology of locust DRA ommatidia, where R7 forms untwisted rhabdomeric microvilli that are oriented at 90 degrees to those of R1,2,5,6,8. J. Outside the DRA, five Svf PRs always co-express blue (B) and green-sensitive (G) Rhodopsins, while two basal Svf cells express only the green Rhodopsin (R1+R4). Additionally, a mosaic of two ommatidial subtypes exists: type I (65%) and type II (35%). Opsin expression in the only PR with long visual fibers (R7) defines the mosaic by choosing between UV (Type I: UV) and blue (Type II: Blue).

Figure 4. Molecular and morphological specializations in the dorsal retina

A. In the dorsal third of the adult Drosophila retina specialized ‘yellow’ ommatidia co-express both UV Rhodopsins, Rh3 and Rh4, creating a fourth ommatidial subtype (DTy). B. Schematic summarizing the retinal mosaic in Drosophila, containing four ommatidial subtypes defined by unique Rhodopsin expression: pale (blue), yellow (green), DTy (orange), and DRA (pink). C. Eye pigmentation of an enhancer trap element inserted in the Iro-C complex of dorsal selector genes (from [122]). D. Summary of the transcription factor network regulating Rh3/Rh4 co-expression in R7 cells of DTy ommatidia. By modulating Spineless levels, Iro-C attenuates repression of rh3 via the transcriptional repressor ‘Defective proventriculus’ (Dve), while Rh4 levels remain unaffected. E. Examples from dragonflies, for different visual behaviors mediated by the dorsal half of the eye (top: prey capture) versus the ventral half of the eye (bottom: detection of water surfaces as habitat or oviposition sites). F. Section through a dragonfly (Sympetrum) eye demonstrating the obvious morphological differences between dorsal and ventral retina. Note the sharp boundary between dorsal retina (expressing yellow pigment) and ventral retina. G. Summary of morphological and molecular features of the dorsal dragonfly retina. Top: ommatidia form fused rhadoms with eight PR cells, two of which have long visual fibers (R6, R7). In the dorsal retina, the Svf PRs most likely all contain a blue Rhodopsin. Bottom: Electroretinogram (ERG) of the dragonfly eye shows prevalence of blue receptors in the dorsal half, whereas the ventral retina is mostly green sensitive. Electrophysiological studies point to a variety of cell types with different spectral sensitivities in the ventral eye, most likely co-expressing different Rhodopsins (UV, B, G, UV+B+G), as well as additional pigments. H. Photograph of a male honeybee drone, summarizing evidence of different behaviors that are mediated by the dorsal and ventral halves of the retina: chasing of the queen (dorsal) and approaching the hive (ventral). I. Left: In situ hybridizations on adult retinas of male honeybee drone visualizing dramatic expression of the Blue Rhodopsin in the dorsal retina, whereas the Green Rhodopsin is found in the ventral half. Note the sharp boundary in expression domains (black arrowhead). Right: schematic proposing an explanation for the specialized DA_drone ommatidial subtype found in the dorsal drone eye (compare to Figure 2E). J. Summary of differences in the retinal mosaic between drones (left) and worker honeybees (right). Note the difference in overall retina size, facet diameter, and ommatidia number. While specialized DA_drone ommatidia occur only in the dorsal retina of drones, it remains unclear whether three stochastically distributed subtypes exist ventrally (question mark). Alternatively, UV- and Blue Rhodopsins could be organized uniformly in Type I ommatidia.

Figure 5. Retinal specializations in the ventral half of the insect retina

A. Visual stimuli influencing the navigational decisions of flying insects, like Drosophila: the sun, sky polarization (double headed arrow), chromatic gradients in the sky, landmarks (trees, bushes), optic flow, and polarized reflections from water surfaces. B. Morphological reconstruction of PR rhabdomere twist in the ventral retina using serial EM sections of R1–6 and R7 revealed a reduced twist of R7 and rhabdomeres in three out of the six Svf PRs (R4–6, symbolized by the green, double-headed arrows; from [123]). C. Behavior in response to ventral stimuli is strongly dependent on PRs with short visual fibers (R1–6), both using UV (R1–6+R7) and green stimuli (R1–6). R7 cells are involved in UV vision only. I contrast, dorsal polarotaxis relies on DRA R7 and R8 alone. D. Ventral eye pigmentation from an enhancer trap inserted into the sloppy paired (slp) locus. E. PR morphology of the mosquito Aedes aegypti, the vector of several pathogens dangerous to humans. F. Whole mounted adult mosquito retina stained for the long-wavelength Rhodopsin LW1/Aaop9 (green) expressed in R7 cells. Note expression in the dorsal region, as well as in a ventral stripe of ommatidia. G. Schematic summary of mosquito opsins co-expressed in R7 cells of non-overlapping regions: ‘dorsal region’ and the ventral stripe co-express the same blue and green Rhodopsin, while the central region in between co-expresses three Rhodopsins (UV+B+G), resulting in broad band sensitivity. H. Summary of the visual system of the backswimmer Notonecta glauca, a water bug (Hemiptera). Top: photograph of the Notonecta eyes. Bottom: Electron micrograph and drawing of a Notonecta ommatidium. Note that the rhabdomeres of R7 and R8 are fused, while R1–6 form an open rhabdom. I. Zonation of the retina of Notonecta in the ventral eye region (only central rhabdomeres are shown). The rhabdomeres of R7 and R8 of dorsalmost ommatidia in this region (shown in blue) are aligned in parallel, as in the rest of the eye (see Figure 5G). R7 and R8 rhabdomeres in the more ventral ommatidia (shown in red and green) are aligned perpendicular to each other. Within a narrow stripe (in between dashed lines), the central rhabdomere pairs are rotated so they align with the dorsoventral axis (red ommatidia), rather than in the mediolateral direction, as the ventral-most ommatidia (shown in green). J. Behavioral significance of the zonation within the ventral Notonecta retina: when hanging below the water surface, ommatidia within the ventral band (shown in red) view the bright region of the water surface, and are perfectly tuned for detecting vertical objects floating on the water surface. K. when the animal is flying, ventral ommatidia with perpendicularly arranged rhabdomeres (red and green) serve as specialized detectors for water surfaces, which reflect horizontally polarized light.