Building a fly eye: terminal differentiation events of the retina, corneal lens, and pigmented epithelia

Abstract

In the past, vast differences in ocular structure, development, and physiology throughout the animal kingdom led to the widely accepted notion that eyes are polyphyletic, that is, they have independently arisen multiple times during evolution. Despite the dissimilarity between vertebrate and invertebrate eyes, it is becoming increasingly evident that the development of the eye in both groups shares more similarity at the genetic level than was previously assumed, forcing a reexamination of eye evolution. Understanding the molecular underpinnings of cell type specification during Drosophila eye development has been a focus of research for many labs over the past 25 years, and many of these findings are nicely reviewed in Chapters 1 and 4. A somewhat less explored area of research, however, considers how these cells, once specified, develop into functional ocular structures. This review aims to summarize the current knowledge related to the terminal differentiation events of the retina, corneal lens, and pigmented epithelia in the fly eye. In addition, we discuss emerging evidence that the different functional components of the fly eye share developmental pathways and functions with the vertebrate eye.

Figure 5.1

Structure of an adult Drosophila ommatidium. Schematic of different regions of an adult ommatidium: the corneal lens region (top), the neural retina (middle), and the retinal floor (bottom). Corresponding regions from toluidine blue-stained semi-thin sections of an ommatidium are provided at the right. Color scheme is as follows: photoreceptor (PR) cell bodies, beige; PR rhabdomeres, dark gray cylinders (outer PRs), dark magenta cylinder (R7), or dark blue cylinder (R8); cone cells, green; primary pigment cells, yellow; secondary pigment cells, gray; tertiary pigment cells, turquoise; mechanosensory interommatidial bristle, purple hexagon; eye unit, longitudinal. The cone cells and primary pigment cells secrete the corneal lens (translucent pink) and a gelatinous pseudocone (translucent white). Each cone cell also extends an “interretinular fiber” between the photoreceptors, eventually expanding just proximal to the rhabdomeres to create a CC feet “plate” at the base of the retina. Based on the position within the ommatidia, the four cone cells are referred to as the apical (a), posterior (p), polar (pl), and equatorial (eq) cone cells. Secondary and tertiary pigment cells and the bristle form a characteristic hexagon around each ommatidia, with the pigment granules easily observed in the toludine blue stainings as reddish-brown (pteridine-containing) and black (xanthommatin-containing) vesicular-like structures. The apical surfaces of the secondary and tertiary pigment cells are tightly restricted, but the basal surfaces of these cells expand at the base of the retina to form a fenestrated membrane through which the axons project into the brain. The six outer photoreceptor rhabdomeres (gray from cells R1 through R6) form a trapezoid at the top of the eye and extend the length of the retina, enwrapping the IPR rhabdomeres—the R7 rhabdomere (Magenta) extends through the top two-thirds of the retina and the R8 rhabdomere (Blue) occupies the bottom third. In addition, the cell body of the R7 is positioned between the R1 and R6 cell, whereas the R8 cell body is located between the R1 and R2 cell, seen by cross section (middle diagrams and thin sections). The interhabdomeric space (white) that is important for preventing rhabdomere fusion is also seen. The entire central portion of the ommatidia is encapsulated by the cone cells—distally, with the rhabdomeres attached by “hemidesmosome-like” contacts, and proximally, with the rhabdomeres attached to the cone cell feet just below the end of the rhabdomere.

