Dissection and Immunohistochemistry of Larval,Pupal and Adult Drosophila Retinas

The compound eye of Drosophila melanogaster consists of about 750 ommatidia (unit eyes). Each ommatidium is composed of about 20 cells, including lens-secreting cone cells, pigment cells, a bristle cell and eight photoreceptors (PRs) R1-R8 ². The PRs have specialized microvillar structures, the rhabdomeres, which contain light-sensitive pigments, the Rhodopsins (Rhs). The rhabdomeres of six PRs (R1-R6) form a trapezoid and contain Rh1 ^{3 4}. The rhabdomeres of R7 and R8 are positioned in tandem in the center of the trapezoid and share the same path of light. R7 and R8 PRs stochastically express different combinations of Rhs in two main subtypes⁵: In the ''p'' subtype, Rh3 in pR7s is coupled with Rh5 in pR8s, whereas in the ''y'' subtype, Rh4 in yR7s is associated with Rh6 in yR8s ^{6 7 8}.Early specification of PRs and development of ommatidia begins in the larval eye-antennal imaginal disc, a monolayer of epithelial cells. A wave of differentiation sweeps across the disc⁹ and initiates the assembly of undifferentiated cells into ommatidia^10-11. The ''founder cell'' R8 is specified first and recruits R1-6 and then R7 ^12-14. Subsequently, during pupal development, PR differentiation leads to extensive morphological changes ¹⁵, including rhabdomere formation, synaptogenesis and eventually rh expression.In this protocol, we describe methods for retinal dissections and immunohistochemistry at three defined periods of retina development, which can be applied to address a variety of questions concerning retinal formation and developmental pathways. Here, we use these methods to visualize the stepwise PR differentiation at the single-cell level in whole mount larval, midpupal and adult retinas (Figure 1).     

Development of the Drosophila retina,a neurocrystalline lattice

Pattern formation in the Drosophila retina proceeds by the recruitment of cells, along a morphogenetic front, into a lattice. At the advancing front, marked by a dorso-ventral furrow in the eye imaginal disc, cells are organized into ommatidial precursors, each containing cells destined to become photoreceptors 2, 3, 4, 5, and 8. Behind the front, a mitotic wave produces photoreceptors 1, 6, and 7, plus the remaining cells needed to complete the ommatidia. During the third larval instar, the front sweeps anteriorly across the eye disc, leaving a highly ordered pattern in its wake. Preceding the dorso-ventral furrow is a groove that bisects the eye disc into dorsal and ventral halves and presumably plays a role in establishing the equatorial symmetry line. Cell lineage plays little role in pattern formation in the eye. Genetic mosaics show that the cells of each ommatidium are not derived from a single mother cell; the cells appear to be recruited at random at the morphogenetic front. Similarly, the mirror symmetry above and below the equator is not established by a clonal mechanism; a single clone can contribute cells to ommatidia on both sides of the equator.     

Preferential adhesion maintains separation of ommatidia in the Drosophila eye

In the Drosophila eye, neighboring ommatidia are separated by inter-ommatidial cells (IOCs). How this ommatidial spacing emerges during eye development is not clear. Here we demonstrate that four adhesion molecules of the Irre cell recognition module (IRM) family play a redundant role in maintaining separation of ommatidia. The four IRM proteins are divided into two groups: Kirre and Rst are expressed in IOCs, and Hbs and Sns in primary pigment cells (1°s). Kirre binds Hbs and Sns in vivo and in vitro. Reducing activity of either Rst or Kirre alone had minimal effects on ommatidial spacing, but reducing both together led to direct ommatidium:ommatidium contact. A similar phenotype was also observed when reducing both Hbs and Sns. Consistent with the role of these factors in sorting ommatidia, mis-expression of Hbs plus Sns within a single IOC led to complete separation of the cell from neighboring ommatidia. Our results indicate mutual preferential adhesion between ommatidia and IOCs mediated by four IRM proteins is both necessary and sufficient to maintain separation of ommatidia.     

