Transcriptional Regulation

The exclusive expression of fringe in the dorsal domain is presumably controlled by the LIM-type homeobox gene apterous (Kim, 1995).

The Drosophila eye is divided into dorsal and ventral mirror image fields that are separated by a sharp boundary known as the equator. Mirror, a homeodomain-containing putative transcription factor with a dorsal-specific expression pattern in the eye, induces the formation of the equator at the boundary between mirror-expressing and non-expressing cells. Evidence is provided that suggests mirror regulates equator formation by two mechanisms: (1) mirror defines the location of the equator by creating a boundary of fringe expression at the mid-point of the eye. mirror creates this boundary by repressing fringe expression in the dorsal half of the eye. Significantly, a boundary of mirror expression cannot induce the formation of an equator unless a boundary of fringe expression is formed simultaneously. (2) mirror acts to sharpen the equator by reducing the mixing of dorsal and ventral cells at the equator. In support of this model, it has been shown that clones of cells lacking mirror function tend not to mix with surrounding mirror-expressing cells. The tendency of mirror-expressing and non-expressing cells to avoid mixing with each other is not determined by their differences in fringe expression. Thus mirror acts to regulate equator formation by both physically separating the dorsal cells from ventral cells, and restricting the formation of a fng expression boundary to the border where the dorsal and ventral cells meet (Yang, 1999).

The formation of an ectopic equator, associated with a fng+/fng- border is restricted to the segments of clonal borders that are located within the anterior one third of the eye, even when the borders extend more posteriorly. A Drosophila eye typically consists of 32-34 vertical columns of ommatidia. No ommatidia of ventral polarity have ever been detected at a mirr mutant clonal border beyond column 12, counted from the anterior edge. Since the ectopic expression of fng-lacZ in mirr mutant discs is also restricted to the dorsal anterior region of the eye, this data is consistent with the hypothesis that ectopic fng expression in mirr mutant clones is required to induce ectopic equator formation at the equatorial borders of clones (Yang, 1999).

It was asked directly if the formation of the ectopic equators at the equatorial borders of mirr dorsal clones is dependent on ectopic fng expression within the clones. Ectopic expression of fng within the mirr mutant clones is required to induce ectopic equators at the equatorial borders of the clones. Thus one way in which mirr regulates equator formation is by repressing fng expression in the dorsal region of the eye. Removal of fng in the dorsal region of the eye causes an ectopic mini-equator to form at the polar border of the clone. One to three ommatidia of ventral polarity are often generated at the polar boundaries of fng dorsal clones. The ectopic juxtaposition of the dorsal and ventral ommatidia at the polar boundary of a fng dorsal clone has been called a ‘mini-equator’ because of its short length. Although the expression of fng transcript is restricted to the ventral region of the eye during the early stages of eye development, it is later transiently present in a narrow band of cells associated with the furrow in the dorsal region of the eye. Thus the formation of the ectopic mini-equators may be in part due to the transient fng+/fng- boundaries created at the polar borders of fng dorsal clones as the furrow passes through the clones (Yang, 1999).

The sharpness displayed by the path of the wild-type equator is a poorly understood aspect of equator formation. Several observations have suggested that mirr is involved in controlling the sharpness of the equator. (1) A reduction in mirr expression causes a reduction in the sharpness of the equator. One role of mirr in sharpening the equator is to create a difference in cell affinities between dorsal and ventral cells. In a clonal analysis, it was noted that cells that reside inside a mirr mutant dorsal clone tend to minimize interactions with the surrounding mirr-expressing cells, resulting in a rounded clone shape. In addition, the border where mirr-expressing and non-expressing cells juxtapose appears to be significantly smoother compared to the border where no difference in mirr exists across the border. (2) mirr mutants that occasionally survive until adulthood display a dramatic dorsal protrusion from the surface of their eyes. Such a protrusion is suggestive of a group of cells attempting to sort out from the epithelium due to differences in cell affinities. (3) When mirr is overexpressed in dorsal regions of the eye through overexpression of wg, a visible indentation of the epithelium is observed at the novel boundary formed between mirr -expressing and non-expressing cells. This suggests that increasing the differences in mirr expression between dorsal and ventral cells causes them to further minimize contacts with each other, forming a physical groove between them. One important aspect of these findings is that the segregation of dorsal and ventral cells appears to be a process that is independent of the difference in their expression of fng. The shapes of mirr::fng dorsal clones remains significantly rounder than those of wild-type clones, thus the ectopic expression of fng within mirr mutant cells is not likely to be the cause for the reduction of cell-cell mixing between mirr-expressing and non-expressing cells. In addition, the shapes of fng mutant ventral clones are irregular and are not significantly different from those of wild-type ventral clones, thus the difference in fng expression between dorsal and ventral cells is unlikely to be the cause for the sharpness of the wild-type equator. Such a finding is in contrast to fng’s role in D/V border formation in the developing wing disc. In wing discs, removal of fng function in a clone of cells in the dorsal half of the wing disc, where fng is normally expressed, results in a very round clone with a smooth clonal border. In addition, ectopically expressing fng in a clone of cells in the ventral part of the dics where fng is typically absent also results in round clones with a very smooth border. It has been suggested that fng might have a role in controlling cell adhesion in the developing wing disc. Although the possibility that fng is also important in regulating cell adhesion in the eye disc cannot be ruled out, the data strongly suggests that additional components regulated by mirr must be involved. One possible model is that mirr might be regulating some adhesion molecules that are differentially expressed between dorsal and ventral cells. It is concluded that mirr regulates equator formation in the eye by two independent yet complementary pathways. mirr acts to sort the dorsal cells from ventral cells by reducing cell-cell mixing at the boundary where the dorsal and ventral cells juxtapose. In addition, it restricts the activation of Notch signaling to the point where the dorsal and ventral cells meet by repressing fng in the dorsal cells. These two functions of mirr lead to a co-ordination of morphology and signaling in the process of equator formation (Yang, 1999).

Dorsoventral axis formation in the Drosophila wing depends on the activity of the selector gene apterous. Although selector genes are usually thought of as binary developmental switches, Apterous activity is found to be negatively regulated during wing development by its target gene dLMO. Apterous-dependent expression of Serrate and fringe in dorsal cells leads to the restricted activation of Notch along the dorsoventral compartment boundary. Evidence is presented that the ability of cells to participate in this Apterous-dependent cell-interaction is under spatial and temporal control. Apterous-dependent expression of dLMO causes downregulation of Serrate and fringe and allows expression of Delta in dorsal cells. This limits the time window during which dorsoventral cell interactions can lead to localized activation of Notch and induction of the dorsoventral organizer. Overactivation of Apterous in the absence of dLMO leads to overexpression of Serrate, reduced expression of Delta and concomitant defects in differentiation and cell survival in the wing primordium. Thus, downregulation of Apterous activity is needed to allow normal wing development (Milan, 2000).

Removing Apterous activity at different stages of wing development shows that Ap is needed throughout larval stages to confer dorsal cell identity, but its role in Notch activation along the DV boundary is temporally and spatially modulated. This can be explained in terms of changes in Serrate and fringe expression. Some of the changes in Serrate and fringe expression are caused by reducing Ap activity, whereas others are Ap independent. In early second instar wing discs, Ap activity is required in the entire dorsal compartment. Removing Ap activity in mitotic recombination clones at this stage induces Notch activation at the interface between wild-type and mutant cells. This response is independent of the position of the clone within the wing pouch. In early third instar wing discs, Ap-dependent expression of Serrate and fringe is reduced by dLMO. Serrate expression gradually becomes restricted to the region near the DV boundary and, subsequently, by mid-third instar is induced only in cells adjacent to the boundary. The effects of removing Ap activity in clones reflects the gradual retraction of Serrate expression toward the DV boundary. Clones of cells lacking Ap activity induced in early third instar activate the Notch pathway and induce Wg if they are located close to the DV boundary. Clones located more proximally do not show this response. This spatial difference can be overcome by providing Serrate in proximal cells (Milan, 2000).

By mid-third instar, new Ap-independent patterns of Serrate and fringe expression are observed. Serrate is expressed on both sides of the DV boundary by the activity of Wg, and fringe is expressed in four quadrants flanking the DV and AP compartment boundaries. Maintenance of Notch activation along the DV boundary is now under control of a feedback loop between Wg and Serrate and Delta. Ap is no longer required for Notch activation at the DV boundary and removing Ap activity no longer leads to activation of the Notch pathway. In the absence of dLMO, Ap activity remains at high early levels as development proceeds. Serrate and fringe expression remain high throughout the dorsal compartment and fail to undergo normal modulation. In addition, Delta is not expressed in dorsal cells. Ap-dependent repression of Delta at early stages is needed to prevent ectopic activation of Notch in dorsal cells, which are inherently Delta-sensitive due to the activity of Fringe. Some of the defects observed in dLMO mutant wings are correlated with excess Serrate activity and insufficient Delta activity. In addition, abnormally high levels of cell death in the dorsal compartment of the dLMO mutant wing disc are due to excess Ap activity and this leads to overall reduction in the size of the wing. These findings indicate the need to downregulate Ap activity to allow normal wing development. However, Ap activity continues to be required for dorsal cell fate specification and for proper adhesion of D and V wing surfaces. Thus it is proposed that different target genes may be controlled at different levels of Ap activity. Serrate, fringe and Delta may be regulated by a higher level of Ap activity than the target genes involved in surface apposition or fate specification. Temporal changes in the level of Ap activity may be required to modulate activity of different genes at different times to allow normal wing development (Milan, 2000).

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

In recent years the key role of the Notch signaling pathway in the segmentation and growth of the Drosophila leg has been established. Notch signaling must be localized within each leg segment to promote the formation of boundaries (joints) that separate each leg segment and to induce leg growth. This requirement for a segmentally repeated pattern of Notch activation is accomplished by restricting the expression of the regulators of Notch activation, Serrate, Delta and fringe, to one ring per segment. By examining the expression of the Notch ligands and fringe during leg development, it has been possible to determine the progressive order in which leg segmentation is established. At early third instar, a single ring of Serrate, Delta and fringe expression is present within the coxa. The next ring to arise is located within the presumptive femur. At mid third instar, expression arises within presumptive tarsal segments 2 and 5. Subsequent expression is observed in the tibia and more tarsal segments, such that ultimately, by the end of third instar, a ring of expression is present in each presumptive leg segment, adjacent to each prospective leg segment border. Thus, segmentation of the Drosophila leg occurs progressively and in a reproducible pattern (Rauskolb, 2001).

Previous studies investigating the expression of a reporter gene [E(spl)mß-CD2] regulated downstream of Notch activation led to the conclusion that the first segment boundary to form was between tarsal segments 4 and 5. Additional rings of expression were then observed in the tarsus and then eventually in all leg segments. This led to the suggestion that the first segmental boundaries to form correspond to the most distal segments. However, further examination of this reporter gene indicates that expression is observed in proximal cells prior to expression within the tarsus. Moreover, temperature shifts of a conditional Notch allele at different stages of development demonstrate that the temperature-sensitive period for Notch in proximal segmentation occurs before that in tarsal segmentation. The conclusion is reached that leg segmentation does not occur in a simple distal to proximal order, nor proximal to distal order, nor are the most proximal and distal segments established first and other segments added by intercalation. Rather, the establishment of Drosophila leg segmentation occurs in a complex sequence (Rauskolb, 2001).

A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).

How do these three transcription factors regulate the formation of nine segments? Since the requirements for and the expression of the leg gap genes encompasses all leg segments, it is unlikely that there are additional leg gap genes yet to be identified. Rather, a collection of distinct combinatorial approaches is used to establish a segmental pattern of Serrate, Delta and fringe expression (Rauskolb, 2001).

