InteractiveFly: GeneBrief

eyegone: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

BIOLOGICAL OVERVIEW

In the process of branching morphogenesis, as seen for example in tracheal development, the final tubular network is formed by repeated branching of large tubes to form ever finer tubes. The seeming reverse of this process is another form of morphogenesis in which small tubes join together to form ever larger and less numerous tubes. This joining, rather than branching, morphogenesis has been little studied, although it is not uncommon. An example of joining morphogenesis occurs during tracheal development: dorsal tracheal branches from each segment fuse with their counterparts from the other side of the embryo to connect the left and right sides of the tracheal system. Similarly, segmental branches fuse along the sides of the embryo to form the lateral tracheal trunks that connect the tracheae of different segments. Formation of the larval salivary glands in Drosophila provides a simple example of joining morphogenesis. During salivary invagination, ducts from the two sides of the embryo meet at the ventral midline and fuse so that continued invagination produces a single common duct that connects to the oral cavity. The Pax homeodomain transcription factor eyegone plays a role in the process of joining morphogenesis that is crucial to the formation of salivary gland ducts (Jones, 1998 and references).

Before a discussion of eyegone in particular, there follows a description of joining morphogenesis in salivary tissues. This process can be separated into three successive events: formation first of the salivary glands, then the individual ducts, and finally the common duct. Initial specification and the primary patterning events of salivary glands involve multiple genes, including Sex combs reduced and Forkhead. Once these patterning events are complete, the cells begin characteristic morphogenetic movements that result in mature salivary glands and ducts. At the end of stage 11 (about 7 hours of development), the most posterodorsal pregland cells begin to invaginate. The site of invagination progresses anteriorly until all of the pregland cells have been internalized, forming a tubular salivary gland with a single layer of secretory cells surrounding a tubular lumen. As the gland cells invaginate, the preduct cells rearrange to form two parallel rows of cells extending across the ventral midline. These preduct cells will form the two types of duct tissue: those cells in the posterior row become the individual ducts and those in the anterior row become the common duct. The lateral ends of the individual ducts remain in contact with the gland cells; when these cells invaginate, they continue the tube that was started during gland invagination. The individual duct invagination continues to the ventral midline where the left and right sites of invagination fuse. Finally, the common duct is formed as the anterior row of duct cells move anteriorly and begin to invaginate. The resulting structure connects the lumen of the individual ducts to the pharynx (Jones, 1998 and references).

In wild-type embryos, the salivary ducts arise as a result of two successive so-called 'convergence and extension' events. Convergence and extension is a common developmental process during which cells intercalate to narrow the tissue in one axis while at the same time lengthening it along the axis perpendicular to the axis along which the cells narrow. As the germ band is retracting in wild-type embryos, the duct primordium narrows from about 6-8 cells to 2 rows of cells in the anterioposterior axis. At the same time, the primordium extends laterally across the ventral midline. The result of this first convergence and extension event is two parallel rows of cells separated by a cleft. The anterior row of cells is fated to become the common duct while the posterior row is fated to become individual ducts. In this process of joining morphogenesis the Pax gene eyegone (eyg) plays a critical role. eyg is required to distinguish individual from common duct domains and is necessary for the morphogenesis of the cells of the individual ducts. It is expressed specifically in individual duct cells and is critical for development of these cells as individual rather than common duct. In eyg-mutant embryos, presumptive individual duct cells are converted to common duct and individual duct cells are defective in convergence and extension. The posterior row of cells expresses eyg and will form the individual ducts. However, the anterior row of non-eyg-expressing cells, however, converges towards the ventral midline and extends anteriorly to form the common duct. In embryos mutant for eyg, the duct primordium never completes the first convergence and extension. Instead of forming the two parallel rows of cells extended across the ventral midline, the preduct cells remain as compact clumps, often slightly separated by the ventral midline. Later, however, the duct cells in eyg-mutant embryos move anteriorly in a process resembling that of wild type, although they do not obviously converge towards the ventral midline. In summary, in eyg-mutant embryos, the duct primordia fail to converge and extend across the midline resulting in the absence of individual ducts. Invagination of the salivary gland cells begins normally but becomes temporarily stalled at the gland/duct boundary until the gland cells finally break loose from the duct cells. In these mutant embryos, many of the presumptive individual duct cells join with the presumptive common duct cells to form an unusually large common duct that does not connect to the glands (Jones, 1998).

eyegone has other roles as well. The eyes absent and eyegone genes encode members of a group of nuclear proteins required to specify the fate of the eye imaginal disc (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). In the absence of eyg, no photoreceptors differentiate and the eye disc does not reach its normal size and shape. Both eyes absent and eyegone are required for normal activation of decapentaplegic expression at the posterior and lateral margins of the disc and also repression of wingless expression in presumptive retinal tissue. The requirement for eyegone can be alleviated by inhibition of the wingless signaling pathway, suggesting that eyegone promotes eye development primarily by repressing wingless. The expression of dpp and wg were examined in eya mutant eye discs. Expression is greatly reduced in early third instar eya mutant discs, prior to the initiation of the morphogenetic furrow, and was completely lost in eya mutant clones, suggesting that eya is required for dpp transcription. Although the initiation of wg expression in early eya mutant eye discs appeared to be normal, ectopic Wg protein was observed in eya mutant clones in late third instar discs. The expression patterns of dpp and wg were also examined in early third instar eyg mutant discs. dpp expression is restricted to the posterior margin of eyg mutant discs, in contrast to its expression around the posterior and lateral margins of wild-type discs. On the contrary, wg expression is expanded, especially on the dorsal side of the disc, where it extends to the posterior margin. eyg thus acts to delimit the domains of dpp and wg expression; since it encodes a Pax-like transcription factor, it is possible that this regulation is direct. Since inhibition of the wg pathway at the posterior margin of eyg mutant discs is sufficient to allow photoreceptor formation, it has been concluded that the misexpression of wg observed at the posterior of the eyg mutant discs is a major cause of the absence of photoreceptor development. Since activated Ras can overcome the effect of ectopic wg, activated Ras has also been shown to be able to rescue photoreceptor differentiation in eyg mutant discs. In summary, the expression patterns of both dpp and wg, and perhaps their cross-regulatory interactions, are determined during early eye development by genes including eya and eyg (Hazelett, 1998).

Differential requirements for the Pax6(5a) genes eyegone and twin of eyegone during eye development in Drosophila

In eye development the tasks of tissue specification and cell proliferation are regulated, in part, by the Pax6 and Pax6(5a) proteins respectively. In vertebrates, Pax6(5a) is generated as an alternately spliced isoform of Pax6. This stands in contrast to the fruit fly which has two Pax6(5a) homologs that are encoded by the eyegone and twin of eyegone genes. This study set out to determine the respective contributions that each gene makes to the development of the fly retina. Both eyg and toe are shown to encode transcriptional repressors that are expressed in identical patterns but at significantly different levels. A molecular dissection of both proteins shows that Eyg makes differential use of several domains when compared to Toe and that the number of repressor domains also differs between the two Pax6(5a) homologs. It is predicted that these results will have implications for elucidating the functional differences between closely related members of other Pax subclasses (Yao, 2008).

An initial analysis of transcriptional patterns indicates that both Pax6(5a) genes are expressed in identical patterns within the retina. However, eyg is expressed at a much higher level than toe. Not surprisingly, while mutations in eyg nearly delete the eye, a reduction in toe via miRNA treatments has no effects on its own. Simultaneous reductions in both genes, in contrast, result in a 'headless' phenotype. Using a set of mini genetic screens and activator/repressor fusion assays, both proteins are demonstrated function as transcriptional repressors. In total, these characteristics suggest that eyg and toe might play redundant roles in during development (Yao, 2008).

