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The EGFR pathway and its roles in eye development

The Epidermal Growth Factor Receptor (EGFR) signal transduction pathway (and its homologs) has been found to play many crucial roles in the regulation of cell cycle, cell fate and patterning in all metazoan animals in which it has been studied (reviewed in 1, 2-11). In particular the EGFR pathway acts in several steps in retinal and other nervous system development in vertebrates and in Drosophila(12-16). We (and others) showed that the EGFR ligand Spitz acts as a non cell-type specific positive signal for photoreceptor and accessory cell differentiation (17-20).

Egfr in the Furrow
We made a new conditional Egfr mutation (Egfr tsla ) and observed the developmental outcome of removing EGFR activity at specific times in retinal development (21). The Extracellular signal Regulated protein Kinase (ERK, also known as the Mitogen Activated Protein Kinase and/or the Microtubule Associated Protein Kinase: MAPK) is the final cytoplasmic molecule in this signal transduction pathway and it has been shown to move to the cell nucleus on phosphorylation (to the active "dp-ERK" form, 22, 23-29). To determine exactly which cells do receive an EGFR mediated signal and when they do so, we used a monoclonal anti dp-ERK antibody (30-32). After removing EGFR function with our conditional Egfr mutation the dp-ERK antigen in the earliest stages of cluster formation is reduced within 15 and eliminated by 30 minutes (21). Furthermore, we have discovered a novel step in ERK regulation: the dp-ERK antigen is first accumulated in the cytoplasm for a period of hours and only later is it detectable in a sub-set of cell's nuclei for much shorter periods of time (minutes, 21). Interestingly we found that even though we can see Egfr dependent dp-ERK in the initial stage of ommatidial development, EGFR signaling has no role in cell patterning or fate at this early stage (its first function is some hours later). Thus we propose that this "cytoplasmic hold" correlates well with the developmental function of EGFR signaling at this stage: the ERK signal is blocked because some other regulatory input is keeping it from the nucleus, until a second signal can be received (21).

Egfr in Eye Specification
Seven "master control" genes have been reported to act in the specification of the compound eye: twin of eyeless (toy), eyeless (ey), eyes absent (eya), sine oculis (so), dachshund (dac), eye gone (eyg) and optix (opt). Where mutants are known, loss of these functions results in the failure of the eye to form, while their ectopic expression (except for so), is sufficient to induce ectopic eyes (33-35). The complexity of the genetic epistatic relationships between these seven genes and the observed in vitro interactions of their protein products suggests that they lie in a regulatory network and not in a simple linear hierarchy (33). The simplest hypothesis based on the published data is that these seven proteins form a multimeric complex that is an eye specifying transcription factor. One might expect them all to be co-expressed at an early time in a limited number of cells and that these cells are thus specified to form the eye.

However, it is clear that these proteins cannot be the sole determinants of eye fate, nor can they lie at the top of the regulatory hierarchy that controls eye specification. In loss of function mutants the presumptive eye disc does not adopt a different fate but degenerates late in development (33). In addition, the ability of these genes to direct eye development in non-eye tissue is limited to subregions of the antennal, wing and leg discs, in which the presence of the patterning genes Hedgehog (Hh) and Decapentaplegic (Dpp) are essential (33). Furthermore, all seven of these eye specification genes are nuclear factors that must receive patterning inputs from one or more signal transduction pathways.

Notch is a trans-membrane receptor activated by "DSL" class ligands that transduces signals to the nucleus by means of a pathway that includes the Enhancer of Split Complex genes (E(spl)C, 36). Notch acts in eye development in setting up the dorsal-ventral compartment boundary, in establishing planar polarity, in the spacing of ommatidial clusters and in cell fate specification (37-39). The Drosophila EGF receptor homolog (Egfr) is a trans-membrane receptor tyrosine kinase (RTK) that acts through the Ras cascade (11, 40). In the developing eye, Egfr signaling has been shown to control cell fate specification, inhibit programmed cell death and modulate cell cycle progression (41-44).

We showed that the Egfr and Notch signaling cascades act antagonistically as homeotic determinants of eye specification: hyperactivation of Egfr signaling or downregulation of Notch activity within the presumptive eye field leads to the complete transformation of the eye into an antenna (45). Under these conditions, the eye specification genes are no longer transcribed in the transformed tissue. We propose that both the Egfr and Notch pathways are genetically upstream of the known complex of eye specification genes and direct the formation of the eye and antenna. To our knowledge this is the first report of a homeotic function in organ specification for any receptor tyrosine kinase.

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