In early third instar leg discs, there are two domains of gene expression: proximal cells express Hth and distal cells express Distal-less. Hth autonomously promotes the expression of Serrate, while Distal-less may prevent expression more distally, giving rise to a ring of expression in the coxa. Additionally, Distal-less-expressing cells may signal to the Hth-expressing cells to restrict Serrate expression to the distal edge of the Hth domain. As the leg disc grows, cells in an intermediate position, lying between the Hth and Distal-less domains, begin to express Dac. Dac, as shown in this study, is both necessary and sufficient to induce the expression of Serrate, Delta and fringe within the femur. Since they are not expressed in all Dac-expressing cells, other factors appear to be required to promote their expression in the proximal femur. The nonautonomous induction of Serrate expression by Hth suggests that this may be accomplished by a signal (X) emanating from the neighboring Hth-expressing cells. By mid third instar stages, expression of Serrate, Delta and fringe is also observed in tarsal segments 2 and 5, within cells expressing Distal-less but not Dac. Given that Distal-less is necessary and sufficient to repress their expression, Serrate, Delta and fringe expression within the tarsus appears to be induced by a mechanism that overrides the repressive effects of Distal-less. Subsequently, expression of Serrate, Delta and fringe is observed within the tibia, in cells expressing both Dac and Distal-less. Dac is necessary for expression of Serrate within the tibia, and its role here may be to overcome the repressive effects of Distal-less. It is also worth noting that the tibia ring of expression is not established at the time when cells first express both Dac and Distal-less. This may be because Dac levels may not be sufficiently high enough to overcome the repression by Distal-less. Clearly levels of Dac expression are critical because simply increasing Dac levels is sufficient to promote Serrate expression in cells already expressing endogenous levels of Dac. This observation can be explained if high levels of Dac expression in cells already expressing Dac override the function of inhibitory regulators of Serrate expression, such as Distal-less, where the expression of these genes overlap. Although late stages of leg segmentation were not investigated in this study, it has been noted that Hth, Dac and Distal-less are co-expressed in the presumptive trochanter late in leg development. It is thus hypothesized that Serrate, Delta and fringe expression is established by the combined activities of the three leg gap genes in the trochanter (Rauskolb, 2001).

Although these here have focused on the regulation of Serrate expression, it is thought that not only Serrate, but also Delta and fringe, receive primary regulatory input from the leg gap genes. Delta and fringe expression, like Serrate, is positively regulated by Dac. Moreover, Dl and fringe mutants have stronger leg segmentation phenotypes than Ser mutants, and thus Delta and fringe expression cannot simply be regulated downstream of Ser. The identification of two separate Ser enhancers, directing expression in the proximal versus distal leg, argues against Serrate being regulated downstream of Dl and fringe. Thus, the simplest model is that expression of all three genes is regulated directly by the leg gap genes. The regulation of Serrate, Delta and fringe expression in each segment appears to occur through independent and separable enhancer elements, supported by the analysis of the Ser reporter genes. This is reminiscent of what occurs during Drosophila embryonic segmentation, where separable enhancer elements direct different stripes of pair-rule gene expression (Rauskolb, 2001).

Importantly, Notch signaling may actually coordinate progressive segmentation of the leg with leg growth. For example, in early leg discs there is a single ring of Serrate expression within the coxa, in Hth-expressing cells immediately adjacent to Dac-expressing cells. However, by the time the femur ring arises, the coxa ring of Serrate expression has been displaced and is no longer within cells immediately adjacent to the Dac-expressing cells; rather, there are Hth-expressing cells lying in between that do not express Serrate. Thus, it is postulated that once Serrate, Delta and fringe expression is established within the coxa, Notch is activated, which promotes local cell proliferation, thereby displacing the coxa ring. This then allows for the femur ring of expression to be established in cells that are not immediately adjacent to the coxa expression ring. This mechanism also requires that once a ring of ligand expression is established in a particular segment, this expression must be maintained such that it is not influenced by later alterations in relation to leg gap gene expression. This maintenance could be accomplished by a feedback loop between Notch activation and ligand expression, similar to what has been observed during late wing development, where Notch activation cell autonomously represses ligand expression and nonautonomously induces ligand expression in flanking cells by regulating the expression of a signaling molecule. Preliminary studies have indicated that Notch activation can influence Notch ligand expression in the developing leg (Rauskolb, 2001).

The establishment of the dorsal-ventral axis of the Drosophila wing depends on the activity of the LIM-homeodomain protein Apterous. Apterous activity depends on the formation of a higher order complex with its cofactor Chip to induce the expression of its target genes. Apterous activity levels are modulated during development by dLMO (Beadex). Expression of dLMO in the Drosophila wing is regulated by two distinct Chip dependent mechanisms. Early in development, Chip bridges two molecules of Apterous to induce expression of dLMO in the dorsal compartment. Later in development, Chip, independently of Apterous, is required for expression of dLMO in the wing pouch. A modular P-element based EP (enhancer/promoter) misexpression screen was conducted to look for genes involved in Apterous activity. Osa, a member of the Brahma chromatin-remodeling complex, was found to be a positive modulator of Apterous activity in the Drosophila wing. Osa mediates activation of some Apterous target genes and repression of others, including dLMO. Osa has been shown to bind Chip. It is proposed that Chip recruits Osa to the Apterous target genes, thus mediating activation or repression of their expression (Milan, 2004).

This study presents evidence that Osa, a member of a subset of Brahma chromatin remodeling complexes, behaves overall as a general activator of Apterous activity in the Drosophila wing. Overexpression of Osa rescues and loss of Osa enhances the Beadex1 phenotype. It does so by modulating the expression levels of Apterous target genes, some of them being activated (e.g. Serrate and probably other unknown target genes) and some repressed (e.g. Delta, fringe). Chip has been shown to bind Osa. The fact that Osa has different effects on the transcription of Apterous target genes suggests that Chip recruits Osa to the promoters and in combination with other unknown factors mediates either transcriptional repression or activation. Osa mediates repression of both Apterous dependent and independent expression of fringe, suggesting a direct and probably Chip independent effect of Osa on fringe transcription (Milan, 2004).

Targets of Activity

The activation of an intronic regulatory element of vestigial in a narrow band of cells centered on the natural dorsoventral boundary is one of the first signs of wing formation. This enhancer is activated by the juxtaposition of fringe-expressing and fringe negative cells within the dorsal compartment. The direct target of Fringe appears to be Serrate, which in turn induces wingless and vestigial in ventral cells, presumably through Notch. Serrate is induced by the juxtaposition of fringe expressing and fringe negative cells (Williams, 1994).

In the developing imaginal wing disc of Drosophila, cells at the dorsoventral boundary require localized Notch activity for specification of the wing margin. The Notch ligands Serrate and Delta are required on opposite sides of the presumptive wing margin; even though activated forms of Notch generate responses on both sides of the dorsoventral boundary, each ligand generates a compartment-specific response. Serrate, which is expressed in the dorsal compartment, does not signal in the dorsal regions due to the action of the fringe gene product. Using ectopic expression, it has been shown that regulation of Serrate by Fringe occurs at the level of protein and not Serrate transcription. Furthermore, replacement of the N-terminal region of Serrate with the corresponding region of Delta abolishes the ability of Fringe to regulate Serrate without altering Serrate-specific signaling (Fleming, 1997).

Polarity of the Drosophila compound eye is established at the level of repeating multicellular units (known as ommatidia), which are organized into a precise hexagonal array. The adult eye is composed of ~800 ommatidia, each of which forms one facet. Sections through the eye reveal that each ommatidium contains eight photoreceptor cells in a stereotypic trapezoidal arrangement that has two mirror-symmetric forms: a dorsal form present above the dorsoventral (DV) midline, and a ventral form below. An axis of mirror-image symmetry runs along the DV midline and is known as the equator. By analogy to the terrestrial equator, the extreme dorsal and ventral points of the eye are referred to as the poles. Differentiation of ommatidia begins during the third instar larval stage when a furrow moves from posterior to anterior over the epithelium of the eye imaginal disc. Each ommatidial unit is born as a bilaterally symmetrical cluster of photoreceptor precursors, that is polarized on its anteroposterior axis. The clusters then become polarized on the DV (or equatorial-polar) axis, by the process of proto-ommatidium rotation via two 45° steps away from the DV midline, forming the equator. It has been suggested that the direction of this rotation is a consequence of a gradient of positional information emanating from either the midline or the polar regions of the disc (Zeidler, 1999 and references).

A number of recent studies have shed light on some of the mechanisms involved in the positioning of the equator on the DV midline of the eye imaginal disc. It is now clear that a critical step is the activation of Notch activity in a line of cells along the midline, and that this localized activation of Notch is a consequence of the restricted expression of the fringe (fng) gene product in the ventral half of the disc and the homeodomain transcription factor Mirror (Mirr) in the dorsal half of the disc. Furthermore, an important role for Wingless (Wg) in polarity determination on the DV axis has been demonstrated. Wg is a secreted protein (and the founder member of the Wnt family of morphogens) that is expressed at the poles of the eye disc. Wg has been shown to act as an activator of mirr expression; increasing the levels of Wg expression in the eye disc shifts mirr expression and the position of the equator ventrally, whereas reduction of wg function shifts mirr expression dorsally. Additionally, it has been shown convincingly that a gradient of Wg signaling across the DV axis of the eye disc regulates ommatidial polarity such that the lowest point of Wg signaling coincides with the equator (Zeidler, 1999 and references).

The JAK/STAT pathway is central to the establishment of planar polarity during Drosophila eye development. A localized source of the pathway ligand, Unpaired/Outstretched, present at the midline of the developing eye, is capable of activating the JAK/STAT pathway over long distances. A gradient of JAK/STAT activity across the DV axis of the eye regulates ommatidial polarity via an unidentified second signal. Additionally, localized Unpaired influences the position of the equator via repression of mirror (Zeidler, 1999).

The data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localized activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, since fng loss of function clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity. Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals (most likely detected by the receptor Frizzled) that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal (Zeidler, 1999 and references).

From these characteristics, the following can be deduced: the nonautonomy of the phenotype produced by removal of the autonomously acting pathway component JAK, and its dependence on clone size and shape, suggests that JAK/STAT affects ommatidial polarity via a secreted downstream signal (which subsequently will be referred to as a second signal, most likely detected by Frizzled). The direction of the nonautonomy (only in a polar direction) and the strict DV nature of the polarity inversions indicates that this second signal must be graded in its activity along the DV axis, with a change in direction of the gradient at the equator. The direction of this gradient would then be the instructive cue to which ommatidia respond when rotating to establish their mature polarity (Zeidler, 1999).

The simplest model would be that there is a single second signal secreted from the equator, which is downstream of mirr/fng/Notch, and that Wg and Upd/JAK/STAT feed into this pathway upstream of Notch. This is consistent with the roles of Wg and Upd as regulators of mirr expression and, thus, in positioning the endogenous equator. However, it is not consistent with the observed ommatidial polarity inversions produced in the eye field both dorsally and ventrally by Wg-pathway and JAK/STAT-pathway LOF and GOF clones. These phenotypes indicate that second-signal concentration is dependent on Wg pathway and JAK/STAT pathway activity across the whole of the eye field, and thus the second signal cannot be only secreted from the DV midline as a consequence of localized Notch activation. It is conceivable that Notch is activated on the polar boundary of JAK/STAT LOF clones, but in this context the only known mechanism of Notch activation is via mirr/fng interactions, and this possibility has been ruled out (Zeidler, 1999).

Instead, the data points to a model in which Upd and Wg first act to define the equator via restriction of mirr expression to the dorsal hemisphere and localize activation of Notch along the DV midline. Definition of the equator is known to occur early in development, while the disc is still small, and divides the disc into two hemispheres separated by a straight boundary that will form the future equator. Such boundaries evidently serve as a source of a second signal that can polarize ommatidia, becausefng LOF clones that induce ectopic regions of activated Notch result in changes in ommatidial polarity (Zeidler, 1999).