However, the high level of sequence divergence within the non-DNA binding domains hints that their functions may only be partially redundant. This study set out to molecularly dissect both Pax6(5a) proteins and determine what, if any, differences exist between the activities of each protein. In two experimental contexts it was possible to demonstrate that such differences between eyg and toe exist. First, a comparison of eyg and toe loss-of-function phenotypes indicated that toe played a greater role in the development of the thorax than the eye. Second, forced expression of both full-length proteins throughout the developing fly identified 43 different instances in which expression of one Pax6(5a) gene induced a different phenotype than the other. Taken together, these results hint that the roles of eyg and toe may be not be completely redundant (Yao, 2008).

Studies were carried out to discover which domain(s) might account for the differences seen in loss-of-function mutants and forced expression assays. A set of deletion and chimeric proteins were generated to dissect the requirement for each domain as well as the level of functional conservation. Attempts were made to rescue eyg¹ mutants as well as generate extra eye fields with these protein variants. The results indicate that Eyg and Toe make differential use of several domains. Many of these differences map to the non-DNA binding domains. One possible mechanism for this is that Toe has only one repressor domain, while Eyg has two. The prediction is that the differences in the non-DNA binding domains are the primary determinants of how each Pax6(5a) protein will influence development. It is less likely that the two DNA binding domains functionally distinguish one protein from another, since there is an extremely high level of sequence conservation within these motifs. Thus the model for how Eyg and Toe function is that both transcription factors bind to similar target genes but can differentially influence transcription through differing levels of repressor activity and/or interactions with disparate binding partners (Yao, 2008).

These results may have broad implications for the activities of other Pax genes in both Drosophila and vertebrates. The fly genome contains two Pax6 genes, eyeless (ey) and twin of eyeless (toy), both of which also arose through a relatively recent duplication. Both share high degrees of homology within the DNA binding domains while having significantly lower levels of sequence conservation in the non-DNA binding regions. Functionally, Ey and Toy have differing abilities to induce eye formation when expressed in non-retinal tissues. Some of these differences have been attributed to the C-terminal tail section of each protein (Yao, 2008).

Mammalian Pax genes are grouped, in part, according to their structure. Individual classes are defined by the presence or absence of the octapeptide and the two DNA recognition (Paired and Homeobox) motifs. Like the fly genes, members within each Pax subclass share a very high degree of sequence conservation within the DNA binding domains thus they are likely to bind to very similar targets. The current results, if extended to these other Pax genes, would suggest that their activity could be distinguished by examining the localization of activation and repressor domains as well as the use of different binding partners (Yao, 2008).

GENE STRUCTURE

The eyg coding region contains two introns and three exons. The first 680-bp intron interrupts the paired domain (PD) in the region preceding the linker region at a gap in the sequence where there seems to be a remnant of the recognition helix of the PAI subdomain (residue 54 of the PD). Although several of the splice sites are conserved among Pax genes belonging to the same class, this splice site is not. The second 200-bp intron found within the homeobox is also found at this position in eve as well as other Prd-class homeobox sequences (Jun, 1998)

Eyg has a characteristic serine at position 50. This residue is only found when the HD is associated with a paired domain (PD). The PD is the defining character of Pax proteins. It consists of two subdomains, the N-terminal PAI and the C-terminal RED subdomains, that can both bind DNA. Interestingly, the PD encoded by the Lune cDNA, originally defining eyg, contains only a partial PAI subdomain and a complete RED subdomain. eyg has an open reading frame of 670 amino acids rather than the 523 amino acids originally reported. These changes involve both N-terminal and C-terminal sequences. 5'-RACE identified two splicing isoforms with their 5' ends identical to that of the Lune cDNA, indicating that the 5' end of the cDNA clone represents the transcription start site. Thus the Lune cDNA represents the full-length transcript, and there is no additional upstream exon to provide a functional PAI subdomain. The two isoforms differ in a 67 bp segment (intron I), which does not affect the coding region. Five introns were identified. All exon-intron junctions conform to the consensus splice site (Bäumer, 2003).

Pax6, like all other Pax proteins, contains a PAIRED DNA binding domain (PD) which itself is comprised of two functionally separable helix-turn-helix motifs, the PAI and the RED domains. In addition Pax6 contains a third nucleic acid recognition motif, the homeodomain (HD). The composition and structure of Pax6 provides for considerable flexibility in its interactions with nucleic acids thereby allowing for the combinatorial use of three functionally independent DNA recognition domains. While vertebrates have only a single Pax6 gene, the fruit fly contains two Pax6 orthologs eyeless (ey) and twin of eyeless (toy). Both play central roles in the specification of the retina. Alternate splicing of vertebrate Pax6 leads to the production of a second isoform, Pax6(5a). This isoform (1) lacks a functional PAI domain; (2) binds to DNA through its RED and HD; and (3) has a different PD binding specificity than canonical Pax6. In vertebrates Pax6 and Pax6(5a) appear to play different roles in retinal development. Pax6(5a) loss-of-function mutants have different phenotypes than those of Pax6. Likewise, overexpression of Pax6(5a) induces different developmental defects and patterns of gene expression than Pax6. Pax6(5a) is also found in Drosophila but, unlike vertebrates, does not result from alternate splicing of Pax6 but rather is encoded by two separate genes, eyegone (eyg) and twin of eyegone (toe). These genes arose from a relatively recent duplication event and together with vertebrate Pax6(5a) represent a novel subclass of Pax genes. Similar to vertebrates, Drosophila Pax6 and Pax6(5a) appear to play different roles in eye development. While ey and toy act primarily in retinal specification, the main function of eyg is to promote cell proliferation. Each isoform exerts its influence on development through different transcriptional mechanisms: Ey acts as an activator while Eyg has the unique property of acting as a dedicated repressor (Yao, 2008 and references therein).

REGULATION

Eyg and Ey Pax proteins act by distinct transcriptional mechanisms in Drosophila development

Drosophila has two pairs of Pax genes, ey/toy and eyg/toe, that play different functions during eye development. ey specifies eye fate, while eyg promotes cell proliferation. This study has determined the molecular basis for the functional diversity of Eyg and Ey. Eyg and Ey act by distinct transcriptional mechanisms. They use different DNA-binding domains for target recognition. Most interestingly, Eyg acts exclusively as a repressor, whereas Ey is an activator. Several vertebrate Pax proteins are known to switch between activator and repressor activities, but none as repressors only. Eyg may be the first Pax protein as a dedicated repressor. Vertebrates produce a Pax6 isoform, Pax6-5a, differing from Pax6 in DNA-binding properties and functions and structurally similar to Eyg/Toe. Pax6-5a acts as an activator like Ey, but has DNA-binding specificity like Eyg (Yao, 2005; full text of article).

The paired domain (PD) can be further divided into two DNA-binding subdomains, PAI and RED, plus a linker (L) region linking the two. Ey specificity, compared with Eyg, is determined by its PAI and L domains. In vitro studies show that when the intact PAI is present, the DNA binding of the RED is masked. However, it was found that in vivo the RED of Ey may still play a role in target recognition or in maintaining the PD structure. The HD is not required for most of Ey functions, although it is required to suppress Dll expression. In contrast, Eyg uses the L, RED, and HD domains for target recognition. The L region is important for the functional specificity of both Ey and Eyg. Although the L region has been shown to contact DNA by X-ray crystallography, its functional role has not been previously demonstrated. The non-DNA-binding B region is not involved in target specificity, but in both Ey and Eyg it is required for the full activity. The different DNA-binding domains may be used independently or in a combinatorial fashion. For example, the Ey PD, but not the HD, is used to induce ectopic eye. The Ey HD is required for the suppression of Dll in order to block the antennal fate during eye development. For Eyg, the PD and HD are redundantly involved in wg suppression. The finding that the Eyg PD and HD could function in trans suggests that the two domains may bind independently to different target genes (Yao, 2005).

