BIOLOGICAL OVERVIEW

sine oculis and three other transcription factors are involved in early eye morphogenesis: eyeless, (a homeodomain protein), optimotor blind (the brachyury homolog) and eyes absent (the cell survival and differentiation gene). Sine oculis is present at the birth of the eye primordium, and has a direct role in this process. In mutants there is an increase in cell death in the region of the primordium. Movement of the primordium into the head is arrested at an early stage. optimotor blind continues to be expressed in the invagination-defective optic primordium of sine oculis mutants. Expressed prior to the development of the morphogenetic furrow, sine oculis may have a role in establishment of the furrow, or may influence the direction and polarity of furrow movement (Cheyette, 1994).

Sine oculis and Eyes absent have been found to form a complex and to regulate multiple steps in Drosophila eye development (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). So and Eya (1) regulate common steps in eye development including cell proliferation, patterning, and neuronal development; (2) they synergize in inducing ectopic eyes and (3) interact in yeast and in vitro through evolutionarily conserved domains. Clones of so and of eya mutant cells overproliferate and fail to differentiate into neurons. Mutant clones retain their epithelial organization and lead to abnormal folding of the disc. The previously reported cell death phenotype is a secondary result of cell overgrowth. It is concluded that both so and eya play a role in controlling proliferation in the eye primordium of the eye-antennal disc and may therefore contribute to regulating the size of the disc (Pignoni, 1997).

Morphogenetic furrow (MF) initiation does not occur in so and eya mutant clones. decapentaplegic expression is not detected in mutant so and eya clones. Development of eye tissue in the posterior and lateral regions but not the anterior region of so and eya mutant discs is rescued by ectopic expression of the respective genes. Rescue is restricted to the most posterior region of the mutant discs, leading to the conclusion that so and eyaare required during MF formation. so and eya are also required for neuronal development. Homozygous mutant cells were produced selectively posterior to the MF. These mutant clones result in absence of a significant number of photoreceptors, precisely those that are known to be born late under normal conditions, corresponding to the time of so and eya mutant clone induction (Pignoni, 1997).

Ectopic expression of so has little or no effect on antennal, wing, or leg disc development, while ectopic eya expression often causes mild growth alterations resulting in extra folds in the epithelium and, rarely, formation of small ectopic ommatidial arrays in the antennal disc. Coexpression of so and eya leads to a dramatic increase in the development of ectopic eye tissue in antennal discs. These ommatidial arrays lead to adult eye structures. Ectopic so/eya induce eyeless expression in the antennal disc. When expressed in eyeless mutant discs, ectopic so/eya produces growth alterations, but ectopic eyes are not observed (Pignoni, 1997).

So and Eya are able to physically interact through their evolutionarily conserved domains. The sequences responsible for interaction are localized to the N-terminal domain of Eya, while the So interaction domain is localized to the Six domain, a conserved sequence shared with vertebrate So homologs. Because of the multiple effects of the So/Eya interaction in MF induction, cell proliferation and neural induction, it is proposed that a So/Eya complex regulates multiple steps in eye development and functions within the context of a network of genes to specify eye tissue identity (Pignoni. 1997).

Evidence is provided for the existence of an extraocular light perception in Drosophila that does not use the same phototransduction cascade as the adult photoreceptors. The Drosophila larva modulates its pattern of locomotion when exposed to light. Modulation of locomotion can be measured as a reduction in the distance traveled and by a sharp change of direction when the light is turned on. When the light is turned off this change of direction, albeit significantly smaller than when the light is turned on, is still significantly larger than in the absence of light transition. Mutations that disrupt adult phototransduction disrupt a subset of these responses. In larvae carrying these mutations the magnitude of change of direction when the light is turned on is reduced to levels indistinguishable from that recorded when the light is turned off, but it is still significantly higher than in the absence of any light transition. Similar results were obtained when these responses were measured in strains where the larval photoreceptor neurons were ablated by mutations in the glass (gl) gene or by the targeted expression of the cell death gene head involution defective (hid). A mutation in the homeobox gene sine oculis (so) that ablates the larval visual system, or the targeted expression of the reaper (rpr) cell death gene, abolishes all responses to light detected as a change of direction. The existence of an extraocular light perception is suggested that does not use the same phototransduction cascade as the adult photoreceptors. These results indicate that this novel visual function depends on the blue-absorbing rhodopsin Rh1 and is specified by the so gene (Busto, 1999). The larval visual system was first described in the house fly Musca domestica by Bolwig and henceforth was named the Bolwig's organ. Similarly, in D. melanogaster, the larval visual system is composed of two bilateral groups of 12 photoreceptor cells located in the more anterior part of the head, juxtaposed to the mouth hooks. The axons of the photoreceptor cells form the larval optic nerve that innervates the optic lobes primordium area of the brain lobes. The early development and the establishment of connectivity in this system has been described previously. In the Drosophila larva a light stimulus modulates the direction of movement as well as quantitative aspects of locomotion such as path length and frequency of turning. Mutations that disrupt phototransduction in the adult eye disrupt aspects of the larval response to light measured in this assay. These results suggest that the larval and adult visual systems are similar from the functional point of view. These mutations, however, fail to abolish all perception of light, suggesting the existence of a light detection mechanism that does not require these gene products. The analysis of developmental mutants and of strains where the cell death genes are ectopically expressed suggests that this novel light detection mechanism is not located in the Bolwig's organ (Busto, 1999).