Subsequently in development, it is surmised that gradients of JAK/STAT and Wg-pathway activity across the DV axis of the eye disc are responsible for setting up a gradient(s) of one or more second signals that can determine ommatidial polarity. These signals might be responsible for maintaining longer range polarization of ommatidia away from the equator and the localized Notch-dependent polarizing signal. A number of observations provide a great deal of support for such a model. (1) It is consistent with the known timing of the events involved. The requirement for fng function has been shown to lie between late first instar and mid second instar, which coincides with the first appearance of high levels of Upd expression at the optic stalk. However, the ommatidia are not formed (and thus do not respond to the polarity signal) until the start of the third instar, a stage when localized Upd expression still persists. Furthermore, extracellular Upd protein can be seen in a concentration gradient many cell diameters from the optic stalk at the early third instar stage, consistent with Upd being at least partly responsible for setting up the long-range gradient of JAK/STAT activity across the DV axis of the eye disc that is revealed by the stat92E-lacZ reporter. (2) This model does not require that a single source of second signal secreted by a narrow band of cells at the equator should be capable of determining ommatidial polarity across the whole of the DV axis of the disc during the third instar stage of development. Instead, the band of activated Notch at the equator would serve to draw a straight line between the fields of dorsally and ventrally polarized ommatidia, and need only secrete a localized source of second signal to polarize ommatidia in this region. Further from the equator, the opposing gradients of Upd and Wg signaling would provide a robust mechanism for maintenance of correct ommatidial polarity across the DV axis. Conversely, without the mirr/fng/Notch mechanism to draw a straight line, it would be impossible to imagine how Upd at the posterior margin and Wg at the poles alone could provide the perfectly straight equator that is ultimately formed. (3) The phenotypes that are observed are consistent with multiple competing mechanisms responsible for determining ommatidial polarity. When inversions of ommatidial polarity are induced by generating hop clones or ectopically expressing Upd, straight equators are not produced, such that two cleanly abutting fields of dorsal and ventral ommatidia are produced. Instead, there is usually some confusion of ommatidial identities as if they might be receiving conflicting signals. Additionally, when upd activity is removed from the optic stalk, an equator still forms (albeit at the ventral edge of the disc), but some ommatidia dorsal to the equator still adopt a ventral fate as if the determination of ommatidial polarity is less robust in the absence of Upd (Zeidler, 1999).

The dorsoventral midline of the Drosophila eye disc is a source of signals that stimulate growth of the eye disc, define the point at which differentiation initiates, and direct ommatidial rotation in opposite directions in the two halves of the eye disc. This boundary region seems to be established by the genes of the iroquois complex, which are expressed in the dorsal half of the disc and inhibit fringe expression there. Fringe controls the activation of Notch and the expression of its ligands, with the result that Notch is activated only at the fringe expression boundary at the midline. The secreted protein Wingless activates the dorsal expression of the iroquois genes. Pannier, which encodes a GATA family transcription factor expressed at the dorsal margin of the eye disc from embryonic stages on, acts upstream of wingless to control mirror and fringe expression and establish the dorsoventral boundary. Loss of pannier function leads to the formation of an ectopic eye field and the reorganization of ommatidial polarity, and ubiquitous pannier expression can abolish the eye field. Pannier is thus the most upstream element yet described in dorsoventral patterning of the eye disc (Maurel-Zaffran, 2000).

Recently, several studies have established that N activation along the dorsoventral midline of the eye disc is critical for eye growth as well as for positioning the equator. This local activation is the consequence of the ventrally restricted expression of fng, which is negatively controlled by the iro-C homeobox genes expressed in the dorsal half of the eye disc. Either loss of fng function, or ubiquitous expression of fng, caup or mirr, abolishes eye growth. The iro-C genes appear to act redundantly, as both ara and caup must be removed from clones of cells to promote the formation of ectopic dorsal eyes similar to those reported for pnr. The similar effects observed for gain or loss of pnr function suggest strongly that pnr might act in the same pathway as the iro-C and fng. To confirm this and to order pnr with respect to these genes, expression of mirr and fng was examined in eye discs mutant for pnr or misexpressing pnr. In eye discs in which pnr function had been removed, mirr expression is greatly reduced, whereas fng is derepressed dorsally. In eye discs expressing constitutively active pnr, mirr expression is expanded ventrally, shifting the point of morphogenetic furrow initiation to the ventral side. fng expression is dramatically reduced in discs overexpressing pnr D4. It thus appears that pnr acts upstream of the iro-C genes, activating their expression dorsally. Consistent with this, it has been found that ubiquitous expression of ara abolishes photoreceptor differentiation, and that removal of pnr function does not restore photoreceptor formation. If pnr were downstream of ara, blocking its function should have induced ectopic eye development even in the presence of ara (Maurel-Zaffran, 2000).

The results above show that pnr acts upstream of the iro-C genes to regulate dorsal eye development. Another molecule that has been shown to act upstream of the iro-C in this context is Wg. wg is required to inhibit the initiation of the morphogenetic furrow at the lateral margins of the eye disc, preventing ectopic eye differentiation there. The dorsal ectopic eyes induced by removing pnr function thus suggest that the functions of pnr and wg may be related. Consistent with this idea, the block in morphogenetic furrow initiation caused by expressing wg throughout the eye disc, like the block caused by expressing pnr D4 , can be rescued by co-expressing an activated form of N. pnr and wg may thus act in the same cascade to prevent eye differentiation (Maurel-Zaffran, 2000).

The role of wg in directing dorsal development is unexpected because wg is also expressed at the ventral anterior margin of the eye disc, although at a lower level than at the dorsal margin; this expression must have an upstream regulator other than pnr. However, the effects of loss of wg are more robust on the dorsal than the ventral side of the eye disc, and misexpression of wg symmetrically at both lateral margins dorsalizes the eye disc. These observations may be explained by the finding that at early stages wg is limited to the dorsal side of the eye disc and may exert its dorsalizing effect at this time (Maurel-Zaffran, 2000).

Recent studies in vertebrates and Drosophila have revealed that Fringe-mediated activation of the Notch pathway has a role in patterning cell layers during organogenesis. In these processes, a homeobox-containing transcription factor is responsible for spatially regulating fringe (fng) expression and thus directing activation of the Notch pathway along the fng expression border. This may be a general mechanism for patterning epithelial cell layers. At three stages in Drosophila oogenesis, mirror (mirr) and fng have complementary expression patterns in the follicle-cell epithelial layer, and at all three stages loss of mirr enlarges, and ectopic expression of mirr restricts, fng expression, with consequences for follicle-cell patterning. These morphological changes are similar to those caused by Notch mutations. Ectopic expression of mirr in the posterior follicle cells induces a stripe of rhomboid (rho) expression and represses pipe (pip), a gene with a role in the establishment of the dorsal-ventral axis. Ectopic Notch activation has a similar long-range effect on pip. These results suggest that Mirror and Notch induce secretion of diffusible morphogens; a TGF-beta (encoded by dpp) has been identified as one such molecule in the germarium. mirr expression in dorsal follicle cells is induced by the EGF-receptor (EGFR) pathway and mirr then represses pipe expression in all but the ventral follicle cells, connecting Egfr activation in the dorsal follicle cells to repression of pipe in the dorsal and lateral follicle cells. These results suggest that the differentiation of ventral follicle cells is not a direct consequence of germline signaling, but depends on long-range signals from dorsal follicle cells, and provide a link between early and late events in Drosophila embryonic dorsal-ventral axis formation (Jordan, 2000).

In oogenesis the expression patterns of mirr and fng are complementary. The expression patterns define borders between cells with specific developmental roles: the encapsulation of 16-cell germline cysts that culminates in their separation from the germarium; the boundary between terminal and lateral follicle cells at stage 6, and the boundary between dorsal anterior and all other follicle cells at stages 8-10. In the germarium, mirr is expressed in the inner sheath cells and the anterior-most follicle cells, whereas fng is expressed in the follicle cells in the posterior part of the germarium. The follicle cells at the expression boundary encapsulate the 16-cell germline cysts and subsequently separate the newly formed egg chamber from the germarium. At stage 6, when the follicle cells in the termini of the egg chamber differentiate from the lateral follicle cells and establish the oocyte anterior-posterior (A-P) polarity, mirr expression is detected in the lateral region, complementary to fng expression in the termini. As the follicle cells migrate posteriorly at stages 8 and 9, mirr expression is detected in the most dorsal anterior follicle cells, whereas fng is expressed in all other follicle cells. At this point, signaling between the oocyte and the follicle cells establishes the dorsal-ventral axis of the follicle-cell layer and the future embryo. The complementary expression patterns of mirr and fng throughout oogenesis are likely to be a consequence of Mirr repression of fng expression. Follicle-cell clones made with a loss-of-function allele of mirr result in an expansion of the fng expression pattern into dorsal anterior follicle cells, and, conversely, overexpression of mirr results in the loss of fng expression (Jordan, 2000).

In a number of developmental systems, regulation of fng by a homeobox gene has a role in establishing a domain in which Notch is activated. Thus the phenotypes observed in mirr and Notch (N) mutants during oogenesis have been compared. In oogenesis, Notch activity is required in the germarium and for the formation of the termini at stage 6. A test was performed to see whether Notch function is also required for dorsal-ventral patterning of follicle cells by analysing the eggs laid by Nts females at the restrictive temperature. The strongest phenotype observed in eggs laid by Nts females is similar to that observed in eggs laid by mirr loss-of-function females: a complete loss of the dorsal appendages. In addition, the ventral pip expression domain is defective in Nts females and restricted due to expression of constitutively active Notch. Thus Notch, like Mirr, functions to restrict pipe expression to the ventral region and to organize dorsal structures; loss of either Mirr or Notch function affects follicle cells on both sides of the Mirr-Fng expression border (Jordan, 2000).

Activation of Notch at a fng expression border has been observed in wing and eye development. In the wing this border acts as an organizing center by producing a morphogen, Wingless, that acts on cells on both sides of the border. At stage 9 in oogenesis the mirr-fng expression border and a region of localized Notch activation are approximately 10 cell diameters from the ventral pip expression border. Nevertheless, reduction of mirr expression expands the pip domain laterally. If a Mirr-Fng border activates Notch locally to produce a morphogen that represses pip, a reduction of pip expression should be seen upon expansion of the mirr expression domain or ectopic activation of Notch. To examine this, mirr was expressed ectopically in anterior follicle cells. pip repression occurs 5-7 cell diameters beyond the mirr expression domain, showing that the effect of Mirr on pip is non-cell autonomous and supporting the idea that a Mirr-Fng border generates a pip-repressing agent. To further test the effect of ectopic Mirr expression, Mirr was expressed in the posterior follicle and the effect on pip and rho, which is normally expressed as two stripes on the dorsal region at stage 10, was tested. Such ectopic mirr expression induces a ring of rho expression and represses pip at a distance. Expression of constitutively active Notch in the posterior follicle cells also represses pip expression at a distance. These results suggest that Mirr and Notch induce secretion of a diffusible molecule that represses pip. Although it is not known what the Notch-dependent diffusible molecule is at stage 9, it was found that dpp is expressed in follicle cells in the mid-germarium near a stripe of cells showing localized Notch activity in a Notch-dependent manner. Furthermore, in follicle cell clones of MAD or MEDEA (downstream effectors of the Dpp pathway), encapsulation defects of 16-cell cysts are seen. This phenotype is similar to Notch- and mirr-mutant phenotypes in the germarium, suggesting that Dpp may be a morphogen induced by Notch activity in the germarium (Jordan, 2000).

Local activation of Notch in a number of developmental systems is achieved by spatially restricted expression of a homeodomain protein that either represses or induces fng expression, generating a border of fng expressing and non-expressing cells. It is less clear how the initial asymmetric expression of the homeobox protein is generated. Because the dorsal anterior expression of mirr is characteristic of a number of genes regulated by the Egfr pathway, mirr expression was analyzed in mutants that lack Gurken, one of the ligands for Egfr. In these egg chambers, the dorsal anterior pattern of mirr expression is reduced or lost, showing that activation of the Egfr pathway is necessary for mirr expression. However, the patterns of oogenesis in the germaria at stage 6 and in the centripetally migrating cells are unaltered, indicating that either another Egfr ligand or another pathway regulates mirr expression at these stages (Jordan, 2000).