Evidence for the priming role of the central retinula cell in ommatidium differentiation of Ephestia kuehniella

Summary In the developing compound eye of Ephestia kuehniella, within the advancing front of differentiation, regular cell clusters arise which consist of a central cell and two flanking cells. The central cell is destined to become the basal retinula cell later in development. Its crucial role in ommatidium formation is confirmed by ³H-thymidine labelling. Eye anlagen labelled early in the pupal stage incorporate thymidine within two distinct zones along the front of differentiation. After the ommatidia are completely differentiated, both zones contain labelled nuclei of all cell types which participate in ommatidia formation. Within the posterior zone, however, the basal retinula cells are always unlabelled, whereas in the anterior they show labelled nuclei. From this observation it must be concluded that the basal retinula cell first terminates proliferation (either alone or together with a few other cells) to become differentiated as the central retinula cell. These results agree with those found in Drosophila and indicate that the ordered stepwise addition of cells to a central founder cell is a widespread principle of ommatidia formation in insects.     

Positive and negative signaling mechanisms in the regulation of photoreceptor induction in the developingDrosophila retina

An ommatidium of aDrosophila compound eye contains eight photoreceptor cells, R1–R8. The fates of the photoreceptors are determined exclusively by inductive interactions between neuronal precursors in the cell cluster from which the ommatidium is formed. R7 induction has been extensively analysed at the molecular level. Activation of a membrane receptor tyrosine kinase (Sevenless) in the R7 precursor by a ligand (Bride of sevenless) present on the surface of R8 triggers a transduction cascade mediated by Ras, establishing the R7 fate of this cell. Other Sev-expressing cells are prevented from taking on the R7 fate by several different mechanisms. Pokkuri-mediated repression represents one such regulatory mechanism. The positive and negative signaling pathways operating in the fate determination of other photoreceptor cells are also discussed.     

Evolution of color vision in pierid butterflies: blue opsin duplication,ommatidial heterogeneity and eye regionalization in <Emphasis Type="Italic">Colias erate</Emphasis>

This paper documents the molecular organization of the eye of the Eastern Pale Clouded Yellow butterfly, Colias erate (Pieridae). We cloned four cDNAs encoding visual pigment opsins, corresponding to one ultraviolet, two blue and one long wavelength-absorbing visual pigments. Duplication of the blue visual pigment class occurs also in another pierid species, Pieris rapae, suggesting that blue duplication is a general feature in the family Pieridae. We localized the opsin mRNAs in the Colias retina by in situ hybridization. Among the nine photoreceptor cells in an ommatidium, R1-9, we found that R3-8 expressed the long wavelength class mRNA in all ommatidia. R1 and R2 expressed mRNAs of the short wavelength opsins in three fixed combinations, corresponding to three types of ommatidia. While the duplicated blue opsins in Pieris are separately expressed in two subsets of R1-2 photoreceptors, one blue sensitive and another violet sensitive, those of Colias appear to be always coexpressed.     

Cell clones and pattern formation: Genetic eye mosaics inDrosophila melanogaster

Summary Genetic eye mosaics ofDrosophila melanogaster have been studied by means of anatomical techniques. Using different cell markers it was found that the ommatidia at the boundaries between phenotypes are composed of cells belonging to different clones. Therefore, the formation of an individual ommatidium does not obey a mechanism based on a common clonal origin of its constituent elements. A statistical analysis of mosaic ommatidia shows that there is a significant tendency for the receptor cellsR2-R5 on the one hand and the receptor cellsR1, R6 andR7 on the other to belong to the same cell clone. The implications of these findings are discussed.     