The results strongly suggest that Eyg acts exclusively as a transcriptional repressor. This is based on three sets of results: (1) Expression of the obligatory repressor Eyg-En in eyg expression domains completely rescued the eyg mutant defects in eye, head, and thorax development, while Eyg-VP16 caused dominant-negative effects. Moreover, ey>eyg554 and ey>eyg-en could rescue headless homozygous eyg^M3-12 to approximately 500-600 ommatidia in size. (2) Ectopic expression of Eyg-En caused phenotypes similar to those of Eyg, while Eyg-VP16 caused opposite effects. Thus, in all functional assays that were tested, Eyg-En behaved just like Eyg, while Eyg-VP16 had the opposite effects. (3) Eyg has two sets of repression domains but has no activation domain (Yao, 2005).

Ey is known to activate directly so and the rhodopsin genes. Ey-VP16 and Ey-En results suggest that, at least for inducing ectopic eye development, Ey functions as an activator. The suppression of Dll transcription by Ey is suggested to be indirect. It was found that Ey-VP16 could repress Dll, while Ey-En could not, strongly suggesting that Ey probably activates a repressor of Dll. These results suggest that Ey in general acts as an activator. Ey uses its C-terminus for the transactivation activity. It was also found that the Toy C-terminus has only weak transactivation activity, providing an explanation on why the Toy C-terminus cannot functionally substitute for the Ey C-terminus (Yao, 2005).

Both Pax6-5a and Eyg have a disrupted PAI and similar DNA-binding preference. It has been proposed that PAX6-5a is functionally equivalent to Eyg. The results ruled out this interesting possibility. This study showed that expression of Eyg and hPAX6-5a cause different effects, and that hPAX6-5a failed to rescue eyg mutant. In addition, hPAX6-5a acted as a transcriptional activator in flies. Thus, hPAX6-5a is not functionally equivalent to Eyg. In contrast, hPAX6-5a functioned like the Ey construct without an intact PAI, suggesting that hPax6-5a uses the RED for DNA binding. Thus, both hPAX6 and hPAX6-5a acted as activators, but induced different target genes through use of different DNA-binding domains (Yao, 2005).

Pax6 genes from a variety of organisms can induce ectopic eyes when expressed in Drosophila, suggesting that the functional mechanism is conserved and they likely act also as activators in flies. In vertebrate development, Pax6 and Pax6-5a can also act as transcriptional repressors. Pax2, Pax3, Pax4, Pax5, and Pax8 can also function as repressors, in addition to being activators. The switch between functioning as activators and repressors may depend on interaction with cofactors. For example, Pax3 interacts with Daxx and HIRA. Eyg is not only the first repressor Pax in Drosophila, but also the first example of a dedicated repressor Pax protein. The results also suggest that Ey may function exclusively as an activator, at least for inducing ectopic eye development. Thus, the activator and repressor roles are delegated to proteins encoded by two different genes, ey and eyg, respectively (Yao, 2005).

Transcriptional Regulation

Based on its pattern of expression, eyegone is thought to play a role in salivary gland organogenesis. Salivary gland primordium (SGP) development responds to positional information. On the anteroposterior axis, Sex combs reduced (Scr) specifies parasegment 2. In Scr minus embryos, no salivary glands are formed and eyg expression is lost, except for a small patch of cells present at the PS1/PS2 border. In a teashirt minus mutation, Scr is expanded to both PS2 and PS3 and results in enlarged SGPs. The SGP expression of eyg is duplicated in PS3, although its appearance and fading are delayed slightly. Along the dorsoventral axis, the SGP is restricted dorsally by decapentaplegic (dpp), and ventrally by the spitz group of genes. In dpp minus embryos, eyg expression expands dorsally to form a ring that is interrupted ventrally. In several spitz-group mutant embryos, such as single minded (sim), the SGPs from each side move ventrally, and eyg expression expands ventrally. Expression in the trunk is also disordered, which may be a secondary effect of the disruption of the mesoderm (Jun, 1998).

Salivary eyegone expression is regulated positively by Sex combs reduced and trachealess (trh) but is regulated negatively by forkhead. Scr, the homeotic gene responsible for patterning parasegment 2, is responsible for the activation of every salivary gene that has been tested. As expected, eyg is not expressed in the salivary primordium of Scr-mutant embryos. The trh gene product is necessary for invagination of all salivary duct cells and it is required for expression of downstream duct markers. Because eyg is also expressed in part of the salivary duct primordium, the relationship between trh and eyg was tested in the pathway for duct determination. In wild-type embryos, both trh and eyg expression in the salivary primordium begin early during stage 10. At this stage, eyg expression in trh-mutant embryos is indistinguishable from expression in wild-type embryos. Therefore, initiation of eyg expression in the salivary primordium is independent of trh. In early stage 12, however, eyg expression becomes dependent on trh. Although eyg is expressed strongly in the posterior preduct cells of wild-type embryos, this expression is completely absent in trh-mutant embryos. Therefore it is eyg maintenance, and not its initiation, that depends on trh. Whether trh expression depends on eyg was also tested and it was found that trh expression is unaffected in eyg null-mutant embryos (Jones, 1998).

forkhead plays an important role in establishing the pregland/preduct border by dorsally limiting duct-specific gene expression. trh, like eyg, is also initially expressed throughout the gland primordium. In fkh-mutant embryos, trh transcript never disappears from the pregland cells (Isaac, 1996). Does fkh play a similar negative regulatory role in eyg transcription? When the wild-type eyg expression pattern is compared to that of fkh-mutant embryos, it becomes clear that fkh indeed negatively regulates eyg. eyg expression persists in gland precursors in fkh-mutant embryos. Thus, fkh represses expression of trh and eyg, both of whose expression disappears from the pregland cells at approximately the same time. eyg plays no role in the regulation of fkh expression (Jones, 1998).

Armed with the knowledge that (1) fkh is responsible for the exclusion of both trh and eyg from the pregland cells and (2) trh is necessary for maintenance of eyg expression in the duct cells, it is possible to ask whether fkh represses eyg in the pregland cells simply by repressing trh or if fkh downregulates trh and eyg independently. To address this question, embryos were generated that were doubly mutant for trh and fkh. If the reason for eyg disappearance from the pregland cells in wild-type embryos is disappearance of trh, then it would be predicted that eyg expression would not persist in trh;fkh-mutant embryos. eyg expression, however, does persist in pregland cells in trh;fkh-mutant embryos, suggesting that trh plays no role in eyg repression by fkh. Thus, after the initial establishment of the salivary primordium by Sex combs reduced, forkhead excludes eyegone expression from the pregland cells so that eyegone's salivary expression is restricted to the posterior preduct cells. trachealess, in contrast, activates eyegone expression in the posterior preduct cells (Jones, 1998).

Localized expression of eyg/toe in the thorax is achieved by the activity of two antagonistic factors: the promoting activity induced by the Iro and pnr genes, and the repressing activities exerted by the Hh and the Dpp pathways. The latter are probably mediated by Hh and Dpp target genes that are yet to be identified (Aldaz, 2003).