Results from several developmental systems have led to the idea that the trio of a homeobox gene, FNG and Notch are fundamental to organogenesis. It is suggested that Mirr, Fng and Notch are part of a conserved mechanism for dividing epithelial cell layers into domains; it is thought that such a mechanism is not restricted to organogenesis. Furthermore, the data suggest that Mirr integrates the Egfr and Notch pathways in oogenesis: mirr transcription is induced by the Egfr pathway, and Mirr in turn spatially regulates fng expression leading to a Notch activation border. Finally, it is proposed that the link between Egfr pathway signaling in the dorsal follicle cells and the differentiation of the ventral follicle cells suggested by genetic studies is mediated by Mirr. The Egfr pathway induces mirr expression, which leads to creation of a Notch-Fng border in lateral follicle cells from which molecules are secreted that repress pipe expression. Pipe regulates the activity of a protease cascade that activates Toll and ultimately determines the dorsal-ventral pattern of the Drosophila embryo. These data show that expression of pip in the ventral follicle cells is not a direct consequence of a graded germline signal by Gurken, but depends on Mirr-dependent long-range signals from dorsal follicle cells. Mirr therefore connects the well-studied events in early and late Drosophila dorsal-ventral axis formation (Jordan, 2000).

At stage 10 of oogenesis, mirror is expressed in anterior-dorsal follicle cells, and this is dependent upon the Gurken signal from the oocyte. The fringe gene is expressed in a complementary pattern in posterior-ventral follicle cells at the same stage. Ectopic expression of mirror represses fringe expression, thus linking the epidermal growth factor receptor (Egfr) signaling pathway to the Fringe signaling pathway via Mirror. The Egfr pathway also triggers the cascade that leads to dorsal-ventral axis determination in the embryo. twist was used as an embryonic marker for ventral cells. Ectopic expression of mirror in the follicle cells during oogenesis ultimately represses twist expression in the embryo, and leads to phenotypes similar to those that occur due to the ectopic expression of the activated form of Egfr. Thus, mirror also controls the Toll signaling pathway, leading to Dorsal nuclear transport. In summary, the Mirror homeodomain protein provides a link that coordinates the Gurken/Egfr signaling pathway (initiated in the oocyte) with the Fringe/Notch/Delta pathway (in follicle cells). This coordination is required for epithelial morphogenesis, and for producing the signal in ventral follicle cells that determines the dorsal/ventral axis of the embryo (Zhao, 2000).

fringe (fng), which encodes a glycosyltransferase-like secreted protein, is involved in different developmental processes, such as the development of the wing and the eye and oogenesis. Its expression at stage 10 of oogenesis is restricted to the ventral and posterior follicle cells, and it is not observed in those cells that express mirr. So the expression patterns of mirr and fng are complementary, and both these patterns are defined by the position of Egfr activation in response to the Grk signal (Zhao, 2000).

To test whether the complementary expression of mirr and fng depend upon Egfr signaling at stage 10, their expression patterns were examined in flies expressing activated or dominant negative forms of Egfr. When the activated form of Egfr (DERAF or lambdatop) is expressed in the anterior follicle cells, all those cells now express mirr, but not fng. When the dominant negative form of Egfr (DER DN) is expressed in those follicle cells surrounding the oocyte, but not the centripetal follicle cells, the anterior-dorsal follicle cells no longer express mirr. The expression domain of fng expands to include the anterior-dorsal cells in these mutants. Thus, the expression of both mirr and fng are either positively or negatively regulated by the activation of Egfr, and a complementary expression pattern is maintained in all these experiments. Experiments show that Grk/Egfr signaling is required to activate the expression of mirr in anterior-dorsal and centripetal follicle cells, which in turn represses the expression of fng in those cells. As a result, fng is only expressed in the posterior and ventral follicle cells, where it is required for the normal morphogenesis of the follicle cell layer (Zhao, 2000).

fng expression is restricted to specific regions of wing discs, eye discs and follicle cells of the egg chamber. The repression of fng expression in anterior-dorsal follicle cells in wild-type egg chambers by mirr possibly uses a similar mechanism to that observed in eye development, where it is required for the modulation of the dorsal-ventral boundary established by Notch activation. As a secreted protein, Fng modulates the binding of Notch to its ligands at the dorsal-ventral boundary. Ectopic expression of fng induces new dorsal-ventral boundaries in the wing disc and can reverse the planar polarity of photoreceptor clusters in the eye disc. Interfering with fng expression using antisense RNA experiments and mitotic clones, causes abnormalities in epithelial development in the egg chamber and defects in the positioning of the chorionic appendages. Vertebrate Fng homologs are similarly involved in mediating the signals between dorsal and ventral cells during limb development. These findings suggest that the boundary between fng-expressing and non-expressing cells is important in pattern formation, and the restricted expression pattern is regulated by mirr, at least in eye development and oogenesis in Drosophila. In this way, mirr is controlling epithelial morphogenesis via fringe and possibly other targets. As yet it is not known whether mirr directly represses fng transcription or whether there are other genes in the pathway between mirr and fng (Zhao, 2000).

fng affects eggshell patterning. The Gal4/UAS system was used to misexpress mirr, and this results in abnormalities of the chorion. When mirr is ectopically expressed at low levels in follicle cells surrounding the oocyte, there are enlargements of the dorsal appendages in the eggs, some of which (2%) become multiple pseudo-dorsal appendages. During oogenesis, when the ectopic expression of mirr is driven by the Gal4 line C710, 95% of eggs laid by these females have no chorion, suggesting that ectopic expression of mirr can repress the expression of the genes in the ventral cells needed for secretion of the chorion over the ventral regions of the egg. Thus mirror is needed for the correct dorso-ventral patterning of the eggshell which is secreted by the follicle cells. How much of this is mediated via fng, and how much through other target genes and pathways is not known (Zhao, 2000).

One of the dorsal group of maternal genes, pipe, is negatively regulated by mirr since pipe expression is expanded in mirr minus clones. Since pipe is required for the activation of twi via Dorsal, it is likely that mirr affects twi expression by repressing the expression of pipe in dorsal follicle cells. The downstream targets of mirr, in addition to pipe, need to be further identified to understand how mirr executes these developmental decisions in response to Grk/Egfr signaling. It is possible that mirr is required for the repression of pipe and windbeutel (wind) along with fng in the anterior-dorsal follicle cells. The ventrally-localised activities of these genes then cooperatively generate an extracellular ventral signal. There is evidence that CF2, which is expressed in the ventral and posterior cells in a pattern similar to fringe, regulates pipe and wind, and is responsible for the ventral signal produced in follicle cells that determines the embryonic axis. One might speculate that mirr could repress CF2 in anterior/dorsal follicle cells. However, it is observed that CF2 transcripts are expressed in the mirr expressing cells, and the ventrally-localized protein distribution of CF2 is translationally regulated. Since mirr encodes a transcription factor it cannot be directly responsible for the lack of CF2 protein in anterior/dorsal follicle cells, but could indirectly cause a repression of its translation in the anterior-dorsal cells via another gene. Alternatively CF2 protein could be upstream of mirror expression and repress transcription in ventral cells (Zhao, 2000).

When the mirr mutant phenotype was examined, some unusual results were found, namely that one or two mirr alleles affect only the eggshell, but not the embryonic cuticle, when heterozygous or heteroallelic. All mirr mutant alleles are homozygous lethal, therefore homozygous mutant adults cannot be obtained for analysis. One of the mirr alleles (mirrP1), when heterozygous, balanced with TM3 (mirrP1/TM3), lays either eggs with no dorsal appendages or with a pair of very tiny dorsal appendages, thus showing that some mirr alleles can have either dominant effects on the egg-shell or they interact with other genes in this genetic background. Since this phenotype is not observed in mirror/+, the latter is more likely. Other stocks carrying this TM3 balancer do not show this phenotype under similar conditions, and TM3/TM6 females also lay eggs with normal chorions. Regardless of how this is controlled, the heterozygous mirrP1/TM3 females show reduced expression of mirr and expanded expression of fng in some egg chambers at stage 10 of oogenesis, and lay eggs showing the normal expression pattern of twi. The resulting embryos have normal denticle patterns, and 99% hatch. Thus the mirr P1 allele in a TM3 background affects epithelial morphogenesis, but not embryogenesis. To test further the effects of mirr mutants on eggshell and embryonic development, different genetic combinations of mirr mutants were investigated. Some heteroallelic combinations are lethal, but others generate some adult females. It is suggested that the effect of mirr on the eggshell and embryo can be separated and this either involves mirr functioning separately in two different genetic pathways, and/or different sensitivities to the level of mirr expression for these two functions. The possibility that there are background mutations in the mirr stocks causing the eggshell phenotype cannot be ruled out. However, the possibility that mirr has more than one function is supported by the recent finding that mirr regulates equator formation in eye development by two mechanisms; creating a boundary of fng expression, and reducing the mixing of dorsal and ventral cells at the equator (Zhao, 2000).

In summary, mirr functions as a transcription factor linking the Grk/Egfr signaling in oogenesis to the formation of the dorsal-ventral axis of the egg chamber and eggshell, by modulating the Fringe/Notch/Delta pathway, and by affecting the establishment of the signal from ventral cells to set up the embryonic dorsal-ventral axis. mirr expression in anterior-dorsal follicle cells is activated by Grk/Egfr signaling at stage 9-10 of oogenesis. It then represses the expression in dorsal follicle cells of those genes that are required only in ventral follicle cells. The activity of these genes in the ventral follicle cells are required either for the formation of the eggshell and its dorsal-ventral pattern, or for the initiation of dorsal-ventral axis formation in embryogenesis (Zhao, 2000).

Protein Interactions

The Notch signaling pathway plays an important role during the development of the wing primordium, especially of the wing blade and margin. In these processes, the activity of Notch is controlled by the activity of the dorsal specific nuclear protein Apterous, which regulates the expression of the Notch ligand, Serrate, and the Fringe signaling molecule. The other Notch ligand, Delta, also plays a role in the development and patterning of the wing. It has been proposed that Fringe modulates the ability of Serrate and Delta to signal through Notch and thereby restricts Notch signaling to the dorsoventral boundary of the developing wing blade. The results are reported of experiments aimed at establishing the relationships between Fringe, Serrate and Delta during wing development (Klein, 1998).

A feedback mechanism exists in which Fringe functions to inhibit Serrate by targeting Notch. In contrast to Delta, the effects of ectopic expression of Ser on wild-type discs are restricted to ventral cells. This has led to the suggestion that there is an inhibitor of Serrate activity in dorsal cells and that this inhibitor is under the control of ap. Consistent with this proposal, ectopic expression of Ser in ap mutants is found to be able to induce the expression of downstream targets of Notch in 'dorsal' cells. A variety of arguments have led to the proposal that the dorsal inhibitor of Serrate function is encoded by the fng gene. For example, ectopic expression of Ser with ptcGAL4 results in the activation of Notch targets in two parallel stripes in ventral cells of the developing wing blade, and this can be observed as early as the beginning of the third instar. When Ser is coexpressed with fng, the anterior stripe, but not the posterior one, is lost completely in late third instar discs. Correspondingly the ectopically induced margin structures are reduced to a posterior stripe with characteristics of the posterior compartment. While Fringe suppresses the function of Serrate cell autonomously, it enhances its signaling ability in a nonautonomous manner. Fringe is thought to dampen Serrate signaling by affecting its interaction with Notch, but no evidence has been presented to support this suggestion. Increasing the concentration of Notch appears to be able to titrate the effects of fng. Furthermore, the effects of ectopic expression of fng are partially suppressed by the expression of Notch with fng and are exaggerated by expressing dominant negative Notch molecules with fng. Altogether, these results strongly suggest that a target of Fringe activity is the Notch molecule itself (Klein, 1998).