In the wild-type strains tested, change of direction when the light is turned off is greater than in the absence of light transitions, suggesting that turning off the light is a transition perceived by the animal. This observation supports the notion that a simple mechanism for the perception of light exists in the Drosophila larva that distinguishes changes in light conditions from absence of light transitions but is unable to distinguish whether the light is being turned on or off. This light response is mediated by the blue-absorbing rhodopsin (Rh1) because it is abolished in part by mutations in the ninaE gene. Interestingly, it does not rely on the same phototransduction pathway as that of the adult visual system as seen by the wild-type response of norpA and ninaC mutant larvae. The results indicate that these hypothetical photoreceptors are not housed within the Bolwig's organ, defined as the larval photoreceptors that depend on the gl gene function for differentiation. However, the observation that the function of this visual system is impaired in larvae where the cell death gene rpr is expressed under the control of the gl promoter demonstrates that these are cells in which the gl transcription factor is functional. Thus it is possible that this novel function is performed by a small number of cells that express Rh1 and the gl gene product but whose differentiation and Rh1 expression are not under the control of the gl gene (Busto, 1999).

Two different groups of cells are likely to be involved in this novel light perception. The observation that the ability of these cells to register this type of light perception is dependent on the so gene function but not gl suggests that these cells are included in the optic lobe primordium. The ablation of this proposed function by expression of the cell death gene rpr under the gl promoter suggests that the central brain neurons that express the gl gene are also involved in this behavior. A precedent for a light detector that does not rely on known elements of the phototransduction machinery in adults is the photic input pathway required for the entrainment of the circadian rhythm. The novel visual system function proposed in this paper presents other parallels with cells involved in the control and generation of circadian rhythms. Mutations in the gl gene do not abolish circadian rhythms. However, the expression of the period (per) gene under the control of the gl promoter is sufficient to restore circadian rhythmicity in per mutant flies. These results strongly suggest that the gl-expressing cells that are not the photoreceptors house the circadian pacemaker. It is possible that this novel visual function, which distinguishes changes in light condition from absence of light transitions but is unable to distinguish whether light is being turned on or off, is also involved in the control of pacemaker oscillation (Busto, 1999).

Dynamic rewiring of the Drosophila retinal determination network switches its function from selector to differentiation

Organ development is directed by selector gene networks. Eye development in Drosophila is driven by the highly conserved selector gene network referred to as the 'retinal determination (RD) gene network,' composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. This study identified a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. Central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. These results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. It is concluded that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development (Atkins, 2013).

This work has found that a switch from high to low levels of Ey expression is required for normal differentiation during retinal development. A mechanism is presented of Ey regulation by the RD gene network members Eya, So, and Dac. Specifically, So switches from being an activator to a suppressor of ey expression, both depending on a So binding site within an ey eye-specific enhancer. It is additionally reported that the So cofactors Eya and Dac are required for ey repression posterior to the furrow but not for its maintenance ahead of the furrow, and are sufficient to cooperate with So to mediate Ey repression within the normal Ey expression domain (Atkins, 2013).

The results support a Gro-independent mechanism for the suppression of target gene expression by the transcription factor Sine oculis (So). An independent study has also shown that So can repress the selector gene cut in the antenna in a Gro-independent process though the mechanism was not determined (Anderson, 2012). It was observed that Ey is expressed at low levels posterior to the morphogenetic furrow. However, when so expression is lost in clones posterior to the furrow, Ey expression and ey-dGFP expression are strongly activated. This is not simply a default response of ey to So loss, as removing So from developmentally earlier anterior cells results in reduced ey expression. Knockdown of So specifically in differentiating cells using RNAi causes a similar phenotype, suggesting that an activator of Ey expression is expressed in differentiating photoreceptors. Mutation of a known So binding site in ey-dGFP results in activation of the reporter posterior to the furrow, supporting a model that binding of So to the enhancer prevents inappropriate activation of ey expression posterior to the furrow. Finally, in vitro it was observed that an excess of So is sufficient to prevent activation of the enhancer; in vivo overexpression of So can also suppress normal Ey expression. The observations are consistent with what in vitro studies have indicated about So function: when So binds DNA without Eya, it can only weakly activate transcription. However, the current work introduces a novel mechanism of regulation for So targets, in which So occupancy of an enhancer prevents other transcription factors from inducing high levels of target gene expression. The results also indicate that suppression of robust ey expression is an important developmental event. It is not yet clear if maintaining basal expression of ey, rather than completely repressing it, is developmentally important; however, it is possible that the ultimate outcome of a basal level of ey transcription may be necessary for the completion of retinal development (Atkins, 2013).