Both Iro genes and pnr act as activators of eyg/toe expression. In Iro gene-mutant clones eyg is abolished, and ectopic Iro gene activity results in ectopic eyg expression. Although pnr^- clones do not lose eyg activity, the probable explanation is that they show Iro gene activity, which upregulates eyg. However, ectopic pnr expression induces eyg activity. Because the Iro gene and pnr expression domains cover the entire notum, in the absence of any other regulation they would induce eyg activity in the whole structure (Aldaz, 2003).

The elimination of eyg activity from the scutellum and lateral notum is caused by the Hh and Dpp pathways. Because the AP compartment border is displaced posteriorly in the notum, these two pathways are active at high levels in the posterior region of the mesothorax. Assuming that the two signals behave as in the wing, Hh activity will be higher in the region close to the AP border, whereas the effect of Dpp will extend further anteriorly. Thus the repressive role of Hh will be greater in the proximity of the AP border and that of Dpp will be greater in more anterior positions. This is precisely what the results indicate. In the region close to the AP border the high Hh levels alone are sufficient to repress eyg. However. in more anterior positions, close to the border of the eyg domain, Hh levels are lower and, although Hh is still necessary, it is not sufficient to repress eyg. Here there is an additional requirement for Dpp activity (Aldaz, 2003).

Thus, the part of the notum that does not express eyg can be subdivided into two distinct zones according to the mode of eyg regulation: a region close to the AP border that requires only Hh, and a more anterior region that requires both Hh and Dpp. In the posterior compartment, the repression of eyg has to be achieved by a different mechanism because neither the inactivation of the Hh pathway in smo^- clones, nor the inactivation of the Dpp pathway, induces ectopic eyg activity. A probable possibility is that en itself may act as repressor (Aldaz, 2003).

The result of the antagonistic activities of the Iro genes and pnr in one case and of the Hh and Dpp signalling pathways in the other, subdivides the notum into an eyg/toe expressing domain and a non-expressing domain. The localized expression of eyg/toe contributes to the morphological diversity of the thorax, as it distinguishes between an anterior-central region and a posterior-lateral one. It provides another example of a genetic subdivision of the body that is not based on lineage. It also provides an example of a patterning gene acting downstream of the combinatorial code of selector and selector-like genes. Its mode of action supports a model in which the genetic specification of complex patterns, such as the notum, is achieved by a stepwise process involving the activation of a cascade of regulatory genes (Aldaz, 2003).

Growth and specification of the eye are controlled independently by Eyegone and Eyeless in Drosophila: Notch promotes growth in eyes by acting through Eyegone

Control of growth determines the size and shape of organs. Localized signals known as 'organizers' and members of the Pax family of proto-oncogenes are both elements in this control. Pax proteins have a conserved DNA-binding paired domain, which is presumed to be essential for their oncogenic activity. Evidence is presented that the organizing signal Notch does not promote growth in eyes of D. melanogaster through either Eyeless (Ey) or Twin of eyeless (Toy), the two Pax6 transcription factors. Instead, it acts through Eyegone (Eyg), which has a truncated paired domain, consisting of only the C-terminal subregion. In humans and mice, the sole PAX6 gene produces the isoform PAX6(5a) by alternative splicing; like Eyegone, this isoform binds DNA though the C terminus of the paired domain. Overexpression of human PAX6(5a) induces strong overgrowth in vivo, whereas the canonical PAX6 variant hardly effects growth. These results show that growth and eye specification are subject to independent control and explain hyperplasia in a new way (Dominguez, 2004).

Localized Notch signal acts through eyg and upd to promote global growth in Drosophila eye

Notch (N) signal is activated at the dorsoventral (DV) border of the Drosophila eye disc and is important for growth of the eye disc. In this study, the Pax protein Eyg is shown to be a major effector mediating the growth promotion function of N. eyg transcription is induced by N signaling occurring at the DV border. Like N, eyg controls growth of the eye disc. Loss of N signaling can be compensated by overexpressing eyg, whereas loss of the downstream eyg blocks the function of N signaling. In addition, N and eyg can induce expression of upd, which encodes the ligand for the Jak/STAT pathway and acts over long distance to promote cell proliferation. Loss of eyg or N can be compensated by overexpressing upd. These results suggest that upd is a major effector mediating the function of eyg and N. The functional link from N to eyg to upd explains how the localized Notch activation can achieve global growth control (Chao, 2004).

Notch is activated at the DV boundary of the early eye disc. This equatorial N signal then activates eyg expression at the transcriptional level. When N signal is reduced, eyg expression is reduced. When N signal is elevated, eyg expression is induced. Induction of eyg expression occurs at the DV border between the dorsal Dl-expressing and the ventral Ser-expressing cells. Furthermore, when the upstream N signal is blocked, overexpression of eyg can rescue the growth defect in the eye, whereas increasing N signaling cannot rescue the eye-growth defect caused by the downstream eyg gene. This analysis shows that the induction of eyg by N is dependent on the ligands Dl and Ser, and involves the effector Su(H) and the antagonist Hairless. Thus, the localized activation of N signal is transmitted to the induction of a transcription factor, Eyg, which then promotes cell proliferation. The Chao study confirms the work of Dominguez (2004), who showed that Notch promotes growth in eyes by acting through Eyegone (Chao, 2004).

Eyg is a transcription factor, so must activate the transcription of some genes that promote cell proliferation. Upd is reported to act through the Jak/STAT signaling pathway to promote cell proliferation. upd expression is dependent on eyg and N signaling. Furthermore, when the upstream N signaling or eyg is reduced, overexpression of upd can rescue the growth defect. The overgrowth effect due to overexpression of the upstream N or eyg is blocked when the downstream upd is defective. The results suggest that upd is a major effector for the growth promotion by N and eyg (Chao, 2004).

These results have demonstrated the functional link from Notch to eyg to upd in the promotion of eye growth. The link to upd solved a long-standing problem. N signaling is activated locally at the border between the dorsal Dl-expressing cells and the ventral Ser-expressing cells. How does a localized activation of N signal promote cell proliferation throughout the entire eye disc? The finding of eyg as the major mediator of N function did not solve the problem, since Eyg is a transcriptional factor and is expected to affect target gene expression autonomously. The link from eyg to upd provided a solution, because Upd is a diffusible signaling molecule. Upd protein can distribute over a long distance and exert long-range non-autonomous effect to promote cell proliferation (Tsai, 2004). So the localized N activation can locally activate eyg, which then turns on upd expression, probably through a short-range signal. The Upd signal then acts over a long range to promote cell proliferation in the early eye disc (Chao, 2004).

Although N activates eyg, and eyg activates upd, these transcriptional activation may be direct or indirect. When novel DV borders were created by ectopic expressing Dl or Ser, eyg is induced non-autonomously at the border of these clones. It is also noted that in Su(H) mutant clones, mutant cells at the border of the clone can still express eyg-lacZ. These observations suggest that N may induce a short-range signal, which then activates eyg expression. Alternatively, the apparent non-autonomous induction may be due to perdurance of the reporter protein in cells that were once close to the clone border. The induction of upd by eyg also may be indirect. Clonal expression of eyg also induced upd expression non-autonomously. In addition, based on RNA in situ hybridization, eyg expression in the eye disc does not extend to the posterior margin, so does not overlap with the expression domain of upd (Tsai, 2004). These observations suggested that the effect of eyg on upd expression may be indirect. However, an eyg enhancer trap line showed reporter expression extending to the posterior margin (Dominguez, 2004). Thus, the possibility that Eyg can directly activate the expression of upd cannot be excluded (Chao, 2004).