Figure 5.2

Time course of Drosophila eye development. A summary of various developmental processes that occur during Drosophila pupal eye development (0–100%). Prior to pupation, in late third instar larva, the antennal/eye disc (A) is easily recognized by strong Cut expression (green) in the antennal portion (anterior, left), and clusters of Elav-positive photoreceptor clusters (blue) in the eye portion (posterior, right) corresponding to individual ommatidial units. Cut-positive cells are also present in the eye-imaginal disc, which represent subretinal glia and CCs precursors. Nonstained cells anterior to the morphogenetic furrow (MF) are retinal progenitors that are still proliferating (see Chapters 1 and 4 for further description). (B) The constricted apical surface of cells within the MF is obvious with E-Cadherin staining (green). In addition, the boundary between the R3 and R4 cell, marked by intense N-cadherin staining (purple), reveals the rotation of the ommatidia relative to the equator that is important for establishing the chiral trapezoid of photoreceptors observed in the adult retina. (C) E-cadherin staining (green) of a whole retina isolated from pupa at ~50% pupation shows the highly regular organization of ommatidia. Inset: A single ommatidium is circled. (D) Photoreceptor-driven Moesin::GFP at 50% pupation shows outer PR axons projecting to the lamina and IPR axons projecting to the medulla. (E) Cut (green) and BarH1(Magenta) specifically recognize the four CC and two primary pigment cell (PPC) nuclei at 50% pupation. (F) Discs Large (Dlg, Purple) highlights the apical contacts of the CCs, PPCs and interommatidial cells in 50% pupal retinas. (G) E-cadherin (green) of the basal surface of the retina shows the petal-shaped distribution of the IOC feet. (H) The bristle cell lineage is composed of four cells which express the transcription factors Cut and Pros, and the neural factor Elav. These nuclei are present at the base of the retina during their development, and eventually move more apically. (I) A scanning electron micrograph of an adult eye pseudocolored to represent the distribution of the pale (blue), yellow(green) and Dorsal Rim Area (DRA; magenta) ommatidia in the eye. (J) Whole mounted adult retina immunostained with Rhodopsin 5 (blue) and Rhodopsin 6 (green) in R8 rhabdomeres. Note the enrichment of Rh6 in the dorsal portion of the retina, corresponding to the dy ommatidia (see text for more detail).

Figure 5.3

Rhabdomere morphogenesis. (A) Coronal TEMs showing the apical membrane elaborations of photoreceptors R1 through R7 at different stages of development. The zonula adherens are marked with blue, the stalk region is highlighted in red, and the interrhabdomeric space (IRS) is the clear space between rhabdomeres that are obvious by 78% pupation (modified from Longley and Ready, 1995, with permission from Elsevier). Some of the interretinular fibers from cone cells, found directly adjacent to the zonula adherens are highlighted in green. (B) Diagram of the 90° turn of the photoreceptor apical surfaces during early pupation and elongation of the rhabdomeres (gray), the stalk region (red), and zonula adherens (blue) at later stages of development. Only two cone cells (green) and two photoreceptors are shown for clarity.

Figure 5.4

Axonal targeting differences between outer and inner photoreceptors. Diagram representing two ommatidia sharing lamina cartridges. The axons from the six outer PRs from each ommatidium turn 180° and project to six different cartridges present in the lamina neuropil present directly underneath the retina. R1–R6 positions within the lamina represent a mirror image of the outer photoreceptor arrangement found in the retina. The R7 (magenta) and R8 (blue) axons bypass the lamina and project to layers M3 and M6 respectively in the adult medulla.

Figure 5.5

Regulatory sequences of the inner photoreceptor Rhodopsin-encoding genes. Schematic of the minimal promoters for Rh3 through Rh6 that recapitulate expression of the endogenous genes. Senseless binding sites (S) are green, Otd binding sites (K50) are light blue, Pax6/RSCI sites (Rhodopsin Conserved Sequence I) are pale pink and Pros sites (seq56) are dark magenta. Rhodopsin Unique Sequences (RUS) 3, 4, 5, and 6 are represented by striped boxes. The summary of the role of each transcription factor is highlighted to the right. Otd activates Rh3 and Rh5, the two Rhodopsins expressed the pale ommatidia, and represses Rh6 in outer photoreceptors (Tahayato et al., 2003). Pros represses the R8 Rhodopsins, Rh5 and Rh6, in R7 photoreceptors (Cook et al., 2003), while Sens represses the R7 Rhodopsins, Rh3 and Rh4, in R8 photoreceptors (Xie et al., 2007). A transcription factor that is predicted to be activated by Spineless in yellow R7 cells to activate Rh4 is indicated by a ? on the Rh4 promoter. In addition, Hazy has recently been shown to be necessary and sufficient for Rh6 expression and bind to the RCSI, making it possible that Hazy, and not Pax6, is responsible for activating the Rh6 promoter in the fly eye (Mishra et al., 2010).