Neuronal development in the Drosophila compound eye: Photoreceptor cells R1, R6, and R7 fail to differentiate in the retina aberrant in pattern (rap) mutant

The compound eye of Drosophila is a reiterated pattern of 800 unit eyes known as ommatidia. In each ommatidium there are eight photoreceptor neurons (R1–R8) and an invariant number of accessory cells organized in a precise manner. In the developing eye, specification of cell fates is triggered by sequential inductive events mediated by cell-cell interactions. The R8 photoreceptor neuron is the first cell to differentiate and is thought to play a central role in the recruitment of the remaining photoreceptor cells. Our previous work demonstrated that mutations in the retina aberrant in pattern (rap) locus lead to abnormal pattern formation in the compound eye. Genetic mosaic experiments demonstrated that for normal retinal patterning to occur, rap gene function is required only in the photoreceptor cell R8. In this study we analyzed the R cell composition of developing as well as the adult eyes of rap mutants employing a variety of R cell specific markers. We show that in rap mutants, although some of the R8-specific markers show normal expression patterns, other aspects of the R8 cell differentiation are abnormal. In addition, the cells R1, R6, and R7 fail to differentiate properly in rap mutants. These results suggest that the rap gene encodes an R8-specific function that plays a role in the determination of the photoreceptor cells R1, R6, and R7. © 1996 John Wiley & Sons, Inc.     

Specialized ommatidia for polarization vision in the compound eye of cockchafers,Melolontha melolontha (Coleoptera,Scarabaeidae)

Summary The superposition eye of the cockchafer, Melolontha melolontha, exhibits the typical features of many nocturnal and crepuscular scarabaeid beetles: the dioptric apparatus of each ommatidium consists of a thick corneal lens with a strong inner convexity attached to a crystalline cone, that is surrounded by two primary and 9–11 secondary pigment cells. The clear zone contains the unpigmented extensions of the secondary pigment cells, which surround the cell bodies of seven retinula (receptor) cells per ommatidium and a retinular tract formed by them. The seven-lobed fused rhabdoms are composed by the rhabdomeres of the receptor cells 1–7. The rhabdoms are optically separated from each other by a tracheal sheath around the retinulae. The orientation of the microvilli diverges in a fan-like fashion within each rhabdomere. The proximally situated retinula cell 8 does not form a rhabdomere. This standard form of ommatidium stands in contrast to another type of ommatidium found in the dorsal rim area of the eye. The dorsal rim ommatidia are characterized by the following anatomical specializations: (1) The corneal lenses are not clear but contain light-scattering, bubble-like inclusions. (2) The rhabdom length is increased approximately by a factor of two. (3) The rhabdoms have unlobed shapes. (4) Within each rhabdomere the microvilli are parallel to each other. The microvilli of receptor 1 are oriented 90° to those of receptors 2–7. (5) The tracheal sheaths around the retinulae are missing. These findings indicate that the photoreceptors of the dorsal rim area are strongly polarization sensitive and have large visual fields. In the dorsal rim ommatidia of other insects, functionally similar anatomical specializations have been found. In these species, the dorsal rim area of the eye was demonstrated to be the eye region that is responsible for the detection of polarized light. We suggest that the dorsal rim area of the cockchafer eye subserves the same function and that the beetles use the polarization pattern of the sky for orientation during their migrations.     

Diversity of the Photoreceptors and Spectral Opponency in the Compound Eye of the Golden Birdwing,Troides aeacus formosanus

The compound eye of the Golden Birdwing, Troides aeacus formosanus (Papilionidae, Lepidoptera), is furnished with three types of ommatidia, which are clearly different in pigmentation around the rhabdom. Each ommatidium contains nine photoreceptors, whose spectral sensitivities were analyzed electrophysiologically. We identified nine spectral types of photoreceptor with sensitivities peaking at 360 nm (UV), 390 nm (V), 440 nm (B), 510 nm (BG), 540 nm (sG), 550 nm (dG), 580 nm (O), 610 nm (R), and 630 nm (dR) respectively. The spectral sensitivities of the V, O, R and dR receptors did not match the predicted spectra of any visual pigments, but with the filtering effects of the pigments around the rhabdom, they can be reasonably explained. In some of the receptors, negative-going responses were observed when they were stimulated at certain wavelengths, indicating antagonistic interactions between photoreceptors.     