The results also show that eya is required for Ey suppression in vivo. However, consistent with its characterization as a transcriptional coactivator, in vitro analysis does not indicate a direct role for Eya in repression. Previous studies, and the current observations, indicate that Eya is required for the expression of So posterior to the furrow in the third instar. Additionally, reporter analysis shows that Eya regulation of ey requires the So binding site. It is proposed that the simplest model for Eya function in the suppression of ey is through its established function as a positive regulator of So expression, as it was observed that overexpression of So alone is sufficient to weakly repress Ey expression and to block reporter activation in vitro. This model could also account for the results reported regarding the inability of this UAS-so construct to induce ectopic eye formation. Briefly, the primary function of So in ectopic eye formation is to repress the non-eye program (Anderson, 2012). Overexpressing the So construct used in this study alone is not sufficient to induce this program, possibly because the transgene expression level is not sufficient; however, co-expression of the so positive regulator Eya is sufficient to induce robust ectopic eye formation. In light of the current findings, it is proposed that Eya co-expression is necessary to induce So expression to sufficient levels to block transcriptional activation of non-eye targets to permit the induction of the ectopic eye program; however other functions of Eya may play a role (Atkins, 2013).

It was further demonstrated that dac expression is required specifically near the furrow for Ey repression. In addition, this study showed that the So binding site is required for strong ey expression in dac clones near the furrow, suggesting that So activates ey in these clones. This suggests that repression by Dac occurs before the transition to repression by So, making Dac the first repressor of ey expression at the furrow, and identifying how the initiation of repression occurs before So levels increase. It was further shown that Eya and So are sufficient to repress ey expression in dac mutant clones anterior to the furrow, though not as completely as in cells that express Dac. This result indicates that Dac is not an obligate partner with Eya and So in ey repression, but is required for the full suppression of ey. One model would be that Dac and So can cooperate in a complex to modestly repress eyeless directly. This would be consistent with loss-of-function and reporter data as well as the observation that Dac and So misexpression can weakly cooperate to repress Ey anterior to the furrow. However, while a similar complex has been described in mammalian systems, previous studies have been unable to detect this physical interaction in Drosophila. An alternative model is that Dac suppresses ey expression indirectly and in parallel to Eya and So. A previous study has shown that dac expression is necessary and sufficient near the furrow to inhibit the expression of the zinc finger transcription factor Teashirt (Tsh). Tsh overlaps Ey expression anterior to the furrow, and can induce Ey expression when misexpressed. Furthermore, tsh repression is required for morphogenetic furrow progression and differentiation. In light of these previous findings, a simpler model is proposed based on current knowledge that Dac repression of tsh at the morphogenetic furrow reduces Ey expression indirectly. Future studies may distinguish between these mechanisms (Atkins, 2013).

In addition to the role of the RD gene network in ey modulation,signaling events within the morphogenetic furrow indirectly regulate the switch to low levels of ey expression. It has been shown that signaling pathways activated in the morphogenetic furrow increase levels of Eya, So and Dac; furthermore, it is proposed that this upregulation alters their targets, creating an embedded loop within the circuitry governing retinal development and allowing signaling events to indirectly regulate targets through the RD network. The identification of ey regulation by So posterior to the morphogenetic furrow represents a direct target consistent with this model (Atkins, 2013).

In conclusion, a model is presented that rewiring of the RD network activates different dominant sub-circuits to drive key transitions in development (see A model for dynamic RD gene network interactions during the third instar). To the interactions previously identified by others, this study adds that strong upregulation of So, dependent on Eya, results in minimal levels of ey transcription. It is proposed that the identification of this novel sub-circuit of the RD network provides a mechanism for terminating the self-perpetuating loop of determination associated with high levels of Ey, permitting the onset of differentiation and the completion of development. Together, these results give a new view into how temporal rewiring within the RD network directs distinct developmental events (Atkins, 2013).

GENE STRUCTURE

Genomic length - 16 kb

cDNA clone length - 2861

Bases in 5' UTR -870

Exons - eight

Bases in 3' UTR - 742

PROTEIN STRUCTURE

Amino Acids - 416

Structural Domains

There is a central homeodomain, a C-terminal stretch rich in glutamine, and an N terminal rich in asparagine (Cheyette, 1994).

date revised: 15 July 98

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