Fringe functions to inhibit Serrate signaling via Notch. The activity of Fringe can inhibit Serrate signaling by enhancing the intrinsic dominant negative activity of Serrate over Notch. Expression of Ser throughout the late wing disc leads to a strong broadening of the wing veins and a moderate increase in the number of bristles in the notum. Both of these neurogenic phenotypes can be suppressed by coexpressing Notch with Ser, indicating that they are due to a dominant negative effect of Serrate. Ectopic expression of fng alone in the same pattern results in nicked wings with normal veins and a reduction of bristles in the notum, which is associated with the loss of sensory organ precursors. Coexpression of fng with Ser suppresses the extra vein phenotype caused by misexpression of Ser and, therefore, supports the notion that Fringe reduces the ability of Serrate to bind Notch. Fringe is shown to impinge on Notch signaling by the observation that the action of Fringe requires the activity of Su(H). Fringe is not able to rescue the defects caused by Su(H) mutants (Klein, 1998).

Delta- and Serrate-mediated signaling can promote the socket cell fate in developing bristle organs. Previous studies have defined roles for Delta in the specification of the sensory organ precursor (SOP), its progeny (pIIa and pIIb), and the daughters of pIIb -- the neuron and thecogen (glia). This paper shows that ectopic expression of Delta or Serrate in neurons within developing bristle organs is capable of non-autonomously inducing the transformation of daughters of pIIa, the pre-trichogen (shaft) cells into tormogen (socket) cells. The frequencies at which Delta can induce these transformations are dependent on the level of ectopic Delta expression and the levels of endogenous Notch signaling pathway components. Delta expression in the cell receiving the Delta signal also has effects on the responsivess of that cell to Delta and Serrate signals. The pre-trichogen cell becomes more responsive to Delta- or Serrate-mediated transformation when the level of endogenous Delta is reduced, and less responsive when the dosage of endogenous Delta is increased, supporting the hypothesis that Delta interferes autonomously with the ability of a cell to receive either Delta or Serrate signal. Thus cell autonomous interactions between Delta and Notch modulate neurogenic signalling in Drosophila. A dominant-negative form of Notch, ECN, is capable of autonomously interfering with the ability of a cell to generate the Delta signal. When the region of Notch that mediates trans-interactions between Delta and the Notch extracellular domain is removed from ECN, the ability of Delta to signal is restored. These findings imply that cell-autonomous interactions between Delta and Notch can affect the ability of a cell to generate and to transduce a Delta-mediated signal (Jacobsen, 1998).

Evidence is presented that the Fringe protein can interfere with Delta- and Serrate-mediated signaling within developing bristle organs, in contrast to previous reports of the converse effects of Fringe on Delta signaling in the developing wing. The fringe gene encodes a pioneer protein, predicted to be secreted, that plays a role in the development of the wing disc by modulating interactions between dorsal and ventral cells that establish the dorsal/ventral boundary and affect specification of the wing margin. One domain of the Fringe product contains motifs similar to the catalytic domain of glycosyltransferases. The primary effect of Fringe on Notch signaling appears to be inhibition of the ability of the Serrate ligand to activate Notch, an effect observed during neuroblast specification within the neuroectoderm and in the developing wing disc. Fringe may act by binding the amino-terminus of Serrate. In the case of Delta and Serrate ectopic expression, coexpression of Fringe with either ligand can interfere with the ability of that ligand to induce trichogen transformation. In this context, Fringe impedes Serrate- and Delta-mediated signaling. The inhibition of Delta and Serrate signaling observed in the developing bristle organ may be context-dependent, i.e., factors present at the wing margin that prevent Fringe from interfering with Delta-mediated signaling may be absent in developing macrochaetae. If Fringe is secreted by the neuron, it could act in a cell non-autonomous fashion to impede the ability of Notch on the pre-trichogen cell to receive ligand-mediated signals. Alternatively, Fringe could function in the neuron in a cell autonomous manner to impede signal generation by interacting with ligand. In either case, Fringe cannot be interfering with Notch-mediated signal reception in a cell autonomous manner in this context. The exact mechanism by which Fringe can operate in the context of bristle development must be the object of future experiments (Jacobsen, 1998).

The activation of Notch is regulated both by the temporal and spatial distribution of the ligands and by the expression of proteins such as Fringe (Fng) that are able to modulate ligand-receptor interactions. This was first evident in the developing wing, where Notch activity results in the expression of genes such as wingless and cut in a narrow 2- to 4-cell-wide domain at the dorsoventral boundary. In this process, Fng influences the effectiveness of the interactions between Notch and its ligands by preventing Ser-mediated activation and potentiating Notch activation by Dl. The localized activation of Notch initially occurs because Apterous promotes the expression of both Ser and Fng in dorsal cells, while the inhibitory effect of Fng on Ser/Notch restricts Ser signaling primarily to ventral cells. At the same time, the effect of Fng on Dl has the consequence that ventral Dl-expressing cells signal primarily to dorsal cells. A similar process occurs in the eye, where again the compartment-specific expression of fng allows localized activation of Notch at the eye dorsoventral boundary (de Celis, 2000 and references therein).

Conventionally Dl and Ser are considered activating ligands of Notch and, in many instances, their elimination has non-autonomous effects on development that are characteristic of a membrane-associated ligand. However, in the Drosophila wing and eye, both Notch ligands have also been shown to have cell-autonomous inhibitory effects on the activity of the receptor. Thus, the elimination of both ligands in clones of cells in the wing can result in Notch activation within the clone, detected as ectopic ct expression, indicating that a normal function of Dl and Ser is to prevent Notch activation within the cells in which they are expressed. In addition, ectopic expression of Dl or Ser in groups of cells causes Notch activation only in the adjacent cells. Consistent with the suggestion that the inhibitory activity of the ligands relies on interactions occurring between molecules within the same cell, the negative effects of ectopically expressed Ser can be alleviated by co-expression of full-length Notch. The negative effect of the ligands could be instrumental in determining the polarity of Notch signaling: cells expressing higher levels of ligand would have reduced Notch responsiveness compared to adjacent cells with lower ligand levels and hence Notch would be more readily activated in the cells with relatively less ligand. The concept that the relative levels of Notch and Dl are important for signaling is also evident from the phenotypes caused by varying the dosage of these genes. Finally, Dl and Notch have been seen to co-localize on the surface of cultured cells, suggesting that they could interact in the plasma membrane. However, the antagonistic interactions could be occurring anywhere within the cell and the functional domain of Notch involved in this process has not been characterised (de Celis, 2000 and references therein).

The extracellular domain of Notch contains an array of 36 EGF repeats, two of which, repeats 11 and 12, are necessary for direct interactions between Notch with Delta and Serrate. An investigation has been carried out of the function of a region of the Notch extracellular domain where several missense mutations, called Abruptex, are localized. These Notch alleles are characterized by complex complementation patterns and phenotypes that are the opposite of those observed with a loss of Notch function. In Abruptex mutant wing discs, only the negative effects of the ligands and Fringe are affected, resulting in the failure to restrict the expression of cut and wingless to the dorsoventral boundary. It is suggested that Abruptex alleles identify a domain in the Notch protein that mediates the interactions between Notch, its ligands and Fringe that result in suppression of Notch activity (de Celis, 2000).

In wild-type discs, the response of Notch to Dl and Ser is affected by the presence of Fng, which is expressed in dorsal cells. Since the domain of Fng expression corresponds to the region where Dl loses its capacity to antagonize Notch in NAx mutants, an analysis was carried out to see whether NAx mutations have an altered sensitivity to Fng by comparing the consequences of ectopic fng expression in wild-type and NAx discs. As with the ectopic ligand expression, clones of cells expressing fng that cross the dorsoventral boundary inhibit expression of ct except at the clone borders. When the Fng-expressing clones lie in the ventral compartment, ct is induced in the cells at the boundary of the clone, with the result that ct is detected in neighboring fng+ and fng- cells. The ability of Fng to prevent ct expression is reduced when Fng-expressing clones are induced in NAx mutant backgrounds. In a weak NAx allelic combination, the expression of ct is still highest at clone boudaries, but significant expression is detected within the clone. In the more severe mutants, the Fng-expressing cells have little or no inhibitory effect on ct, and there are high levels of Ct throughout the clone. Similar effects are seen when fng misexpression is driven by Gal4-sal. Normally this causes an inhibition of ct expression at the dorsoventral boundary; in NAx mutant discs, however, Ct is detected throughout most of the domain of ectopic Fng-expression. If the NAx domain is significant in the interactions between Notch and Fng, the NAx mutations should modify phenotypes caused by alterations in fng expression. In the allele fngD4, fng is expressed throughout the wing pouch, causing severe scalloping of the wing margin. This correlates with the loss of ct and wg expression at the dorsoventral boundary and the expansion of vvl/drifter expression. In NAx heterozygous flies, the phenotype of fngD4 is reduced both at the level of wing scalloping and the expression of dorsoventral boundary markers. In hemizygous NAx males, both the expression of ct and vvl and the adult phenotype are similar to the expression and phenotype typical of NAx. Taken together, these results suggest that NAx proteins are also deficient in some activity related to the capability of Fng to restrict Notch activity (de Celis, 2000).

The amino-acid sequence of Fng indicates that it could be a glycosyltransferase. Since NAx mutations affect the extracellular domain of Notch, the fact that the NAx alleles have altered behavior with respect to Fng suggests that the mutated domain could be a target for Fng-mediated glycosylation. If the NAx mutations perturb glycosylation of Notch by Fng, this might explain why they only affect the activity of Notch in the imaginal discs and not in the early embryo, since fng is only required at later stages of development. NAx alleles also affect several processes, such as sensory organ development and vein cell differentiation, that do not seem to require fng activity. This indicates that the NAx domain also affects negative interactions between Notch with Dl and Ser independent of fng function (de Celis, 2000).

The results shown here indicate that the NAx domain of Notch is only necessary to mediate the functions of Fng and the ligands that result in the suppression of Notch activity. A comparison between the effects on Fng, Dl and Ser indicates that the interactions between these molecules and Notch are affected to different extents by NAx mutations. For example, although the dominant negative effects of Dl and Fng are dramatically reduced in NAx alleles, these mutations do not appear to compromise the potentiating effect of Fng on Dl activation, since there is still a strong bias towards Dl activity in the dorsal domain where Fng is present. Similarly high levels of ectopic Ser can efficiently suppress Notch activity in NAx backgrounds, even though the phenotype of NAx mutant discs indicates that NAx mutations perturb the dominant negative effects of Ser when it is expressed at normal levels. Each NAx allele has a characteristic strength that is reflected in its phenotype and in the extent of ectopic ct activation. Furthermore, heteroallelic combinations between NAx alleles often result in synergistic phenotypes, a phenomenon called negative complementation. This suggests that the correct conformation of the NAx domain in Notch multimers is critical for the efficiency of the interactions between Notch, its ligands and Fng that determine suppression of Notch activity (de Celis, 2000).