Figure 5.6

Ommatidial subtypes express different inner photoreceptor Rhodopsins. (A) A whole-mount staining of an adult retina stained with phalloidin (gray) shows the trapezoidal arrangement of the actin-rich rhabdomeres of the six outer photoreceptors and the random distribution of the pale and yellow ommatidia are revealed by immunostaining for Rh5 (blue) and Rh6 (green) that are expressed in the central R8 cells. (B) Diagram of the Dorsal Rim Area (DRA), dorsal yellow, pale, and yellow subsets found in the Drosophila eye, defined by the Rhodopsins expressed in the R7 and R8 inner photoreceptors. All outer photoreceptors express the same Rhodopin, Rhodopsin 1. (C) Transverse sections of adult eyes, dorsal left, stained with R7 Rhodopsins (left), Rh3 (cyan) and Rh4 (red), or R8 Rhodopsins (right), Rh5 (blue) and Rh6 (green). Note that two rows of ommatidia at the dorsal side of the eye express Rh3 in the R7 and R8 layers, representing the DRA ommatidia. Rh3 and Rh4 expression in the dy ommatidia are weaker than in the remainder of the eye. (D) Schematic representing the factors that direct inner photoreceptor identity, differentiation, and rhodopsin expression. The relative position of the nuclei that would be in the cell body for the different cell types are also indicated. See text for detail.

Figure 5.7

Events leading to Drosophila corneal lens formation. (A) A third instar imaginal disc, stained with Elav (blue) to mark specified photoreceptors and the transcription factor Prospero (green), to mark the R7 photoreceptor and the cone cell precursors. The side view shows that the nuclei of cell move from a basal to apical position as they are recruited. (B) A high magnification of the cone cell layer from a single ommatidium shows that distinct subpopulations of cells that express different levels of Prospero (green), dPax2 (magenta), and Cut (blue) exist. This also is represented diagrammatically, with high Pros expression in equatorial (eq) and polar(pl) CCs, and high dPax2/Cut expression in anterior (a) and posterior (p) CCs. (C) Drosocrystallin (magenta) begins to be made in CCs, marked with Cut (green) at 50% pupation and is secreted from the cells by 75%. Drosocrystallin is also expressed at lower levels in the interommatidial bristle lineage (arrows). (D) A transmission electron micrograph of an adult ommatidium, pseudocolored to highlight the striated corneal lens (magenta), the clear pseudocone (gray), the primary pigment cells (PPCs, yellow), the cone cells (CCs, green), and the secondary/tertiary pigment cells (IOCs, purple). Note the abundant, large pigment granules in the IOCs, that the PPCs outline the CCs and pseudocone, and that the CCs lie between the pseudocone and the tips of the photoreceptor rhabdomeres. (E) Top and side view schemata of lens development, beginning from the imaginal disc through different stages of pupation using the same color scheme as in Fig. 5.1. The apical surface contacts change between the a/pCCs and eq/pl CC during pupation, patterning, and pruning of the IOCs occur prior to 30% pupation, and the corneal lens is secreted by ~75%. Afterward, the pseudocone is secreted and pushes the cone cells away from corneal lens.

Figure 5.8

Scanning electron micrographs of adult eyes from wild type (A,E, E′), dPax2 spa^pol mutants (B,G, G′), whole eye prospero (pros) mutants (C,H), pros/spa^pol double mutants, and BarH1 mutants (F,F′; modified from (Higashijima et al., 1992), with permission from Genes and Development). The spapol mutant is a dPax2 hypomorph that shows differences in lens phenotypes between the anterior (G) and posterior (G′) portions of the eye. All images are oriented with anterior to the left. A summary of the role for these various CC and PPC expressed transcription factors is shown in J. See text for more detail.