eyegone (eyg) is required for eye development. Loss-of-function eyg mutations cause reduction or absence of the eye. Similar to the Pax6 eyeless (ey) gene, ectopic expression of eyg induces extra eye formation, but at sites different from those induced by ey. Several lines of evidence suggest that eyg and ey act cooperatively: (1) eyg expression is not regulated by ey, nor does it regulate ey expression; (2) eyg-induced ectopic morphogenetic furrow formation does not require ey, nor does ey-induced ectopic eye production require eyg; (3) eyg and ey can partially substitute for the function of the other, and (4) coexpression of eyg and ey has a synergistic enhancement of ectopic eye formation. These results also show that eyg has two major functions: to promote cell proliferation in the eye disc and to promote eye development through suppression of wg transcription (Jang, 2003).

Two enhancer trap lines (Eq-1, Eq-2) have been identified with the P[lacW] construct inserted near the eye gone (eyg) gene in 69C on the third chromosome. The two lines show no eyg phenotype. Starting from these lines, a large number of derivative lines were generated (by gamma-irradiation induced chromosomal aberrations, P-element imprecise excisions and local transpositions), some of which showed eye reduction phenotypes and failed to complement eyg¹ (Jang, 2003).

Weak loss-of-function eyg mutations result in the reduction or absence of the adult eyes. In late third instar larvae of the hypomorphic eyg¹ mutant, the eye discs are significantly reduced in size, while the antennal discs appeared normal. In strong loss-of-function mutants, the adults fail to emerge from the pupal case. Their heads are severely reduced in size, but they appear normal. In a null mutant eyg^M3-12, the adults have a headless phenotype and all structures derived from the eye-antennal discs are missing. The prominent remaining structure is the labellum derived from the labial discs. The fish-trap bristles, derived from the clypeo-labral disc, are also present. The headless phenotype is similar to those reported for ey and toy null mutants. In flies with eyg alleles of different strengths, the size reduction of third instar eye discs is proportional to the severity of the adult eye phenotype. Strong alleles do not affect the morphology and size of the antennal discs. However, no eye-antennal discs can be found in eyg^M3-12 larvae. These observations suggest that the antennal disc requires only a low Eyg level or activity, so the antennal phenotype is manifested only in the null mutant, a situation similar to those of ey and toy mutants. Alternatively, the effect of eyg may be specific to the eye disc, and the loss of antennal disc-derived structures in null mutants may be a secondary effect due to the missing eye disc (Jang, 2003).

The effect of eyg is already apparent in earlier stages. Strong eyg alleles result in no eye disc or only a rudimentary stub of a disc in early third instar. Weaker allelic combinations produce a smaller eye disc than normal in early third instar, and in mid-third instar excessive cell death could be detected anterior to the morphogenetic furrow by staining with the dye Acridine Orange. Similar apoptosis anterior to the furrow has been observed in other small eye or eyeless mutants, e.g. eya, so, dac and ey. In late third instar, there is no more excessive apoptosis (Jang, 2003).

The small size of early third instar mutant eye discs indicates either that early cell proliferation is affected, or that there is excessive apoptosis prior to third instar, or both. Misexpression of the anti-apoptosis baculoviral protein P35, driven by the dpp-GAL4 or ey-GAL4, fails to rescue the 'no eye' phenotype in the eyg¹/eyg^M3-12 mutant. The eyg¹/eyg^M3-12 mutant has complete absence of the adult eyes and has rudimentary eye discs. The adult eyes and the larval eye discs are not rescued by misexpression of P35, suggesting that apoptosis is not the major cause of the eye phenotype (Jang, 2003).

A second Pax gene was identified about 30 kb downstream of eyg, based on the fly genome sequence. The predicted gene is represented by an EST clone. Its encoded protein is most homologous to Eyg in its paired domain and homeodomain, so the gene was named twin of eyg (toe). In the eye disc, the expression pattern of toe is very similar to that of eyg. Three independent lines, 37-1, 22-2 and 94-4, were generated from mobilization of P[GawB]^EM458, which has the insertion 124 bp upstream of the Lune transcription start site and 527 bp upstream of the first ATG. The P[GawB] has transposed 8 bp downstream in all three lines, and is accompanied by an 86 bp, 159 bp and 224 bp deletion, respectively, of the flanking genomic region. Thus eyg^22-2 and eyg^94-4 have deletions extending into the 5' untranslated region. eyg^37-1 is homozygous viable and results in a very weak small eye phenotype. eyg^22-2 is homozygous viable and produces a small eye (about 500 ommatidia) phenotype. The eyg^94-4 homozygote dies at the pharate adult stage. The phenotypes of pharates range from nearly headless, to complete absence of eye, to small eyes. These mutations fail to complement eyg¹, so they are eyg alleles as defined by genetic complementation. In these mutants, eyg mRNA is strongly reduced in the eye disc and in the antenna disc, while toe mRNA level is not significantly affected. The eyg^M3-12 mutant has a large deletion starting at 23 bp upstream of the eyg transcription start site and extending to about 13 kb downstream of eyg. The molecular nature of these mutations, together with the rescue results, strongly suggest that the eyg gene is a Pax gene represented by the Lune cDNA (Jang, 2003).