The activation of eyg and upd are context dependent. N^act does not induce eyg expression in antenna and wing discs. In the eye disc, N^act induces eyg expression only in the region anterior to the MF, and not within the wg expression domain in the lateral margin. Similarly, N^act and eyg can only induce upd expression at the margin, but not in the center of the eye disc. N^act induces upd at the posterior margin but not lateral margins, while eyg can induce upd in the lateral margins but not in the posterior margin. The context dependence indicates that additional factors are involved to determine the specificity of induction (Chao, 2004).

In a late third instar eye disc, eyg is expressed in an equatorial domain that does not overlap with the disc margin, so eyg cannot induce upd. In early eye disc, eyg expression domain comes closer to the posterior margin. Thus, the induction of upd by eyg is likely at second instar, which is consistent with the timing of upd expression (Chao, 2004).

Although eyg plays an important role in mediating the growth-promoting N signal, it is probably not the only effector. In the eyg^M3-12 null mutant, ey>N^act does not rescue the endogenous eye field, but can still induce proliferation to provide the antennal disc and an extra eye field. Thus, N can induce proliferation by an eyg-independent mechanism. The effect on antenna and on eye seems to be separate, because ey>N^act can induce a large antenna disc with duplicate or triplicate antennal field without rescue of the eye disc. Because N can induce upd, but not eyg, in the posterior margin, the induction of upd can also be through an eyg-independent mechanism (Chao, 2004).

N^act can induce overgrowth in the central domain of the eye disc. In this case, eyg, but not upd, is induced. In addition, the overgrowth does not extend much beyond the clone. Ectopic eyg in the central domain also induces proliferation without inducing upd. In eyg¹ mutant, there is no upd-lacZ expression in eye disc, but the eye is only slightly reduced. These results suggest that the N signaling and eyg can induce local proliferation independent of upd (Chao, 2004).

Drosophila Pax6 promotes development of the entire eye-antennal disc, thereby ensuring proper adult head formation

Paired box 6 (Pax6) is considered to be the master control gene for eye development in all seeing animals studied so far. In vertebrates, it is required not only for lens/retina formation but also for the development of the CNS, olfactory system, and pancreas. Although Pax6 plays important roles in cell differentiation, proliferation, and patterning during the development of these systems, the underlying mechanism remains poorly understood. In the fruit fly, Drosophila melanogaster, Pax6 also functions in a range of tissues, including the eye and brain. This report describes the function of Pax6 in Drosophila eye-antennal disc development. Previous studies have suggested that the two fly Pax6 genes, eyeless (ey) and twin of eyeless (toy), initiate eye specification, whereas eyegone (eyg) and the Notch (N) pathway independently regulate cell proliferation. This study shows that Pax6 controls eye progenitor cell survival and proliferation through the activation of teashirt (tsh) and eyg, thereby indicating that Pax6 initiates both eye specification and proliferation. Although simultaneous loss of ey and toy during early eye-antennal disc development disrupts the development of all head structures derived from the eye-antennal disc, overexpression of N or tsh in the absence of Pax6 rescues only antennal and head epidermis development. Furthermore, overexpression of tsh induces a homeotic transformation of the fly head into thoracic structures. Taking these data together, this study demonstrates that Pax6 promotes development of the entire eye-antennal disc and that the retinal determination network works to repress alternative tissue fates, which ensures proper development of adult head structures (Zhu, 2017).

In contrast to vertebrates that have a single Pax6 gene, the Drosophila genome contains two Pax6 homologs, ey and toy. Both genes are expressed broadly throughout the entire eye-antennal disc but are later limited to a far more restricted domain within the undifferentiated cells of the eye field. Whereas most studies on Pax6 in the eye-antennal disc have focused on the developing compound eye, several reports have hinted at a role for both genes outside of the eye. However, the underlying mechanism of how Ey/Toy promote eye-antennal disc development has been elusive. This is, in part, because of the use of single Pax6 mutants to study development. The phenotypes associated with individual mutants are variable and often restricted to the eye. Several studies have suggested that Ey and Toy function redundantly to each other. This finding most likely explains the variability of phenotype severity and penetrance. Thus, the combined loss of both Ey/Toy may be a more accurate reflection of the effect that Pax6 loss has on Drosophila development. Indeed, this appears to be the case as it is reported that the combined loss of both ey and toy leads to the complete loss of all head structures that are derived from the eye antennal disc. This study attempted to determine the mechanism by which Ey/Toy support eye-antennal disc development (Zhu, 2017).

Previous studies in the fly eye proposed that Pax6 is concerned solely with eye specification, whereas Notch signaling and other retinal determination proteins, such as Eyg, Tsh, and Hth, control cell proliferation and tissue growth. This study proposes an alternate model in which Ey/Toy are in fact required for cell survival and proliferation in addition to eye specification. The data indicate that Ey/Toy regulate growth of the eye-antennal disc through Tsh, N/Eyg, and additional N-dependent proliferation promoting genes. It is proposed that on simultaneous removal of Ey and Toy the eye-antennal disc fails to develop, in part, because the expression of eyg and tsh is lost in complete absence of Pax6. Expression of tsh and activation of the N pathway are sufficient to restore tissue growth to the eye-antennal disc. Support for this model linking Ey/Toy to cell proliferation via Eyg and Tsh comes from studies showing that eyg loss-of-function mutants display a headless phenotype identical to that seen in the ey/toy double knockdowns, that cells lacking eyg do not survive in the eye disc, and overexpression of Tsh causes overproliferation (Zhu, 2017).

The results also show that the combined loss of Ey and Toy affects the number of cells that are in S and M phases of the cell cycle. This observation directly supports the model that Ey/Toy control growth of the eye-antennal disc and is consistent with studies in vertebrates that demonstrate roles for Pax6 in the proliferation of neural progenitors within the brain. Earlier studies observed cells undergoing apoptosis in Pax6 single-mutant eye-antennal discs and showed that blocking cell death alone can partially rescue the head defects of the ey^D and toy^hdl mutants. Although this study shows that retinal progenitor cells lacking both Pax6 proteins undergo even greater levels of apoptosis, blocking cell death does not restore the eye-antennal disc. What accounts for the differences in the two experiments? In the ey^D and toy^hdl rescue experiments, each genotype contained wild-type copies of the other Pax6 paralog, but this study has knocked down both Pax6 genes simultaneously. Another possible difference is that Pax6 levels are being reduced while the ey^D and toy^hdl mutants are likely functioning as dominant negatives. It is concluded from these results that a reduction in cell proliferation but not elevated apoptosis levels is the proximate cause for the complete loss of the eye-antennal disc (Zhu, 2017).

Although the activation of Tsh and the Notch pathway can restore antennal and head epidermal development, neither factor is capable of restoring eye development to the ey/toy double-knockdown discs. This is most likely because both Pax6 genes are also required for the specification of the eye. In particular, Ey/Toy are required for the activation of several other retinal determination genes, including so, eya, and dac. Thus, the results suggest that Notch signaling, Eyg, and Tsh can restore nonocular tissue growth to the eye field but cannot compensate for the Pax6 requirement in eye specification (Zhu, 2017).

Finally, the results using the double knockdown of ey/toy are consistent with the dosage effects that are seen in mammalian Pax6 mutants. Although mutations in ey have just eye defects, the combined loss of ey/toy lacks all head structures. Mice that are heterozygous for Pax6 mutations have small eyes, whereas those that are homozygous completely lack eyes, have severe CNS defects, and die prematurely. Similarly, human patients carrying a single mutant copy of Pax6 suffer from aniridia, whereas newborns that are homozygous for the mutant Pax6 allele have anophthalmia, microcephaly, and die very early as well. As a master control gene of eye development, Pax6 appears to initiate both retinal specification and proliferation. These data demonstrate that the functions of Ey and Toy in the eye-antennal disc are redundant and dependent upon gene dosage, thereby making the roles of Pax6 in the Drosophila similar to what is observed in vertebrates where Pax6 controls both specification and proliferation of the brain and retina in a dosage-sensitive manner (Zhu, 2017).

