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1.4.1 Introduction to Drosophila eye development

The Drosophila adult eye is made up of about 800 units called ommatidia, which

contain eight photoreceptor cells, called R l-8, as well as cone cells and pigment cells (Wolff and Ready 1993, figure 1.6). Drosophila eyes develop from two eye-antennal imaginai discs which give rise to most of the adult head. The eye part of the disc forms the eye and most of the head capsule and the antennal part forms the antenna, rostral membrane and maxiallary palpus (Cavodeassi et al. 2000). The cells that make up the eye- antennal disc are specified during embryogenesis and the disc develops as a monolayer epithelium during larval stages (Wolff and Ready 1993). The eye and antennal parts of the disc appear morphologically different early in development, but eye or antennal fate is not pre-determined and depends on a number of signalling cascades and restricted expression

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of transcription factors. Until recently it was thought that the specification of the eye field was completely under the control of the so-called “master control” genes: twin o f eyeless

{toy), eyeless (ey), eyes absent (eya), sine oculis (so), dachshund {dad), and eye gone {eyg)

(Treisman and Heberlein 1998). The absence of any of these genes leads to reduction or loss of the eye and ectopic expression can induce ectopic eyes in other imaginai discs (except so). However, ectopic eyes can only be induced in specific parts of other imaginai discs suggesting that the presence (or absence) of other factors is necessary to allow eye development. The seven genes encode nuclear factors and form a complex regulatory network which involves both regulation of each others expression and direct protein- protein interactions. The Pax6 homologues toy and ey seem to be at the top of the hierarchy as expression of these genes occurs first in the eye-antennal disc primordium. However, specification of eye versus antennal fate apparently occurs much later in development.

The EGFR and Notch signalling pathways seem to be upstream of the master control genes for eye development in specifying the difference between eye and antennal fates (Kumar 2001 figure 1.7). Activation of the EGFR is required for the subdivision of the wing disc into notum and wing regions and it seems that it is also important for subdivision of the eye-antennal disc. Hyperactivation of the EGFR as well as expression of some of the downstream components of the pathway, leads to the transformation of eye into antenna. In addition, inactivation of the Notch signalling pathway also induces this transformation. The transformation to antennal fate is accompanied by the loss of expression of toy, ey, eya, os, and eyg suggesting that EGFR and Notch signalling repress and activate these genes respectively. The critical time for specification of eye versus antennal fates seems to be during the second larval instar. This is also the time at which the expression of all the “master control” begin to overlap at the posterior portion of the eye disc (Kumar and Moses 2001).

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Figure 1.6 The Drosophila adult eye

Figure 1.6 Figure courtesy of Helen McNeill. A) Scanning electron microscopy picture of the Drosophila adult eye showing the external structure of the -800 ommatidia and the inter-ommatidial bristles. B) Section through adult eye (dorsal is up) showing the trapezoid arrangement of the rhabdomeres. The ommatidia in the dorsal half of the eye have opposing polarity and chirality compared to those in the ventral half. C) Schematic of an individual dorsal ommatidium indicating the positions of each of the photoreceotors.

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Investigations into the specification of antennal fate has been mostly focused on the specification of antenna versus leg and not the distinction between the antennal and eye fates. Early patterning events in antennal and leg discs are similar involving the establishment of reciprocally exclusive domains of Wg and Dpp expression (Theisen et a l

1996). Accumulation of both Wg and Dpp activates the expression of distalless (dll) in both discs. However, the presence of homothorax (hth) in the antennal disc ensures the specification of antennal versus leg fates (Dong et a l 2000). The results described above suggest that activation of the EGFR pathway as well as the absence of Notch signalling is necessary to specify antennal versus eye fate. This could involve the regulation of one or more of the genes mentioned above (figure 1.7).

Early patterning of the eye disc involves establishment of the dorso-ventral and anterior-posterior axis (Treisman and Heberlein 1998. figure 1.7). During early development, possibly before the second larval instar, the eye disc is divided into dorsal and ventral compartments. There has been some debate concerning the characterisation of the dorsal and ventral regions in the eye disc as true compartments. Although there is a general restriction for clones to cross the D/V border, progeny from a single cell can give rise to ommatidia in both the dorsal and ventral halves of the eye. However, the discovery of the restricted expression of factors in the dorsal and ventral halves of the disc indicates that they are indeed true compartments. As with the wing disc, growth and patterning of the eye disc depends on the establishment of a signalling centre at the boundary of the D and V compartments. In the eye disc the D/V compartment boundary is also marked by a boundary of Fringe expression (Cho and Choi 1998; Dominguez and de Celis 1998). Fringe is a glycosyltransferase and is expressed in the ventral half of the eye. Fringe affects the interactions of the Notch receptor with its ligands Delta and Serrate and this causes activation of Notch signalling in a narrow band around the midline. The Iro-C is essential for the D/V patterning of the eye and this will be discussed in detail below.

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Figure 1.7 Models for early and late patterning of the eye disc

Egfr Notch

Hh W g

Figure 1.7 A) From Kumar and Moses 2001, EGFR and Notch signaling pathways are suggested to be upstream of the “master control” genes for eye development in the specification of eye versus antennal fate. Notch activation induces eye development and represses antennal development and EGFR activation represses eye development and induces antennal development. B) From Cho and Choi, 1998. The third instar eye disc is patterned in the anterior-posterior and the dorso-ventral axis. Wg and Dpp divide the disc into anterior and posterior regions. Mirror and the Iro-C are involved in setting up the D/V boundary which becomes a signaling center. The intersection of Notch activity at the midline with the posterior margin leads to localised activation of Flh and the differentiation of photoreceptors.

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The eye disc is also patterned in the anterior-posterior axis although there is no A/P compartment boundary (Treisman and Heberlein 1998). As mentioned above, the posterior part of the disc is where differentiation of the photoreceptors of the eye will begin. This part of the disc is marked by the expression of the eye specification genes and by Dpp expression at the margin. The most anterior parts of the eye disc will give rise to the dorsal and ventral head capsule (Pichaud and Casares 2000). In the third instar, this part of the disc is marked by the expression of Wg and Homothorax. Both Wg and Hh are upstream of the Iro-C in D/V patterning (see below) and the establishment of mutually exclusive Wg and Dpp domains is important for A/P patterning. (Treisman and Heberlein

1998, figure 1.7).

The differentiation of photoreceptors is marked by an indentation called the morphogenetic furrow (MF) which moves across the disc from posterior to anterior (Treisman and Heberlein 1998 , figure 1.8). The MF is induced at the posterior margin of the disc where it is intercepted by the D/V boundary. The initiation of differentiation is dependent on the presence of Dpp and Notch signalling and is marked by the localised upregulation of Hh. The progression of the furrow is dependent on continuous Hh signalling. As the furrow moves across the disc, the differentiating photoreceptors express Hh which induces Dpp in cells within the furrow. Neuronal differentiation is a pre­ requisite for Hh expression ensuring that the movement of the furrow is linked to the progression of photoreceptor specification. Hh and Dpp signalling control the rate of furrow progression by inducing proneural genes just ahead of the furrow and antineural genes in a region more anterior to the furrow where signalling activity is lower. In addition to the morphological changes occurring in the MF, the progression of the furrow is also marked by synchronisation of the cell cycle (Baker 2001). In response to Dpp signalling, cells just anterior to the furrow are arrested in G l. The differentiation of photoreceptors is thereby also coordinated with regulation of the cell cycle.

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Figure 1.8 The morphogenetic furrow and photoreceptor development