Rescue experiments (hs-eyg, ey>eyg and E132>eyg) suggest that the critical time for eyg function is in the late second instar. Excessive apoptosis occurrs in the mid-third instar eye disc, but is not the major cause of the eye phenotype because blocking apoptosis does not rescue the eye phenotype. Ectopic expression of eyg can lead to ectopic MF initiation in the ventral side of the eye disc. Thus, the loss-of-function and gain-of-function phenotypes suggest that eyg acts as an important regulator of eye development (Jang, 2003).

eyg appears to have two major functions. The first is to promote cell proliferation in the eye disc. eyg loss-of-function mutants have reduced eye discs, already apparent in early third instar, before photoreceptor differentiation. In clonal analysis, eyg^M3-12 mutant clones induced in first or second instar are undetectable in late third instar eye disc. Ectopic eyg expression causes local overgrowth, a phenotype opposite that of the loss-of-function phenotype. The overgrowth does not always develop into photoreceptor cells. These results indicate that eyg promotes cell proliferation independent of photoreceptor differentiation. The second function of eyg is to promote eye development or MF initiation. If the eyg-induced proliferation occurs at the ventral margin of the eye disc, ectopic MF can initiate. The induction of ectopic MF is probably mediated by the suppression of wg, which is known to repress MF initiation along the lateral margins (Jang, 2003).

Since eyg is a Pax gene that shares sequence similarity with ey and toy in the PD and HD domains, its relationship with ey is of particular interest. The results indicate that eyg and ey are transcriptionally and functionally independent for two reasons. (1) Except for a small amount of eyg expression ventral to the equator of the eye disc, eyg and ey do not regulate each other's expression. In this respect, eyg is different from dac, so and eya, whose expression is strongly regulated by ey (and can induce ey expression in some cases). Thus eyg transcription is neither downstream of ey, nor does eyg participate in the ey/eya/so/dac positive feedback loop. This transcriptional independence is similar to that of optix. (2) eyg and ey can each function (to induce ectopic eyes) in the absence of the other. Again, this is similar to optix, which can induce ectopic eyes in ey² mutant. Whether optix is required for ey function has not been tested, because of the lack of optix mutants (Jang, 2003).

However, other evidence indicates that the functions of eyg and ey must converge at some point in the pathway leading to eye development: (1) eyg; ey double hypomorphic mutants show a much stronger eye-loss phenotype; (2) coexpression of ey and eyg causes synergistic enhancement of the ectopic eye phenotype; (3) eyg and ey are able to substitute functionally for each other. Overall, the results suggest that these two Pax genes may act cooperatively. This genetic cooperativity might mean that eyg and ey interact and cooperate as proteins in the same pathway or that they act in parallel pathways. eyg and ey are coexpressed in the eye disc primordium in the embryo. Their expression domains also overlap in the eye disc, especially in the early eye disc when eyg function is critically required. So it is possible that the two Pax proteins act within the same cell, although the possibility they act in different cells to achieve a functional cooperativity has not been ruled out (Jang, 2003).

If eyg and ey are both required for eye development, how could ectopic expression of either one be sufficient for ectopic eye development? One possibility is that the two Pax proteins form heterodimers, directly or indirectly via other proteins, to activate target genes. When the level of either one is low, the target genes that lead to eye formation cannot be induced. However, when either one is strongly expressed ectopically, the high level of homodimer can partially substitute for the heterodimer. Since both genes are required for normal eye formation, this model predicts that the Eyg-Ey heterodimer is more effective than either homodimer in inducing eye formation. As expected by this model, coexpression of eyg and ey causes enhanced ectopic eye formation (Jang, 2003).