Fringe (Fng), an extracellular protein, determines the boundary of two different cell populations during the development of diverse structures, not only in Drosophila, but also in vertebrates. The identification of the proteins that physically interact with Fng is important to understand the molecular mechanisms of Fng function. Since most known Fng-mediated developmental processes require Notch signaling, Notch is a strong candidate for Fng-interacting proteins. To test whether Fng binds Notch, a series of biochemical experiments were performed focusing on the EGF-like repeats 22 to 36 and Lin-Notch repeats (LNRs) of Notch, to which most Ax mutations and antineurogenic mutations map. Expressed either separately or together in transgenic Drosophila were a chimaeric Notch, named NSG, which includes the region from EGF repeat 22 to the transmembrane domain of Notch fused to an S tag and green fluorescent protein (GFP), and Fng, tagged with the Myc epitope (FngM). The extracts of the transgenic larvae were immunoprecipitated with anti-GFP or anti-Myc antibody and then purified by S-protein affinity column chromatography. This ensured that all of the positive bands in Western blots with anti-GFP antibody contained an S tag as well as a GFP tag. Because Notch is processed into two polypeptides linked by disulphide bonds in the trans-Golgi, the NSG protein could be expected to yield two polypeptides by SDS-polyacrylamide gel electrophoresis. The first product is an amino-terminal peptide of relative molecular mass 89,000 (Mr 89K), which is untagged. The second polypeptide is a carboxy-terminal peptide (NSG-C) of Mr 43K that contains an S tag and GFP. The eluents of the S-protein affinity column from extracts of animals expressing 2xNSG or 2xNSG plus 2xFngM all yield a positive 43K band as expected. When the immunoprecipitations were carried out with anti-Myc antibody, which specifically binds to FngM, the extract from the animal co-expressing NSG and FngM yields the co-precipitated 43K NSG-C peptide (Ju, 2000).

To map the domains of Notch that interact with Fng, various forms of Notch derivatives were expressed with an S tag and GFP in stably transfected S2 cell lines with or without Fng-GST (Fng fused to glutathione S-transferase). AxM1 is a missense mutation of Cys 999 to tyrosine. The extracts from these cell lines were loaded onto glutathione-Sepharose column. If NSG, or its derivatives, form a complex with Fng, it should be retained on the column with Fng-GST. NSG, LNRSG, N22-36SG and AxSG were retained with the Fng-GST protein on gutathione-Sepharose, but N1-21SG and Ax22-36SG were not. These data suggest that Fng specifically binds to Notch through the LNRs as well as the EGF22-36 and that AxM1 mutation abolishes the Fng interaction with EGF22-36 of Notch but does not affect the Fng interaction with the LNRs (Ju, 2000).

The subcellular colocalization of Fng-GST with the Notch derivatives also supports the specific Fng-Notch interaction. Examined were the subcellular localization of Fng-GST and the Notch derivatives, which were co-expressed in S2 cells. The subcellular distribution of Fng mostly overlaps with that of the Notch derivatives, which interacted with Fng in biochemical experiments (NSG, N22-36SG and LNRSG). In contrast, N1-21SG and Ax22-36SG are mainly not colocalized with Fng and do not interact. These data indicate that Fng may be present as a complex with Notch even before its secretion (Ju, 2000).

Consistent with these observations, when Fng-GST and the Notch derivatives are expressed separately and mixed in vitro, none of the Notch derivatives co-purified with Fng-GST on a glutathione-Sepharose column. In addition, when FngM and NSG are expressed separately and mixed in vitro and then analysed by glycerol-gradient centrifugation, FngM is mainly found in fractions 16-19, whereas NSG is found in fractions 2-7. This implies that Fng and Notch do not form a complex if they are not expressed in the same cells simultaneously. These data are consistent with the finding that Fng is not detected on the surface of cells expressing Notch when Fng protein is added exogenously. Therefore, Fng-Notch complex formation may occur before secretion, probably within the secretory pathway. This explains why the secretary Fng protein acts cell-autonomously in Notch-expressing cells. It is unlikely to modify Notch found on the surface of neighboring cells or cells that may be reached by secreted Fng. Fng has been proposed as a glycosyltransferase on the basis of sequence similarity. Fng may bind Notch and may modify the glycosylation of Notch during its secretion in the endoplasmic reticulum (ER) or Golgi. A comparison of the glycosylation status between the Fng-Notch complex and Notch alone will be of considerable interest (Ju, 2000).

Genetic evidence from Ax mutants suggests that the Notch EGF 22-36 are involved in the Notch-Fng interaction in vivo. In wild-type mid-third Drosophila wing imaginal discs, the expression of Serrate (Ser), the vestigial (vg) boundary enhancer-lacZ (vgBE) and wingless (wg) are upregulated by Fng and Notch near or at the dorsoventral (D-V) boundary but not in dorsal interior cells. In contrast, in AxM1 mutants Ser and vgBE expression are expanded into all dorsal cells, including interior cells. This expansion may be due to disturbance of the Fng-Notch interaction through the EGF22-36 of Notch by the Ax mutation (Ju, 2000).

To test this, Fng was ectopically expressed in AxM1 mutant wing discs using UAS-fng and a patched (ptc)-Gal4 driver. In wild-type imaginal discs, ectopic expression of Fng in the ptc pattern induces vgBE, Wg and Delta (Dl) only in ventral cells in a single stripe along the A/P boundary. In AxM1 mutants, however, Ser, Wg, vgBE and Dl expression are strongly induced by ectopic Fng even in dorsal cells. The level of Ser expression in the dorsal cells expressing ectopic Fng is much higher than that in other dorsal cells. The effects of ectopic Fng expression on Ser, Wg, vgBE, and Dl expression are also similar in Ax28 homozygous and Ax16172/Ax28 heterozygous mutant wing discs, although the extent of the effect varies. Ax mutations do not affect the response of dorsal or ventral cells to Ser or Dl as significantly as their response to Fng. This implies that the induction of Ser, Dl and vgBE by Fng in Ax mutant dorsal cells may not be mediated by Ser (Ju, 2000).

To confirm the effects of Fng on Ser expression in AxM1 mutants, homozygous fng null mitotic clones were generated in the AxM1 mutant background. Notably, homozygous fng- dorsal cells of AxM1 mutant wing discs do not express Ser. Ser expression in the interior dorsal cells of AxM1 mutant therefore requires Fng function, and the AxM1 mutation disrupts the normal downstream regulatory effects of the Fng-Notch interaction (Ju, 2000).

The LNRs of Notch function as a repressor domain in Notch signaling. Since Ax mutations prevent EGF22-36 of Notch from binding to Fng but do not affect Fng binding to the LNRs of Notch, one of the possible mechanisms of Fng-dependent Notch target gene activation in Ax mutants is that the Ax mutation allows Fng to bind to LNRs alone rather than to EGF 22-36. This may dampen or silence the repressor function of the LNRs, and might, in turn, make Notch more sensitive to Delta. Alternatively, Ax mutations may mimic the conformation of Notch bound with a ligand and Fng may help this form of Notch to be converted to a fully activated form by antagonizing the repressor function of the LNRs (Ju, 2000).

Fringe has been proposed to execute its boundary determining function by inhibiting the Notch response to Ser and potentiating the Notch response to Dl. Because Fng does not bind Ser or Dl, modulation of Notch signaling by Fng is directly mediated by the complex formation of Fng and Notch during their secretory transits. Notch bound to Fng may have preferential affinity or sensitivity to Delta, whereas free Notch may have a higher affinity or sensitivity to Ser. Upon Dl binding to the Fng-Notch complex, Fng may also antagonize the repressor function of the Lin-Notch repeats and facilitate the activation of Notch signaling (Ju, 2000).

Signaling via the Notch receptor is a key regulator of many developmental processes. The differential responsiveness of Notch-expressing cells to the ligands Delta and Serrate is controlled by Fringe, itself essential for normal patterning in Drosophila and vertebrates. The mechanism of Fringe action, however, is not known. The protein has an amino-terminal hydrophobic stretch resembling a cleaved signal peptide, which has led to the widespread assumption that it is a secreted signaling molecule. It also has distant homology to bacterial glycosyltransferases, although it is not clear if this reflects a shared enzymatic activity, or merely a related structure. A functional epitope-tagged form of Drosophila Fringe is localized in the Golgi apparatus. When the putative signal peptide is replaced by a confirmed one, Fringe no longer accumulates in the Golgi, but is instead efficiently secreted. This change in localization dramatically reduces its biological activity, implying that the wild-type protein normally acts inside the cell. Fringe specifically binds the nucleoside diphosphate UDP, a feature of many glycosyltransferases. Furthermore, specific mutation of a DxD motif (in the single-letter amino acid code where x is any amino acid), a hallmark of most glycosyltransferases that use nucleoside diphosphate sugars, does not affect the Golgi localisation of the protein but completely eliminates in vivo activity. These results indicate that Fringe does not exert its effects outside of the cell, but rather acts in the Golgi apparatus, apparently as a glycosyltransferase. They suggest that alteration in receptor glycosylation can regulate the relative efficiency of different ligands (Munro, 2000).

The suggestion that Fringe is secreted came from the amino-terminal hydrophobic stretch, which was predicted to be a leader peptide. Golgi glycosyltransferases are, almost without exception, Type II membrane proteins with a single transmembrane domain within the first 5-50 residues of the amino terminus. Because the transmembrane domains of Golgi proteins are usually shorter than those of plasma membrane proteins, their amino-terminal regions can appear similar to signal peptides. Indeed, the best current signal-peptide prediction programs incorrectly predict known mammalian glycosyltransferases to have cleaved amino termini. The fact that, when attached to a confirmed signal peptide, Fringe is secreted and has a smaller apparent size, strongly suggests that the amino-terminal hydrophobic stretch is not normally cleaved, but rather is a transmembrane domain typical of Golgi glycosyltransferases. A previous examination of Fringe expressed in transgenic flies concluded that the protein was being secreted on the basis that it could be detected outside of the expression domain of the GAL4 expression driver being used. This 'extracellular' Fringe appeared in a punctate pattern strikingly similar to the Golgi localization described here, raising the possibility that it was in fact weak ectopic expression of the protein, caused by leakiness of the Gal4 driver. Furthermore, even if a small amount of Fringe is secreted, forcing its secretion dramatically reduces its activity, indicating that it is the intracellular form, rather than the secreted form that regulates Notch signaling. An intracellular site of action for Fringe is also easier to reconcile with the protein's observed cell autonomy in regulating Notch (Munro, 2000 and references therein).

Specific modification by Fringe of one or more of the O-linked structures on the Notch EGF repeats could alter the binding affinity of the ligands Delta and Serrate. Specifically, genetic evidence implies that Serrate binding and/or activation of Notch would be inhibited by Fringe-mediated glycosylation, whereas Delta binding and/or activation would be enhanced. Delta and Serrate both bind EGF repeats 11 and 12 of Notch, the latter of which contains a well conserved site for O-linked glucosylation. However, the situation may be more complex, as Abruptex mutations in Notch that map to EGF repeats 24, 25, 27 or 29 have interestingly been found to be insensitive to modulation by Fringe. It may be that recognition or modification of many of the Notch repeats is critical for affecting ligand binding (Munro, 2000 and references therein).

This study establishes the biochemical mechanism of Fringe action. Fringe is a secreted protein that both positively and negatively modulates the ability of Notch ligands to activate Notch signaling. Drosophila and mammalian Fringe proteins possess a fucose-specific beta1,3 N-acetylglucosaminyltransferase activity that initiates elongation of O-linked fucose residues attached to epidermal growth factor-like sequence repeats of Notch. Biological evidence that Fringe-dependent elongation of O-linked fucose on Notch modulates Notch signaling has been obtained by using co-culture assays in mammalian cells and by expression of an enzymatically inactive Fringe mutant in Drosophila. The post-translational modification of Notch by Fringe represents a striking example of modulation of a signaling event by differential receptor glycosylation and identifies a mechanism that is likely to be relevant to other signaling pathways (Moloney, 2000).

A site-specific mutation was generated in Drosophila Fng, D236-D237-D238 to D236-E237-E238 [D-Fng(DEE)], to show that the GlcNAc-transferase activity of Fringe is essential for it to modulate Notch signaling in vivo. This aspartic acid patch is highly conserved among galactosyltransferases and several GlcNAc-transferases. Conservative changes within the DDD motif disrupt the catalytic activity of these enzymes without destabilizing their overall structure. Indeed, affinity-purified D-Fng(DEE) is enzymatically inactive (Moloney, 2000).