Targets of Activity

In salivary ducts, breathless (btl) but not Serrate is activated by eyegone. btl codes for a Drosophila FGF receptor homolog that is critical to tracheal and midline glial cell development, while Ser encodes a Notch ligand containing a single EGF repeat. Although neither is required for duct morphogenesis, they are expressed during most of duct development and therefore serve as useful duct markers. Ser and btl expression have been shown to depend on trh (Kuo, 1996). Since trh also regulates eyg, whether eyg might act as an intermediate in the regulation of btl and Ser by trh was also tested. btl expression in duct cells is strongly reduced in embryos lacking eyg. In contrast, expression of Ser is not downregulated in the duct cells of eyg-mutant embryos even though the duct primordia are not fused as in wild-type embryos (Jones, 1998).

Protein Interactions

In Drosophila, the morphological diversity is generated by the activation of different sets of active developmental regulatory genes in the different body subdomains. This study investigates the role of the homothorax/extradenticle (hth/exd) gene pair in the elaboration of the pattern of the anterior mesothorax (notum). These two genes are active in the same regions and behave as a single Hox independent functional unit. Their original uniform expression in the notum is downregulated during development and becomes restricted to two distinct, alpha and ß subdomains. This modulation appears to be important for the formation of distinct patterns in the two subdomains. The regulation of hth/exd expression is achieved by the combined repressing functions of the Pax gene eyegone (eyg) and of the Dpp pathway. hth/exd is repressed in the body regions where eyg is active and that also contain high levels of Dpp activity. Evidence is presented for a molecular interaction between the Hth and the Eyg proteins that may be important for the patterning of the alpha subdomain (Aldaz, 2005).

This study deals with a novel hth/exd function: its patterning role in the notum. It is not related to the specification of notum identity because notum identity is not affected by alterations of hth/exd activity. For example, in the absence of hth/exd, the cells still differentiate as notum, if an abnormal one. Conversely, high and uniform Hth levels also produce notum tissue but with abnormal pattern. This function is only required in part of the notum and is therefore linked to the modulation of hth expression during the development of the disc. The final result of this modulation is the appearance of the alpha and ß subdomains of hth that is reported in this study. These two subdomains differentiate distinct notum patterns, suggesting that Hth/Exd interact with other localised products to generate these patterns (Aldaz, 2005).

Thus, there are two principal aspects in the patterning function of hth/exd: (1) the spatial regulation, that eventually results in the restriction of its expression to the alpha and ß subdomains, and (2) the local interactions of Hth/Exd with other products in either of the subdomains (Aldaz, 2005).

Although hth and exd form a single functional unit, their mode of regulation is different: exd is expressed ubiquitously but is regulated at the subcellular level by hth, which promotes Exd nuclear transport. Therefore, the key element of hth/exd regulation is the transcriptional control of hth (Aldaz, 2005).

Originally, hth is expressed in all the notum cells and later becomes restricted to the alpha and ß subdomains. Consequently, the principal aspect of hth regulation is the mechanism(s) leading to its repression in the regions outside the alpha and ß subdomains. Two negative regulators have been identified, the eyg gene and the Dpp pathway, which probably acts through some unidentified downstream gene. In the notum hth behaves as a downstream target of both the Dpp pathway and eyg (Aldaz, 2005).

The role of Eyg as a negative regulator of hth is based on the following observations: (1) the beginning of the modulation of hth expression in the notum at the early third instar coincides with the initiation of eyg expression; (2) in eyg mutants the hth domain is expanded, extending to most of the notum; (3) mutant eyg clones show hth derepression in the inter-subdomains region, and conversely, ectopic eyg activity in the ß subdomain represses hth. The fact that this ectopic activity fails to affect hth in the alpha subdomain was expected since eyg and hth are normally co-expressed in this subdomain. In conclusion, eyg suppresses hth in the inter-subdomains region and also acts as a barrier for hth in the eyg/ß-hth border (Aldaz, 2005).

The role of the Dpp pathway as a negative regulator of hth is based on results showing that Mad ^- mutant clones in the inter-subdomains region show activation of hth. This is in contrast to the behaviour of those clones in the alpha subdomain, where they have no effect, or in the ß subdomain, where they show suppression of hth. It is believed that the reason for the latter effect is that eyg is up regulated in those clones, and in turn Eyg suppresses hth. The lack of effect of Mad ^- clones in the alpha subdomain is probably due to the low activity of Dpp in that region. In principle, the observation that the high activity levels generated in the Tkv^QD clones suppress hth in this subdomain supports this view. Expectedly, Tkv^QD clones do not affect hth expression in the ß subdomain, because it normally possesses high Dpp activity levels (Aldaz, 2005).

Taking all the results together, the following model of hth regulation is proposed. Since hth is originally expressed in all trunk embryonic cells and in all the notum cells in the early disc, the regulation of hth during wing disc development essentially reflects local repression in specific parts of the disc. The basic idea is that hth is repressed by the joint contribution of eyg and high/moderate levels of the Dpp pathway. Neither of these elements can repress hth individually. Although eyg appears to act uniformly in its domain, the repressing activity of Dpp is concentration dependent. Within the eyg domain, the hth alpha subdomain is located in the anterior region, in which the Dpp levels are too low to be effective and Eyg alone cannot repress hth/exd. In the inter-subdomains region the Dpp levels are high enough to repress hth, since here it acts together with Eyg. The ß subdomain is outside the eyg domain and therefore in the absence of Eyg even the high Dpp levels are not capable of repressing hth/exd. The model is also supported by the experiments of overexpressing eyg. The eyg-expressing clones in the ß subdomain suppress hth because the two repressors are active in the clones, while they have no effect in the alpha subdomain because it normally contains high eyg levels. In principle the experiments overexpressing the Dpp pathway (Tkv^QD clones) appear to support the model. These clones have no effect in the ß subdomain, which normally possesses high Dpp activity levels, but they suppress hth in the alpha subdomain. However, these clones are known to suppress eyg and therefore hth should not be repressed according to this model. It is possible that in certain circumstances the very high Dpp activity levels induced by these clones may be sufficient to down regulate hth, even in the absence of eyg (Aldaz, 2005).

The presence of two distinct repressors may suggest that the hth promoter region contains binding sites for Eyg and for Mad/Medea that would be responsible for the transcriptional repression. The ubiquitous expression in the absence of these two repressors may be due to a constitutive promoter (Aldaz, 2005).

The second aspect of the late patterning function of hth/exd arises from the observation that the alpha and ß subdomains form different patterns with similar levels of hth. This suggests the existence of interactions between Hth/Exd and products specifically localised to the different subdomains. In the case of the alpha subdomain, the obvious candidate for the interaction is Eyg. The joint activity of hth/exd and eyg specifies a notum pattern that is different from those specified by each of these genes alone (Aldaz, 2005).