Dpp and Wg are two signaling molecules important for the initiation of eye differentiation: Dpp activates MF initiation while Wg suppresses it. Does eyg exert its effect on eye development by activating Dpp signaling or by suppressing Wg signaling? dpp is expressed at two stages in the eye disc: an early expression along the posterior and lateral margins, and a later expression in the propagating MF. The early expression in the margins is required for MF initiation. It was found that dpp expression along the lateral margins is absent in early third instar eyg¹ eye disc, suggesting that dpp expression in the lateral margins is regulated by eyg. However, activating DPP signaling at the lateral margin does not rescue the eyg¹ phenotype, suggesting that eyg has other functions in addition to activating dpp expression (Jang, 2003).

wg is expressed uniformly in the eye disc of second instar larvae. In the third instar eye disc, wg is expressed in the lateral margins and acts to prevent MF initiation from the lateral margins. The wg-expression domain expands in eyg¹ eye discs. The results further show that ectopic eyg expression (dpp>eyg) can suppress wg expression at the transcriptional level. The suppression of wg is functionally significant, because expression of the wg-activated omb gene is similarly suppressed in dpp>eyg. Blocking of the Wg signaling pathway can partially rescue the eyg mutant phenotype. These results indicate that the suppression of wg transcription by eyg may be a major mechanism by which eyg induces MF initiation, hence eye development. This is consistent with the finding that ectopic eyg induces ectopic eye formation primarily in the ventral margin of the eye disc, where wg expression is weaker and most easily suppressed by dpp. wg is normally expressed in the entire eye disc during second instar. It has been shown that Wg signaling can suppress the expression of so and eya. It is possible that in the late second instar eye disc, eyg expression in the central domain of the eye disc suppresses wg expression in the central domain, thus allowing the expression of eya and so, hence eye development (Jang, 2003).

As predicted by the eyg and ey interaction, ey also suppresses wg expression. Suppression of wg expression by eyg (and ey) is also seen in the wing disc. However, suppression does not occur in all cells expressing eyg, suggesting that additional factors are required for the wg suppression. The relationship of eyg/ey and wg may be mutually antagonistic, since ectopic ey cannot induce eya and so expression in regions of high wg expression (Jang, 2003).

The eyegone (eyg) gene is involved in the development of the eye structures of Drosophila. eyg and its related gene, twin of eyegone (toe), are also expressed in part of the anterior compartment of the adult mesothorax (notum). The anterior compartment is termed the scutum and consists of the part of the notum from the anterior border to the suture with the scutellum. In the absence of eyg function the anterior-central region of the notum does not develop, whereas ectopic activity of either eyg or toe induces the formation of the anterior-central pattern in the posterior or lateral region of the notum. These results demonstrate that eyg and toe play a role in the genetic subdivision of the notum, although the experiments indicate that eyg exerts the principal function. However, by itself the Eyg product cannot induce the formation of notum patterns; its thoracic function requires co-expression with the Iroquois (Iro) genes. The restriction of eyg activity to the anterior-central region of the wing disc is achieved by the antagonistic regulatory activities of the Iro and pnr genes, which promote eyg expression, and those of the Hh and Dpp pathways, which act as repressors. It is argued that eyg is a subordinate gene of the Iro genes, and that pnr mediates their thoracic patterning function. The activity of eyg gives rise to a new notum subdivision that acts upon the pre-extant one generated by the Iro genes and pnr. As a result the notum becomes subdivided into four distinct genetic domains (Aldaz, 2003).

A significant functional feature of eyg/toe is that it is unable to induce notum structures by itself, but requires co-expression of its activator the Iro gene, and probably pnr. For example, whereas ectopic eyg/toe activity induces scutum-like structures in the scutellum (which is also part of the notum and which expresses pnr), it fails to do so in most of the wing. Interestingly, it only induces notal structures in the middle of the wing, precisely the place where there is Iro gene activity in normal development. This mode of action is unlike that of selector or selector-like genes, such as the Hox genes, en, Dll, pnr or the Iro genes, which are able to induce, out of context, the formation of the patterns they specify. This indicates that eyg/toe is not of the same rank, but that it is developmentally downstream of the Iro genes and pnr, and appears to mediate their 'thoracic' function. The restriction of eyg/toe activity to the thorax, unlike the Iro genes and pnr, which are also expressed in the abdomen, is fully consistent with this role. eyg/toe is also expressed in a similar domain in the metathorax, suggesting that it may perform a parallel role in this segment (Aldaz, 2003).

The eyg/toe expression domain occupies the larger part of the notum, extending from the anterior border to the suture between the scutum and the scutellum. This domain coincides with the region affected in the eyg mutations, and is consistent with the gain-of-function experiments. Thus the eyg/toe expression domain corresponds to the zone where eyg/toe function is required. Since it is only a part of the notum, a question of interest is to find out how eyg/toe expression is restricted to this zone. This restriction is necessary for the appearance of distinct anterior-central and posterior-lateral subdomains, for if eyg/toe is expressed uniformly, as in ap-Gal4 > UAS-eyg flies, the entire notum develops as the anterior-central domain (Aldaz, 2003).