The biological activity of fngDEE was examined by using an ectopic expression assay. The influence of fng on Notch signaling results in establishment of a stripe of Notch activation along the borders of fng expression. Ectopic expression of wild-type Fringe proteins under the control of patched regulatory sequences eliminates the normal Fringe expression border in the middle of the wing and induces an ectopic Fringe expression border in ventral cells. This results in corresponding changes in the pattern of Notch activation, as judged by Wingless expression in developing wing imaginal discs and by formation of margin bristles in adult wings. Strikingly, ectopic expression of fngDEE fails to cause any discernible effect on Notch activation in the wing, or on any other aspects of Drosophila development. Thus, the GlcNAc-transferase activity of Fringe is essential for it to modulate Notch signaling (Moloney, 2000).

Glycosylation modifies protein activities in various biological processes. This study reports the functions of a novel UDP-sugar transporter (UST74C, an alternative name for Fringe connection (Frc), which is localized to the Golgi apparatus in cellular signalling of Drosophila. Mutants in the frc gene exhibit phenotypes resembling wingless and Notch mutants. Both Fringe-dependent and Fringe-independent Notch pathways are affected, and both glycosylation and proteolytic maturation of Notch are defective in mutant larvae. The results suggest that changes in nucleotide-sugar levels can differently affect Wingless and two distinct aspects of Notch signalling (Goto, 2001).

The precise regulation of growth factor signalling is crucial to the molecular control of development in Drosophila. Post-translational modification of signalling molecules is one of the mechanisms that modulate developmental signalling specificity. A new gene, fringe connection (frc), is described that encodes a nucleotide-sugar transporter that transfers UDP-glucuronic acid, UDP-N-acetylglucosamine and possibly UDP-xylose from the cytoplasm into the lumen of the endoplasmic reticulum/Golgi. Embryos with the frc mutation display defects in Wingless, Hedgehog and fibroblast growth factor signalling. Clonal analysis shows that fringe-dependent Notch signalling is disrupted in frc mutant tissue (Selva, 2001).

Notch modulation by O-fucosyltransferase 1 is essential for Notch interaction with its ligands and for Fringe function

Notch and its ligands are modified by a protein O-fucosyltransferase (O-fut1, also known as Neurotic or Ofut1) that attaches fucose to a serine or threonine within EGF domains. By using RNAi to decrease O-fut1 expression in Drosophila, it has been demonstrated that O-linked fucose is positively required for Notch signaling, including both Fringe-dependent and Fringe-independent processes. The requirement for O-fut1 is cell autonomous, in the signal-receiving cell, and upstream of Notch activation. Therefore, O-fut1 activity is required for the cell's ability to receive ligand signals, and would thus be consistent with the hypothesis that the key substrate of O-fut1 is Notch. The transcription of O-fut1 is developmentally regulated, and surprisingly, overexpression of O-fut1 inhibits Notch signaling. Together, these results indicate that O-fut1 is a core component of the Notch pathway, one that is required for the activation of Notch by its ligands, and whose regulation may contribute to the pattern of Notch activation during development (Okajima, 2002).

A mutation has been isolated in the gene encoding O-fucosyltransferase, and analysis of the mutant phenotype confirms the RNAi studies and reveals an unprecedented example of an absolute requirement of a protein glycosylation event for a ligand-receptor interaction. A novel maternal neurogenic gene, neurotic, is essential for Notch signalling. neurotic functions in a cell-autonomous manner, and genetic epistasis tests reveal that Neurotic is required for the activity of the full-length but not an activated form of Notch. neurotic has been shown to be required for Fringe activity. fringe encodes a fucose-specific ß1, 3 N-acetylglucosaminyltransferase that modulates Notch receptor activity. Neurotic is essential for the physical interaction of Notch with its ligand Delta, and for the ability of Fringe to modulate this interaction in Drosophila cultured cells. These results suggest that O-fucosylation catalysed by Neurotic is also involved in the Fringe-independent activities of Notch and may provide a novel on-off mechanism that regulates ligand-receptor interactions (Sasamura, 2003).

Since O-fucose on Notch has been shown to act as a molecular scaffold for GlcNAc that is elongated by Fng, one would expect that the phenotypes of O-fucosyltransferase mutant might be the same as those of fng. Unexpectedly, however, the nti and fng mutant phenotypes are quite different. Strikingly, the embryonic neurogenic phenotype that is evident in nti mutant, and is an indication of its essential role in Notch signalling, is not evident in fng mutants. Furthermore, it is thought that Fng does not have a significant role in lateral inhibition, while it is involved in the generation of the cell boundary between cells expressing Fng and cells not expressing Fng. Additionally, an in vitro binding assay revealed that Nti is essential for binding between Notch and Delta. Based on the previous findings and the present results, it is proposed that O-glycosylation of Notch EGF repeats has two distinct roles for binding to Delta. (1) O-fucosylation catalysed by Nti is an absolute requirement for binding between Notch and the ligand, and this binding is sufficient to accomplish lateral inhibition. For this function, no additional glycosylation to O-fucose residue is required. This idea is also supported by the observation that in the tissues and organisms that do not express fng, Delta is competent to activate the Notch receptor. In this respect, it is worth noting that in the C. elegans genome there is a highly conserved nti, while a fng homolog is not found. (2) Addition of GlcNAc to the O-fucose residue by Fng enhances the interactions between Notch and Delta, modulating the receptor-ligand interactions. In fact, the expression of fng shows a high degree of regional specificity, and the boundary of the cells expressing and not expressing Fng often defines the border of distinct tissue structures. Thus, the region-specific expression of fng allows modulation of Notch signalling, resulting in generation of complex structure of organs. As expected from the second function of Nti, its function is essential for Fng-dependent modulation of Notch signalling as well as Fng-independent function. In the wing disc, nti is epistatic to fng, and fng requires nti to induce Wg at the dorsal and ventral compartment boundary. Additionally, in the in vitro binding assay, Fng depends on Nti to enhance the binding between Notch and Delta. These lines of evidence indicate that Nti is involved in Fng-dependent modulation of Notch signalling, which is consistent with an O-glycan structure of the Notch EGF repeats (Sasamura, 2003).

To investigate the molecular basis for the requirement for O-linked fucose on Notch, an assay was carried out of the ability of tagged, soluble forms of the Notch extracellular domain to bind to its ligands, Delta and Serrate. Downregulation of O-fut1 by RNAi in Notch-secreting cells inhibits both Delta-Notch and Serrate-Notch binding, demonstrating a requirement for O-linked fucose for efficient binding of Notch to its ligands. Conversely, over-expression of O-fut1 in cultured cells increases Serrate-Notch binding but inhibits Delta-Notch binding. These effects of O-fut1 are consistent with the consequences of O-fut1 overexpression on Notch signaling in vivo. Intriguingly, they are also the opposite of, and are suppressed by, expression of the glycosyltransferase Fringe, which specifically modifies O-linked fucose. Thus, Notch-ligand interactions are dependent upon both the presence and the type of O-fucose glycans (Okajima, 2003).

The requirement for O-Fut1 in Notch signaling has been demonstrated by RNAi in Drosophila (Okajima, 2002), and by a targeted mutation in the murine Pofut1 gene. One line from a large scale screen for lethal transposable element insertions in Drosophila has an insertion in the 3' end of O-fut1, and is predicted to result in replacement of the seven C-terminal amino acids of O-fut1 with four different amino acids followed by a stop codon (Oh, 2003). To confirm that this insertion creates an O-fut1 mutation, animals in which patches of cells were made homozygous mutant for this allele were examined by mitotic recombination. These animals exhibit classic Notch mutant phenotypes, such as wing notching, thickened wing veins, and loss of sensory bristles on the notum, consistent with the phenotypes generated by RNAi of O-fut1 (Okajima, 2002). In developing wing imaginal discs, the expression of targets of Notch signaling, such as Wingless, is lost in cells mutant for O-fut1. This mutation (referred to hereafter as O-fut1SH) thus provides an independent demonstration of the requirement for O-fut1 for Notch signaling in Drosophila, and indicates that the seven C-terminal amino acids of O-fut1 are essential for function in vivo. The last four amino acids of O-fut1 conform to a consensus signal for retention in the endoplasmic reticulum, and experiments are in progress to determine whether the loss of function in O-fut1SH is due to loss of enzymatic activity or to mislocalization (Okajima, 2003).

The studies presented here indicate that O-fucosylation is required for the physical binding of Notch to its ligands Dl and Ser. These binding studies are consistent with prior genetic studies, which positioned a requirement for O-fucosylation in signal receiving cells, upstream of the cleavages associated with Notch activation (Okajima, 2002). Although the current results do not exclude the possibility that O-fucose glycans could also act at other steps, and indeed some influence of O-fut1 RNAi on secretion of Notch extracellular domain fusion proteins is detected, the requirement for O-fucose for Notch-ligand binding can in principle account for the requirement for O-fut1 in Notch signaling (Okajima, 2003).

Notably, O-fut1 is required for efficient binding of Notch to both Ser and Dl. This is consistent with the severe Notch phenotypes observed in vivo when O-fut1 is impaired by mutation or RNAi. By contrast, elongation of O-fucose by the GlcNAc transferase Fringe exerts opposing influences on the ability of Notch to bind to Ser and Dl. Fringe has clear and reproducible effects on both Dl-Notch and Ser-Notch binding. Importantly, these effects of Fringe on Notch-ligand binding recapitulate its effects on signaling by these two ligands in Drosophila. The ability of both the O-fucose monosaccharide and elongated forms of O-fucose to influence Notch-ligand binding, the influence of O-fucosylation on binding by both ligands, and the consistent correlations between the effects of O-fucosylation on binding in vitro and its effects on signaling in vivo all argue that O-fucose glycans act at the ligand binding step of Notch signaling (Okajima, 2003).

Beyond their importance to understanding regulation of Notch signaling, these observations thus provide a striking example of glycosylation as a mechanism for modulating protein-protein interactions. With the determination that O-fucosylation affects Notch-ligand binding, attention must now be turned to elucidating the mechanistic basis for this effect (Okajima, 2003). O-fut1 and Fringe always act in Notch-expressing cells to influence Notch signaling and Notch-ligand binding: this implicates Notch itself as the relevant substrate. However, the actual sites of glycosylation on Notch that mediate the effects of these glycosyltransferases remain to be identified. It is also not yet clear whether the importance of O-fucosylation reflects a role for lectin-like recognition of Notch by its ligands or other co-factors, or whether instead O-fucose glycans influence Notch-ligand binding indirectly, by altering the conformation or oligomerization of Notch (Okajima, 2003).

By contrast to the positive requirement for O-fut1 demonstrated by RNAi, over-expression of O-fut1 enhances Ser-Notch binding but inhibits Dl-Notch binding. It is intriguing that elevated O-fut1 expression provides a mechanism for differentially modulating the ability of different Notch ligands to interact with the Notch receptor. Previously, Fringe was the only factor known that could discriminate between the ability of Delta to activate Notch and that of Serrate to activate Notch. Indeed, elevated O-fut1 expression might be a mechanism for increasing the sensitivity of cells to the presence or absence of Fringe. In vivo, Fringe only affects a subset of Notch signaling events, and it remains unclear why certain processes are sensitive to Fringe whilst others are insensitive. Although O-fut1 action is the opposite of Fringe, its effects can be blocked by Fringe; therefore, the relative impact of Fringe on Dl-Notch or Ser-Notch interactions is expected to be greater in tissues where O-fut1 is expressed at higher levels. Indeed, even though expression of Fringe alone has no obvious effect on the patterning of notal bristles, it has a strong effect when O-fut1 is also overexpressed. Overexpression of O-fut1 inhibits Dl-Notch signaling, resulting in the formation of excess sensory bristles, but this effect is partially inhibited by co-expression with Fringe (Okajima, 2002). In addition to increasing the sensitivity of Notch signaling events to the presence or absence of Fringe, elevated O-fut1 expression presents a potential mechanism for modulating Notch signaling independently of Fringe. Although the in vivo relevance of Notch-ligand modulation by increased expression of O-fut1 at endogenous levels of expression remains uncertain, it is noted that certain tissues, such as the lymph gland, express much higher levels of O-fut1 than surrounding cells (Okajima, 2002). Intriguingly then, in most Drosophila tissues Dl is the sole or major Notch ligand. However, in the larval lymph gland, a role for Notch signaling in regulating cell fate decisions during hematopoeisis has recently been described, and Ser, rather than Dl, is the ligand that regulates Notch in this tissue. These observations provide some support for the possibility that transcriptional regulation of O-fut1 might provide a mechanism for Notch pathway regulation (Okajima, 2002), and suggest developmental contexts in which this issue may be investigated further (Okajima, 2003).

