BIOLOGICAL OVERVIEW

Optix is a new Drosophila member of the Six/sine oculis gene family that contains both a Six domain and a homeodomain. Because of its high amino acid sequence similarity with the mouse Six3 gene, Optix is considered to be the orthologous gene from Drosophila, rather than sine oculis as was previously believed. Whereas Sine oculis belongs to the Six1 subclass of the Six/so gene family, Optix belongs to the Six3 subclass. Optix expression is detected in the eye, wing and haltere imaginal discs. Ectopic expression of Optix leads to the formation of ectopic eyes, suggesting that Optix has important functions in eye development. Although Optix and sine oculis both belong to the Six/so gene family and share a high degree of amino acid sequence identity, there are a number of factors that suggest that their developmental roles are different: (1) the expression patterns of Optix and sine oculis are clearly distinct; (2) sine oculis acts downstream of eyeless, whereas Optix is expressed independent of eyeless; (3) sine oculis functions synergistically with eyes absent in eye development whereas Optix does not; (4) ectopic expression of Optix alone, but not of sine oculis, can induce ectopic eyes in the antennal disc. These results suggest that Optix is involved in eye morphogenesis by an eyeless-independent mechanism (Seimiya, 2000).

In order to determine a possible epistatic relationship between Optix and eyeless, Optix expression was examined in the ey² mutant. In ey², no ey transcripts can be detected, either in the embryonic eye primordia or in the larval eye disc. In ey² eye discs, Optix expression is not affected (Seimiya, 2000). In contrast, so expression is no longer observed in the early third instar eye discs of ey² mutants (Halder, 1998).

Optix is expressed in front of the morphogenetic furrow, strongly suggesting that Optix may play an important role in early eye disc development. Since to date no mutant for Optix has been identified, the potential for Optix to induce the formation of ectopic eyes was examined using a gain-of-function strategy. The GAL4 system was used to target Optix expression to various imaginal discs where Optix is normally not expressed. UAS-optix was crossed to dpp^blink-GAL4 that expresses GAL4 along the anteroposterior compartment boundary in leg, wing and antennal imaginal discs. Targeted expression of Optix cDNA induces ectopic eye structures just in the antenna and the anterior medial region of the head, but not in the legs or in the wings. The normal eyes are reduced in size and only rarely detected are extra ocelli and interocellar bristles, around the vertex region. The efficiency of induction of ectopic eyes is relatively low (i.e. 20% as compared to 100% in ey). In contrast to Optix, ectopic expression of so alone cannot induce ectopic eyes. UAS-Optix was crossed to E132-GAL4 which can induce ectopic eyes in combination with UAS-ey. However, the UAS-Optix x E132-GAL4 flies die as embryos, whereas the UAS-ey x E132-GAL4 controls survive and formed ectopic eyes (Seimiya, 2000).

Since ectopic expression of eyes absent, dachshund, eya plus sine oculis and eya plus dac requires eyeless to form ectopic eyes, an examination was performed to see whether ey expression is also induced during ectopic eye formation by Optix. In the eye discs of UAS-Optix dpp-GAL4 flies, no ectopic ey expression was detected. Therefore attempts were made to induce ectopic eye formation with Optix in an ey² mutant background. Targeted expression of the Optix gene in an ey² background results in ectopic eye formation. The efficiency of occurrence of ectopic eyes does not change from the wild-type background situation, but extra ocelli are induced more often than in a wild-type background. From these results, it is concluded that Optix does not require ey expression for the induction of ectopic eyes (Seimiya, 2000).

Since ey is expressed much earlier in the eye anlagen than Optix, the fact that Optix can induce ectopic eyes only in the eye disc while ey can induce ectopic eyes in other discs as well suggests that ey induces a larger set of target genes than Optix, and that the activity of some of those genes are required for eye induction by Optix. This interpretation is supported by the observation that Optix cannot induce ectopic eyes in a so or eya mutant background. Furthermore, the ectopic expression of ey is sufficient to induce ectopic Optix expression, although in normal eye development Optix transcription is not regulated by ey. Since all these results come from an ectopic situation it will be necessary to analyze the relationship of Optix and ey in an Optix mutant background (Seimiya, 2000).

A Sine oculis/Eyes absent complex regulates multiple steps in eye development and functions within the context of a network of genes to specify eye tissue identity. Ectopic expression of so alone does not induce ectopic eyes, and ectopic expression of eya alone induces ectopic eyes just in the antenna at low frequency (10%), but coexpression of so and eya leads to an increase in the induction of ectopic eyes in the antenna both in frequency (76%) and size. This synergistic effect is probably due to the capability of So and Eya to form a protein complex. The domains required for complex formation are the evolutionarily conserved Six and Eya domains. Since Optix has a Six domain as well, a test was performed to see whether Optix and Eya also synergize and enhance ectopic eye induction. UAS-eya;UAS-Optix was crossed to dpp ^blink-GAL4 and the frequency of induction of ectopic eyes was examined. Optix can induce ectopic eyes (22%) but so cannot (0%); so has a synergistic function with eya (0% and 10% individually, to 60% when coexpressed), but coexpression of Optix and eya does not lead to an increase in frequency (20%) nor in size of ectopic eyes. Therefore, although Optix has a Six domain, no synergistic interaction with Eya has been demonstrated (Seimiya, 2000).