The photoreceptor localization confirms the spectral heterogeneity of ommatidia in the male small white butterfly,<Emphasis Type="Italic"> Pieris rapae crucivora</Emphasis>

The compound eye of Pieris rapae crucivora contains ventrally three types of histologically distinct ommatidia. An ommatidium contains nine photoreceptors, four of which (R1-4) construct the distal tier of the rhabdom. We determined the sensitivity spectra of the R1-4 distal photoreceptors in each type of ommatidia by intracellular electrophysiology and identified UV, blue, double-peaked blue, green, and a green receptor with depressed sensitivity in the violet. We localized these receptors in each type of ommatidia by injecting dye after the recording. In type I ommatidia the R1 and R2 cells are UV and blue receptors. When R1 is UV sensitive, R2 is always blue sensitive, or vice versa. R3 and R4 in type I are both green receptors. In type II, R1 and R2 are both double-peaked blue receptors and R3 and R4 are both green receptors with depressed sensitivity in the violet. In type III, R1 and R2 are both UV, and R3 and R4 are green receptors. The double-peaked blue, and green receptors with depressed sensitivity in the violet in type II ommatidia have depressed sensitivity at 420 nm, which is probably due to the filtering effect of a fluorescing material present in the type II ommatidia. Spectral heterogeneity of ommatidia seems to be a common design of insect compound eyes.     

Septate junctions are required for ommatidial integrity and blood-eye barrier function in Drosophila

The anatomical organization of the Drosophila ommatidia is achieved by specification and contextual placement of photoreceptors, cone and pigment cells. The photoreceptors must be sealed from high ionic concentrations of the hemolymph by a barrier to allow phototransduction. In vertebrates, a blood-retinal barrier (BRB) is established by tight junctions (TJs) present in the retinal pigment epithelium and endothelial membrane of the retinal vessels. In Drosophila ommatidia, the junctional organization and barrier formation is poorly understood. Here we report that septate junctions (SJs), the vertebrate analogs of TJs, are present in the adult ommatidia and are formed between and among the cone and pigment cells. We show that the localization of Neurexin IV (Nrx IV), a SJ-specific protein, coincides with the location of SJs in the cone and pigment cells. Somatic mosaic analysis of nrx IV null mutants shows that loss of Nrx IV leads to defects in ommatidial morphology and integrity. nrx IV hypomorphic allelic combinations generated viable adults with defective SJs and displayed a compromised blood-eye barrier (BEB) function. These findings establish that SJs are essential for ommatidial integrity and in creating a BEB around the ion and light sensitive photoreceptors. Our studies may provide clues towards understanding the vertebrate BEB formation and function.     

UV photoreceptors in the compound eye of Daphnia magna (Crustacea,Branchiopoda). A fourth spectral class in single ommatidia

Summary The spectral sensitivities of individually stimulated ommatidia in the compound eye of Daphnia magna were measured using a fast spectral scan voltage-clamp technique with extracellular recording. Chromatic adaptation was used to reveal the contributions of individual spectral classes of photoreceptors to the ommatidial sensitivity. Ommatidia in the dorsal and ventral regions of the compound eye were tested. Four spectral classes of photoreceptors were found in each ommatidium, among them a previously undetected class with peak sensitivity in the ultraviolet. The wavelengths of peak sensitivity were at 348, 434, 525, and 608 nm for the dorsal ommatidia. The three longer wavelength classes agreed well with those found previously by intracellular recording (Schehr 1984). Only small differences in wavelength and magnitude of peak sensitivity were found between the four classes in the dorsal versus ventral ommatidia.     