The finding that the Eyg and Hth proteins associate to form a complex in vitro suggests a mechanism to achieve the pattern difference between the alpha and the ß subdomains. As it has been shown to be the case for the in vivo specificity of the Hox genes, the association of Hth/Exd with the different Hox products results in higher affinity and specificity for target sites. Here, the formation of an Eyg/Hth/Exd complex in the alpha subdomain may result in a constellation of gene activity different from that in the ß subdomain where Eyg is not present. In the latter subdomain hth/exd may act alone, for after all the two genes encode transcription factors. Alternatively, the Hth/Exd products may interact with some other yet unidentified co-factor (Aldaz, 2005).

An interesting aspect of the interaction of hth/exd and eyg is that it acts in two different ways. At the gene regulation level, eyg participates in the spatial control of hth/exd activity, but where the two genes are co-expressed their proteins interact, presumably to contribute to the in vivo affinity and specificity for target genes (Aldaz, 2005).

DEVELOPMENTAL BIOLOGY

Embryonic

eyegone has an unique spatial and temporal expression pattern, unlike that of other Pax genes in Drosophila. eyg seems to play a role in later stages of embryonic development, more specifically in salivary gland organogenesis and eye development. In situ hybridization showed a dynamic pattern of expression. eyg is first expressed ventrally in the labial lobe, in a cluster of cells that correspond to the region of the salivary gland progenitor cells that form the salivary gland placode (SGP). At first, eyg is expressed nonuniformly in the placode, but expression then becomes more uniform; eventually, eyg is expressed strongly in all of the SGP cells. In embryos that have been double-stained with Engrailed antibody, eyg overlaps the anterior but not the posterior En stripe flanking the SGP. Thus, eyg is expressed in PS2. This expression is transient and fades away before the cells invaginate to form the salivary gland. Fading begins in the dorsoposterior regions of the SGP and progresses in a ventroanterior direction, forming a crescent shape before disappearing, hence the original name, lune. eyg expression in the subantennal region of the head begins at the same time as in the SGP and is persistent throughout embryonic development. It is reminiscent of the expression of the eyeless gene in the primordium of the eye. In the trunk, eyg is expressed in a segmental pattern, initially in single cells within each segment, but then the expression expands to a patch of cells that are restricted ventrally, forming a striped pattern. Double-stained embryos show that this expression alternates with the En stripes. This expression fades to a narrow stripe during germ-band retraction and disappears. It was determined, by using markers for the tracheal placodes, that eyg is expressed near, but not in, the tracheal placodes (N. Hacohen and M. Krasnow, personal communication to Jun, 1998). Finally, eyg expression is also found in the region around the posterior spiracle. This signal seems coincident with segmental expression and persists late in embryonic development after germ-band retraction (Jun, 1998 and Jones, 1998).

Eyegone mRNA is first detected in the salivary primordium at the beginning of stage 10. By stage 13, expression can be seen in the dorsal ectoderm in segmentally repeated stripes. These stripes span the anterior edge of each thoracic and abdominal segment, with sharp anterior but diffuse posterior borders. There are also ventral stripes of eyg expression in each thoracic segment that extend ventrally across the ventral midline. eyg is also expressed in two pairs of sensory organ primordia. In stage 17 embryos, eyg is expressed in precursors of the eye-antennal disc in what appear to be the same cells that express eyeless. The precursors of the larval antennal organ (AO), which is thought to function in olfaction, not only strongly express eyg but require it for their development. Although the cells of the AO are present in egy-mutant embryos, the characteristic finger-like projections known as sensillae are often absent, suggesting a role for eyg in their morphogenesis. Finally, there are three additional pairs of ectodermal staining areas in the head and three ventral thoracic spots that are either next to or associated with the cells of the leg discs (Jones, 1998).

Beginning in stage 10, both the pregland and the posterior preduct cells strongly express eyg. At this stage, gland precursors express eyg more strongly than their duct counterparts. As the germ band retracts during stage 12, eyg transcript disappears from the gland precursors, beginning with the posterior regions, leaving a crescent-shaped staining pattern. As germ band retraction continues, eyg transcript begins to be restricted to the most posterior of the preduct cells and expression finally completely disappears from the pregland cells. Later in stage 12, as the individual duct cells are invaginating, eyg transcript completely disappears from the duct cells as well. eyg expression, however, reappears and, by stage 14, eyg is expressed in the mature individual ducts and also in the anterior cells of each salivary gland. In contrast to trachealess, eyg expression is never seen in the anteriorly extending common duct. In summary, eyg expression is initially seen throughout the salivary primordium (except the common duct primordium) and then disappears from all cells, mimicking the order of their invagination. The individual duct cells are the last cells to lose expression but eyg expression is reinitiated in them and in anterior gland cells once morphogenesis is complete (Jones, 1998).

In the embryo, Eyg is also expressed in the antennal organ, salivary gland, and in a segmentally repeated lateral pattern (Bäumer, 2003).

Larval

In situ hybridization shows that in the eye disc of late third instar larvae, Lune is expressed in the central anterior region, well ahead of the morphogenetic furrow. Expression is stronger dorsal to the equator. In the early eye disc, the expression domain is broader. It is also expressed in the central region of the antennal disc, in the anterior notum, dorsal hinge and in an arc at the posterior periphery of the wing pouch of the wing disc, and is weakly expressed in several arcs in the leg discs. In the embryo it is expressed in the eye-antennal disc primordium, similar to ey and toy (Bäumer, 2003).

eyg transcripts are distributed within several embryonic tissues as well as the leg, wing and eye-antennal imaginal discs. eyg and twin of eyeless (toe) transcripts are first detected in stage 9 embryos within the salivary gland precursor (SGP) and a small cluster of cells within the dorsal head. The expression of toe transcripts in the SGP will persist through the rest of embryonic and larval development while eyg expression is terminated in late stage embryos and reinitiated later. By late stage 10 both transcripts are also found in identical patterns within the posterior spiracle (PS) and within a cluster of cells at the anterior edge of each thoracic and abdominal segment. Expression of eyg and toe expands to the larval antennal organ (AO) as well as the leg disc primordia by stage 12. During the latter stages of embryogenesis both eyg and toe transcripts accumulate in the presumptive eye-antennal imaginal disc. Only two other members of the eye specification cascade, ey and toy, share this expression pattern. The remaining members are added sequentially during the larval development. The only discernable difference between the expression patterns of either Pax6(5a) gene during embryogenesis is found within the SG: eyg expression is eliminated while toe transcriptional levels are maintained (Yao, 2008).

Within the developing larval eye-antennal discs both eyg and toe transcripts accumulate in identical patterns. Within the antennal segment both transcripts localize to the medial and distal segments while in the eye disc expression of both genes is found anterior to the morphogenetic furrow. Unlike the similarities found in the embryo, eyg and toe expression is somewhat different from that of ey and toy. The Pax6 transcripts are expressed broadly ahead of the advancing furrow. However, eyg and toe expression is restricted to a narrow domain of cells that straddle the dorsal-ventral compartment boundary and does not extend laterally. This difference in expression is likely due to the requirements of eyg (and possibly toe) in Notch mediated control of cell proliferation at the organizing center versus the role of ey and toy in tissue specification. Within the developing wing primordium both transcripts are expressed broadly within the notum and in two discrete regions within the presumptive wing. It is interesting that one of those areas is particularly susceptible to being transformed into retinal tissue in response to forced expression of ey. Both eyg and toe transcripts are also found within identical patterns of the leg primordium and the anterior duct cells of the salivary gland. The results from this and other studies of eyg and toe expression suggest at first glance that these genes may play redundant roles within several developing tissues including the compound eye. It is unlikely, however, that these genes play completely surplus roles (at least in the eye) as eyg loss-of-function mutants show near complete loss of retinal tissue and forced expression of toe is insufficient to restore eye development to these flies (Yao, 2008).

odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C₂H₂ zinc finger genes, which has been implicated in notum growth and patterning. This study shows that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. These data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior–posterior (AP) axis using both autonomous and non-autonomous mechanisms (Del Signore, 2012).

