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).

PROTEIN STRUCTURE

Amino Acids - 523

Structural Domains

Pax proteins, characterized by the presence of a paired domain, play key regulatory roles during development. The paired domain is a bipartite DNA-binding domain that contains two helix-turn-helix domains joined by a linker region. Each of the subdomains, the N-terminal PAI and the C-terminal RED domains, have been shown to be a distinct DNA-binding domain. The PAI domain is the most critical, but in specific circumstances, the RED domain is involved in DNA recognition. The PAI domain is necessary and sufficient for DNA binding in vitro. In fact, the Drosophila Paired protein does not need the RED domain to confer full function to the protein in vivo. There are, however, situations in which the RED domain may play a role. The bipartite organization of the PD allows for the recognition of composite sites by both PAI and RED domain. Furthermore, there is an isoform of the Pax-6 protein (Pax-6 5a) that contains, in the middle of the recognition helix of the PAI domain, an 11-residue insertion that inactivates DNA binding to sequences normally bound by the Pax-6 PD. Pax-6 5a, however, is able to bind to a recently described sequence called 5aCON. Other Pax proteins also contain insertions in the PAI domain that inactivate DNA binding and uncover RED-domain DNA-binding activity. However, to date, there has been no report of a Pax protein containing only a PAI or a RED domain (Jun, 1998 and references).

A Pax protein, originally called Lune, is the product of the Drosophila eyegone gene (eyg). It is unique among Pax proteins, because it contains only the RED domain. Most PDs are located in close proximity to the N terminus of Pax proteins: the RED domain of Eyg starts at position 36 of the protein. It exhibits very limited homology with the end of the PAI domain until residue 47 of the full-length PD. At position 48, the homology becomes higher and extends 7 aa to position 54. The sequence is then interrupted by a 6-aa gap, at the position of a splice site that is not found in other PDs. The homology then continues from position 61 in the PD and remains uninterrupted to include the entire RED domain. There is a proline residue inserted after position 65 that is not found in other PDs. Alignment of the Eyg RED domain with that of other complete PDs indicates that it is distinct from other known Pax classes. The linker region between PAI and RED domains is most related to that of Pax-2, Pax-5, and Pax-8 (Drosophila homolog: Sparkling/Shaven), whereas the HTH region of its RED domain is most related to Pax-6 (Drosophila homolog: Eyeless). The most closely related PDs are those of Pax-6 and Pax-8. Eyg also contains a Prd-class HD with a serine at position 50. Overall, the HD is most closely related to that of Pax-3 (Drosophila homolog: Paired) and Pax-7, although it is significantly distinct. The numbers of identical residues between Eyg and other HDs are 40 for Pax-7; 38 for Pax-6, and 36 for Gsb-p. The rest of the Eyg protein has few distinguishing features other than a glutamine-rich region found frequently in Drosophila proteins (Jun, 1998).

A high-affinity binding site for the Eyg RED domain was identified by using systematic evolution of ligands by exponential enrichment techniques. This binding site is related to a binding site previously identified for the RED domain of the Pax-6 5a isoform. Eyg also contains another DNA-binding domain, a Prd-class homeodomain (HD), whose palindromic binding site is similar to other Prd-class HDs. The ability of Pax proteins to use the PAI, RED, and HD, or combinations thereof, may be one mechanism that allows them to be used at different stages of development to regulate various developmental processes through the activation of specific target genes (Jun, 1998).

The Eyg protein has two DNA binding domains: the RED subdomain of its truncated PD and the Prd-class HD. It probably functions as a transcription factor by binding DNA targets through these domains, singly or in combination. In addition, its interaction with other proteins may affect this DNA binding (Jang, 2003).

The PD consists of two independent subdomains: the N-terminal PAI and the C-terminal RED subdomains. Based on crystal structure of the human Pax6 PD, the linker region connecting the two subdomains also contacts DNA. In Eyg, the PAI subdomain is largely missing and most likely cannot bind DNA. One interesting possibility is that the truncated Eyg PD has a dominant negative effect, competing with other PD proteins. In addition, truncation of the PAI subdomain in the Pax6-5a and Pax8(S) isoforms probably exposes the RED subdomain to recognize a distinctly different DNA sequence. Thus the Eyg PD may bind DNA through its RED domain, similar to the Pax6-5a and Pax8(S) isoforms and distinct from the Pax6 PD. This prediction has in fact been proven by site-selection using the Eyg RED domain. Through its RED domain, Eyg can probably regulate different target genes than those regulated by Ey. This ey-independent function of eyg is also shown by its involvement in salivary gland development, and in bristle formation when ectopically expressed. Vertebrate homologs of Eyg have not yet been identified. It is possible that Eyg plays a role equivalent to the vertebrate Pax6-5a isoform (Jang, 2003).

In addition to the PD, many Pax proteins (including Ey and Eyg) also contain a Prd-class homeodomain. Two Prd-type HDs can bind cooperatively to a palindromic site composed of two inverted TAAT motifs separated by 2 or 3 bps. The Prd-type HD of Eyg can form heterodimers with the Prd-type HD of Prd upon binding to a consensus DNA target. It is possible that Ey and Eyg also form heterodimers via their HDs. This would be consistent with the findings that they act synergistically. However, although the HD of Eyg is required for its functions, the HD of Ey has been shown not to be required for its function in eye development. Thus the HD of Eyg is required, not for direct interaction with the HD of Ey, but may be for DNA binding or for interacting with other proteins (Jang, 2003).

date revised: 7 February 99

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