The color-vision circuit in the medulla of Drosophila

BACKGROUND: Color vision requires comparison between photoreceptors that are sensitive to different wavelengths of light. In Drosophila, this is achieved by the inner photoreceptors (R7 and R8) that contain different rhodopsins. Two types of comparisons can occur in fly color vision: between the R7 (UV sensitive) and R8 (blue- or green sensitive) photoreceptor cells within one ommatidium (unit eye) or between different ommatidia that contain spectrally distinct inner photoreceptors. Photoreceptors project to the optic lobes: R1-R6, which are involved in motion detection, project to the lamina, whereas R7 and R8 reach deeper in the medulla. This paper analyzes the neural network underlying color vision into the medulla. RESULTS: We reconstruct the neural network in the medulla, focusing on neurons likely to be involved in processing color vision. We identify the full complement of neurons in the medulla, including second-order neurons that contact both R7 and R8 from a single ommatidium, or contact R7 and/or R8 from different ommatidia. We also examine third-order neurons and local neurons that likely modulate information from second-order neurons. Finally, we present highly specific tools that will allow us to functionally manipulate the network and test both activity and behavior. CONCLUSIONS: This precise characterization of the medulla circuitry will allow us to understand how color vision is processed in the optic lobe of Drosophila, providing a paradigm for more complex systems in vertebrates.     

Cell clones and pattern formation: On the lineage of photoreceptor cells in the compound eye ofDrosophila

Summary The generalogical relationships of photoreceptor cells within the compound eye ofDrosophila have been studied using cell labelling, with either³H-thymidine or recessive mutations, during the third larval stage. It has been found that photoreceptor and secondary pigment cells arise from different precursor cells. Under the present experimental conditions, precursors of receptor cells give rise to about 8 elements which differentiate as R cells of two different groups. One of the cells differentiates as R7 and the remaining as any one of the R1 to R6. The last cells behave initially as equivalent, and can differentiate within the same or within different, but neighbouring, ommatidia. The class of R1 to R6 cell in which each one of these elements differentiates, seems to depend on the time of its origin. The implications of these findings for the formation of the ommatidial pattern are discussed.     

Microvillar orientation in the photoreceptors of the ant Cataglyphis bicolor

Polarization sensitivity in arthropod photoreceptors is crucially dependent on the arrangement of the microvilli within the rhabdom. Here, we present an electron-microscopical study in which the degree of microvillar alignment and changes in the cross-sectional areas of the rhabdoms along their length were studied in the compound eye of the desert ant, Cataglyphis bicolor. Serial cross-sections through the retina were taken and the orientation of the microvilli was determined in the photoreceptors of individually identified ommatidia. The reconstructions of microvillar alignment were made in the three anatomically and functionally distinct regions of the Cataglyphis compound eye: the dorsal rim area (DRA), the dorsal area (DA), and the ventral area (VA). The following morphological findings are consistent with polarization sensitivities measured previously by intracellular recordings. (1) The microvilli of the DRA photoreceptors are aligned in parallel along the entire length of the cell from the distal tip of the rhabdom down to its proximal end, near the basement membrane. The microvilli of the retinular cells R1 and R5 are always parallel to each other and perfectly perpendicular, with only minor deviation, to the microvillar orientation of the remaining receptor cells. (2) In the DA and VA regions of the eye, the microvillar tufts of the small receptors R1, R3, R5, R7, and R9 change their direction repetitively every 1-4 7m for up to 90°. In contrast, the large receptor cells R2, R4, R6, and R8 maintain their microvillar orientation rigidly. (3) In the DRA ommatidia, the cross-sectional areas of the rhabdomeres do not change along the length of the rhabdom, but substantial changes occur in the DA and VA ommatidia.     

Neuronal differentiation in the Drosophila ommatidium

Using monoclonal and polyclonal antibodies as differentiation markers, we have found that the eight photoreceptors of the Drosophila ommatidium differentiate in a fixed sequence. The foundation photoreceptor, R8, expresses neural antigens first. The paired photoreceptors R2/5 are next to express, followed by the pair R3/4, followed by the pair R1/6; R7 is the final photoreceptor to differentiate. From previous studies it is known that Drosophila photoreceptors use local, positional cues to select their identities. Together with the morphological picture of ommatidial development, the sequential order of photoreceptor differentiation demonstrated here suggests that these cues may be encoded in the particular combination of cells an undetermined cell finds itself in contact with.