In many developmental processes, signals that emanate from field borders play a crucial instructive role in patterning morphogenetic fields. The early Drosophila embryo is patterned by opposing gradients of Bicoid and Nanos that are generated from localized translation of corresponding mRNAs at the anterior and posterior poles of the embryo. In the embryonic epidermis, the pattern of cell differentiation across each segment is regulated by the secreted Wg and Hh signals that emanate from localized sources at the anterior and posterior borders of each segment. Similarly, the dorsoventral axis of the vertebrate spinal cord is organized by Shh ventrally, and BMP and Wnt signals that emanate from localized dorsal sources. By contrast, current models of notum AP patterning focus mainly on the organizing influence of Dpp, which is secreted from the posterior border of the notum. Previous work has found that odd-skipped genes are expressed along the anterior border of the notum, and that broadly inhibiting their function in early wing discs caused a severe reduction or complete loss of the notum. As this reduction occurred despite the maintenance of dpp expression (Nusinow, 2008), whether the odd-skipped genes might define a second organizing center within the developing notum was investigated. The current findings indeed suggest that signals that emanate from the anterior border of the notum act reciprocally to Dpp to promote expression of anterior notum genes and repress expression of posterior genes. Through loss- and gain-of-function clonal analyses, it was demonstrated that the odd-skipped genes pattern the notum AP axis both locally through regulation of Eyg, Bar, and Dl, and broadly through the regulation of Eyg and Tup. Finally, it was shown that lines acts antagonistically to bowl in this process (see Model of the role odd-skipped genes in notum AP patterning) (Del Signore, 2012).

drm overexpression was sufficient to promote Eyg accumulation non-autonomously within the notum. This activity suggests that drm controls expression of an unidentified signal that spreads from the drm domain to induce Eyg accumulation non-autonomously. Alternatively, drm could initiate the propagation of a cascade of local inductive interactions to induce Eyg at a distance. Recent studies have shown that recruitment of cells to the wing field is accomplished by the propagation of a feed forward signal from the DV compartment boundary. In this process signaling at the border between Vestigial (Vg) and non-Vg expressing cells is used to recruit non-Vg expressing cells to the expanding wing field, a process dependent on signaling through the Fat-Dachsous pathway. Though a functional relationship between odd-skipped genes and Ft-Ds signaling has yet to be characterized, it is interesting to note that Ds accumulates in a complex graded AP pattern across the notum, consistent with such a role (Del Signore, 2012).

In addition to the broad induction of Eyg accumulation, it was surprising to find that drm overexpression also induced Bowl in cells just adjacent to clones. Though the effect was subtle, it is noted that this pattern of activation recapitulated the endogenous nested pattern of drm and Bowl expression in the presumptive prescutum. It is unclear whether the nested expression of odd-skipped genes plays a functional role in notum AP patterning. Despite this, the concordance of endogenous and ectopic expression patterns supports the hypothesis that ectopic drm induces a physiologically relevant program of anterior gene expression in the notum. One possible clue as to the relevance of this nested pattern may come from the observation that only drm was able to promote Bowl non-autonomously. In contrast, lines^−/−, odd⁺, and sob⁺ clones each induced only cell-autonomous accumulation of Bowl. Notably, these clones rounded up and segregated from the epithelium, while drm expressing clones remained integrated with the surrounding epithelium. One interpretation of these data is that abrupt discontinuities in the level of Odd-skipped proteins may alter epithelial morphology. This interpretation is further supported by the observation that bowl mutant clones within the Bowl domain adopt a compact, round morphology relative to clones outside the Bowl domain. It is hypothesized that drm promotes lower levels of Bowl in nearby cells to dampen otherwise sharp discontinuities in Bowl activity to regulate local buckling of the epithelium (Del Signore, 2012).

Alternatively, differences in the total levels or ratios of Odd family proteins along the anterior border of the notum could elicit different transcriptional outcomes. Since Odd and Bowl have been shown to interact with the transcriptional co-repressor Groucho, variation in the levels of the Odd-skipped proteins could titrate Groucho and affect Groucho-dependent transcriptional outputs. Alternatively, given their distinct structure outside the zinc finger domain, the Odd-skipped proteins could interact with distinct sets of target genes to pattern the anterior border of the notum. Though additional experiments will be required to ascertain whether such mechanisms are active in the prescutum, this study provides evidence that bowl is strictly required for the early autonomous induction of Eyg, the later expression of Bar genes, and the repression of Dl. These results provide evidence that odd-skipped genes act both independently and redundantly to organize the notum AP axis (Del Signore, 2012).

bowl is essential for patterning the prescutum, but not for broadly patterning the notum AP axis. Previous studies have revealed a variety of essential and redundant functions for odd-skipped family genes in patterning embryonic and larval tissues. In the embryo, drm and bowl antagonize lines function to pattern the dorsal embryonic epidermis, foregut, and hindgut, while odd functions as a pair rule gene to promote embryonic segmentation. In the leg imaginal disc, bowl is essential for patterning the tarsal proximodistal axis at early stages, but acts redundantly with other odd-skipped genes to control leg segmentation later in developmen. In the eye, bowl is essential for the initiation of retinogenesis from the eye margin, while odd and drm have been proposed to activate Bowl redundantly (Del Signore, 2012).

Loss-of-function analysis revealed that neither drm nor odd is necessary to stabilize Bowl. At present the possibility cannot be excluded that sob is necessary to promote Bowl accumulation because a null sob mutant is not yet available. Biochemical and genetic analysis demonstrates that not only Drm, but also Odd and Sob can each outcompete the interaction of Lines with Bowl and stabilize the Bowl proteins in S2 cells and in vivo. These results suggest that different combinations of Odd-skipped proteins could be used to activate bowl depending on context (Del Signore, 2012).

Previous work suggested reciprocal roles for lines and odd-skipped genes in subdividing the early wing disc into disc proper and peripodial epithelium. The loss-of-function analysis described in this study suggests that the odd-skipped genes act redundantly to control the early specification of the PE and the subsequent expansion of the notum, while revealing an essential role for bowl in specification of the anterior prescutum. Redundancy can increase the robustness of essential developmental processes and provide a buffer against fluctuations in activity of single genes. The redundant role of the odd-skipped genes in PE specification and notum expansion could therefore serve to ensure the optimal growth of the wing disc at early stages and that of the notum at later stages and protect these critical processes from perturbations (Del Signore, 2012).

It is concluded that the growth and patterning of the wing field are coordinated with the elaboration of the wing PD axis. The developing notum lacks an obvious PD axis, and instead is subdivided into a series of AP and mediolateral domains. The establishment of organizers that act antagonistically from opposing field borders is a robust strategy to subdivide the notum AP axis. This work demonstrates that the odd-skipped genes act autonomously at the anterior border of the notum to specify the prescutum, and non-autonomously at short and long range to control the expression of transcription factors that prefigure the differentiation of the notum AP axis. Though further experiments will be required to characterize the mechanism by which this putative organizer acts, these studies provide evidence that the anterior border of the notum exhibits the functional attributes of an organizer (Del Signore, 2012).

REFERENCES

Search PubMed for articles about Drosophila eyegone

Aldaz, S., Morata, G. and Azpiazu, N. (2003). The Pax-homeobox gene eyegone is involved in the subdivision of the thorax of Drosophila. Development 130: 4473-4482. 12900462

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date revised: 22 December 2017

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