The isolation and functional analysis of Optix provides new insights into the evolution of the Six/so gene family. [table below]

Subclasses
six3	six1	six4
Optix Six3 Optx2	so Six1 Six2	Six4 Six5

Optix belongs to the Six3 subclass, whereas so has been assigned to the same subclass as Six1; Six4 and Six5 form a third subclass.

The mouse genes, Six3 and Optx2, which are in the Six3 subclass, the same as Optix, are expressed in the optic vesicles and the lens, i.e. in eye morphogenesis (Oliver, 1995; Toy, 1998). In contrast Six1 and Six2, members of the Six1 subclass, are expressed in phalangeal tendons, skeletal and smooth muscle, i.e. primarily in myogenesis. Although [Six1 and Six2] and [Six4 and Six5] are assigned to different subclasses on the basis of their amino acid sequences, both Six1 and Six5 seem to control early steps of myogenesis, and Six1 and Six4 are able to transactivate a reporter gene containing a myogenin promoter fragment. These Six genes seem to act at a high level in the hierarchical cascade controlling myogenesis. Based on these reports, it is conceivable that genes in subclasses Six1 and Six4 share the same functions and are controlling muscle formation. In contrast, Six3 subclass genes have an important function in eye development. Therefore, it seems that these two groups of Six genes might have diverged to serve different functions. This also applies to the interactions with Eya genes. In the mouse, Six2, Six4 and Six5 induce nuclear translocation of Eya1, Eya2 and Eya3, which are all localized in the cytoplasm, but Six3 does not. Furthermore, Six1/Eya2 and Six2/Eya1 genes are widely coexpressed in many tissues during organogenesis. Moreover, the Pax3 gene is also required for the same steps. These findings suggest the possibility that Pax, Six and Eya proteins, all of which are coexpressed during vertebrate somitogenesis, cooperate during vertebrate muscle development. In addition to their major roles in myogenesis, Six2, Six4 and Six5 are expressed in the retina, but the gene that plays a major role in eye development is Six3. For this reason, it had been thought that so is the Drosophila ortholog of Six3, but this assignment needs to be revised. Optix is the putative Six3 ortholog; so clearly belongs to the Six1 subclass. This phylogenetic relationship is also supported by the fact that so interacts with eya, whereas Optix does not (Seimiya, 2000 and references therein).

GENE STRUCTURE

Optix maps to position 44A on chromosome 2, a position relatively close to the so locus (43C). This suggests that Optix and so arose by a tandem gene duplication event (Seimiya, 2000). Similarly, the mouse Six2 and Six3 genes are closely linked on chromosome 17 (Oliver, 1995), as are the human SIX1 and OPTX2 genes on chromosome 14 (Toy, 1998).

PROTEIN STRUCTURE

Amino Acids - 292 (Seo, 1999)

Structural Domains

The vertebrate Six genes are homologs of the Drosophila homeobox gene sine oculis (so), which is essential for development of the entire visual system. Two new Six genes in Drosophila, D-Six3 and D-Six4, are described that encode proteins with strongest similarity to vertebrate Six3 and Six4, respectively. In addition, the partial sequences of 12 Six gene homologs from several lower vertebrates are described. The class of Six proteins can be subdivided into three major families, each including one Drosophila member. Comparisons of the sequence identities between the homeodomain (HD) and Six domain (SD) of the individual Drosophila proteins relative to their respective vertebrate homologs show clear differences in the degree of conservation. The HDs of both so and D-Six3 are almost identical (95% and 97%) to the corresponding sequence of their murine homologs Six1/Six2 and Six3, respectively. So and D-Six3 belong to separate subclasses of Six genes: subclass Six1 and Six3 respectively (Seimiya, 2000). D-Six3 is almost identical in sequence to Optix, a second Six3 subclass protein in Drosophila (Seimiya). The similarity between the SD and so of Six1/Six2 (84%) is significantly higher than between D-Six3 and Six3 (77%). In the case of D-Six4 and its murine homolog Six4 (both members of the Six4 subclass), both the HD (82%) and SD (57%) have considerably lower sequence identities. These values show that the HDs are generally the most conserved domains of these homologous proteins, which have more variable divergences in their SDs (Seo, 1999).

The N- and C-terminal regions of the three Drosophila proteins are highly variable in length and sequence. Such differences also exist in the vertebrate homologs Six3 and Six4. One characteristic feature of the so protein is the presence of homopolymorphic regions in both the N- and C-terminal parts. Similarly, D-Six3 has a few short homopolymers in the C-terminus, whereas the D-Six4 protein does not exhibit this type of amino acid repeat (Seo, 1999).

The contents of Pro-Ser-Thr (PST) in the C-terminal regions of the three Drosophila proteins (21%-23%) is considerably lower than in many other related vertebrate Six proteins. However, the N-terminal part of D-Six3 is quite rich in PST, suggesting the presence of transactivating functions. In the case of D-Six4, which has a much larger N-terminal domain, the average PST level is significantly lower (24%). However, a subregion of 18 amino acids (residues 96-113) within the N-terminus is remarkably PST-rich (72%), and this may correspond to a transactivating domain. Another notable feature of this protein is the presence of many amino acid doublets (and a few triplets), particularly Ser-Ser and Gly-Gly, for which the significance is not known (Seo, 1999).

Alignment of the Optix and other Six/so family homeodomain amino acid sequences reveal that Optix is the putative ortholog of mouse Six3, rather than so, as had been previously assumed (Seimiya, 2000).

date revised: 22 April 2000

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.