Mutational alteration of sites of Fringe mediated O-fucosylation of Notch: Influence on Notch signaling

Two glycosyltransferases that transfer sugars to EGF domains, OFUT1 and Fringe, regulate Notch signaling. However, sites of O-fucosylation on Notch that influence Notch activation have not been previously identified. Moreover, the influences of OFUT1 and Fringe on Notch activation can be positive or negative, depending on their levels of expression and on whether Delta or Serrate is signaling to Notch. This study describes the consequences of eliminating individual, highly conserved sites of O-fucose attachment to Notch. The results indicate that glycosylation of an EGF domain proposed to be essential for ligand binding, EGF12, is crucial to the inhibition of Serrate-to-Notch signaling by Fringe. Expression of an EGF12 mutant of Notch (N-EGF12f) allows Notch activation by Serrate even in the presence of Fringe. By contrast, elimination of three other highly conserved sites of O-fucosylation does not have detectable effects. Binding assays with a soluble Notch extracellular domain fusion protein and ligand-expressing cells indicates that the NEGF12f mutation can influence Notch activation by preventing Fringe from blocking Notch-Serrate binding. The N-EGF12f mutant can substitute for endogenous Notch during embryonic neurogenesis, but not at the dorsoventral boundary of the wing. Thus, inhibition of Notch-Serrate binding by O-fucosylation of EGF12 might be needed in certain contexts to allow efficient Notch signaling (Lei, 2003).

To begin to identify EGF domains whose O-fucosylation influences Notch activation, S was substituted at the O-fucose attachment site for A, and T for V. A or V can be found at this position in other EGF repeats of Notch or its ligands, and hence are unlikely to cause disruptions of EGF structure. Focus was placed on four EGF repeats of Notch: 12, 24, 26 and 31. EGF24, EGF26 and EGF31 were chosen because they lie in or near the region of Notch to which the NAx alleles map, and because they contain highly conserved O-fucose sites that conform to the original consensus sequence. EGF12 was chosen because it corresponds to one of two EGF repeats identified as necessary and sufficient for Notch-ligand binding in a cell aggregation assay, and because it contains a potential O-fucose site in all cloned Notch receptors with 36 EGF repeats. Although this site does not conform to the original consensus for O-fucosylation, EGF12 of Notch1 has been shown to be glycosylated by O-FucT-1 and Fringe in CHO cells. O-fucosylation of Drosophila Notch EGF12 in Drosophila cells was confirmed by assessing the ability of a fragment of Notch isolated from S2 cells to serve as an in vitro substrate for Fringe (Lei, 2003).

Therefore, EGF12 is a biologically relevant site of O-fucosylation. O-fucose is attached to an S or T. Consequently, when that amino acid is changed to one that lacks a terminal hydroxyl group, O-fucosylation of the EGF domain cannot occur. Consistent with this, the S to A mutation eliminates the ability of a Notch fragment including EGF12 to serve as a substrate for Fringe. For several reasons, the observed differences between N-EGF12f and wild-type Notch can be attributed to this absence of glycosylation, rather than to the amino acid change per se. Substitution of an S with an A is a conservative change, and the two amino acids differ only by an oxygen atom. A is found at this location in other EGF repeats (e.g., EGF36 of Drosophila Notch, and EGF7 and EGF19 of mammalian Notch1), and hence is unlikely to disrupt the EGF structure. Indeed, this same mutation in EGF26 does not result in a detectable phenotype. A distinct amino acid change in EGF12, the E491V mutation in NM1, results in a strong loss-of-function phenotype, as would be predicted for a gross structural change in the ligand-binding domain (Lei, 2003).

By contrast, the phenotype of N-EGF12f is consistent with that which would be expected of a Notch receptor that had lost a functional site of glycosylation by Fringe. Expression of N-EGF12f results in an ectopic activation of Notch in dorsal wing cells that is insensitive to Fringe, yet dependent upon endogenous ligand expression. Binding studies further show that Serrate is able to bind to this mutant form of Notch even in the presence of Fringe, which contrasts with the lack of detectable Serrate binding to wild-type Notch expressed in the presence of Fringe. Based on these observations, it is concluded that EGF12 is an essential site for inhibition of Serrate-to-Notch signaling by the Fringe glycosyltransferase (Lei, 2003).

Although the O-fucose site in EGF12 is essential for Fringe inhibition of Serrate signaling in the wing, Fringe still reduces N-EGF12f:AP-Serrate binding. The decrease in binding is not sufficient to prevent N-EGF12f activation, but there must nonetheless be multiple sites that can contribute to the inhibition of Serrate signaling by Fringe. There must also be distinct sites that mediate the potentiation of Delta-Notch signaling by Fringe, because N-EGF12f:AP-Delta binding is potentiated almost as effectively as N:AP-Delta binding. Importantly then, the effects of Fringe on Delta versus Serrate signaling appear to be mediated, at least to some extent, through distinct sites of O-fucosylation (Lei, 2003).

The importance of additional O-fucose sites is further underscored by the distinct consequences of removal of O-fucose only at EGF12 by the S to A mutation, compared with removal of O-fucose at all sites by Ofut1 mutation or RNAi. Using a cell aggregation assay, EGF11 and EGF12 of Notch have been shown to have a key role in ligand binding. Deletion of EGF11 and EGF12 prevents aggregation between Notch-expressing cells and Delta-expressing cells, and a construct including only EGF11 and EGF12 of Notch is able to confer Delta-binding activity upon cells, albeit with decreased efficiency compared with full-length Notch. Although a role for other EGF repeats in ligand binding has been suggested based on the consequences of expressing fragments of Notch in the wing imaginal disc, and by cell aggregation experiments with mutant Notch proteins, EGF11 and EGF12 have generally been considered to be the key EGF domains for ligand binding. However, because RNAi of Ofut1 in S2 cells indicates that O-fucose is required on Notch for binding to its ligands, yet O-fucosylation of EGF12 is not required for ligand binding, other O-fucosylated EGF domains must also be required for Notch-ligand interactions. Thus, multiple sites are subject to O-fucosylation, but with different phenotypic consequences (Lei, 2003).

Among the 15 Notch receptors with 36 EGF repeats in sequence databases, an average of 20 of the 36 EGF repeats contain potential sites for O-fucosylation. However, only three EGF repeats contain O-fucose sites in all of these 15 Notch receptors: EGF12, EGF26 and EGF27. Thirteen other EGF domains contain sites that are somewhat conserved (i.e., an O-fucose site is found in that repeat in 11 or more of the 15 Notch protein sequences), including EGF24 (13/15 Notch receptors) and EGF31 (14/15 Notch receptors). These conserved sites for O-fucosylation cluster in an N-terminal region, and in a more C-terminal region centered around the NAx mutations. This general pattern of conservation (most Notch receptors have many sites, but only a few sites are absolutely conserved) suggests that at least some aspects of OFUT1 and Fringe regulation might be achieved through glycosylation of regions of Notch, rather than through glycosylation of specific EGF repeats. The lack of effect of mutation of individual, highly conserved EGF repeats in the NAx region is consistent with this suggestion, and experiments to analyze the consequences of mutation of arrays of O-fucose sites are in progress (Lei, 2003).

Notch ligands activate Notch receptors expressed by neighboring cells, but inhibit Notch receptors expressed by the same cell. Elevated expression of the Notch extracellular domain can also inhibit the ability of ligands to signal to neighboring cells. Thus, one apparent consequence of the transmembrane nature of Notch ligands is that Notch activation depends not simply on the ability of ligand to bind receptor, but also on a competition between intracellular and intercellular interactions. Previously, most attention has focused on the impact of different levels of expression on this competition. But the balance in this competition can also be shifted by adjusting the affinity between Notch and its ligands. Indeed, even though most studies have focused on the ability of Fringe to inhibit the response of a cell to Serrate, the ability of cells to send a Serrate signal appears to be enhanced by co-expression with Fringe, which is consistent with the idea that decreasing intracellular Serrate-Notch interactions increases the amount of Serrate available to signal to neighboring cells (Lei, 2003).

Cell-based binding assays indicate that the O-fucose site in EGF12 is not just important for Fringe-dependent inhibition: even the presence of the O-fucose monosaccharide at this site inhibits Serrate binding. The presence of an inhibitory site of O-fucosylation in EGF12 was unexpected given the general positive requirement for O-fucose in Notch signaling. However, the presence of an inhibitory site can be rationalized in terms of a competition between intracellular and intercellular Notch-ligand interactions. The competition model implies that it is important, at least in certain contexts, for Notch not to bind too strongly to its ligands. One such context is probably the DV boundary of the Drosophila wing, because Notch ligands are expressed on both sides of the compartment boundary, and Notch is activated on both sides of the compartment boundary. Thus, it is suggested that N-EGF12f is unable to rescue normal Notch activation at the DV boundary because its increased affinity for ligands enhances intracellular binding to a degree that interferes with the ability of a cell to send and receive Notch signals. Notably, EGF12 is apparently essential for both intercellular and intracellular Notch-ligand interactions (Lei, 2003).

The highly conserved presence of an O-fucose site in EGF12 suggests that inhibition of ligand binding by the O-fucosylation of EGF12 might be of widespread importance. However, if O-fucosylation of EGF12 was constitutive, it would simply counteract the positive influence of O-fucosylation at other sites. If, by contrast, O-fucosylation of EGF12 was regulated, then differential O-fucosylation of EGF12 could occur, and could serve as a mechanism of Notch regulation. Intriguingly then, EGF12 is distinguished from other potential O-fucose sites by the presence of an acidic amino acid (E or D) at the -2 position relative to the O-fucose attachment site. None of the other EGF repeats in Notch contain an acidic amino acid at this position, yet 13/15 Notch receptor proteins contain an acidic amino acid at this position in EGF12. It is not yet known what fraction of Notch receptors in a cell are modified at any of the potential sites of O-fucosylation, but the presence of this conserved sequence difference suggests that EGF12 might be O-fucosylated under different conditions, or with a different efficiency, than other EGF domains, and hence that differential fucosylation of this site might serve as a regulatory mechanism (Lei, 2003).

Distinct functional units of the Golgi complex in Drosophila cells

A striking variety of glycosylation occur in the Golgi complex in a protein-specific manner, but how this diversity and specificity are achieved remains unclear. This study shows that stacked fragments (units) of the Golgi complex dispersed in Drosophila imaginal disc cells are functionally diverse. The UDP-sugar transporter Fringe-Connection (Frc) is localized to a subset of the Golgi units distinct from those harboring Sulfateless (Sfl), which modifies glucosaminoglycans (GAGs), and from those harboring the protease Rhomboid (Rho), which processes the glycoprotein Spitz (Spi). Whereas the glycosylation and function of Notch are affected in imaginal discs of frc mutants, those of Spi and of GAG core proteins are not, even though Frc transports a broad range of glycosylation substrates, suggesting that Golgi units containing Frc and those containing Sfl or Rho are functionally separable. Distinct Golgi units containing Frc and Rho in embryos can also be separated biochemically by immunoisolation techniques. Tn-antigen glycan is shown to be localized only in a subset of the Golgi units distributed basally in a polarized cell. It is proposed that the different localizations among distinct Golgi units of molecules involved in glycosylation underlie the diversity of glycan modification (Yano, 2005).

The pattern of glycosylation is extremely diverse, yet is highly specific to each protein. How can this specificity (and diversity) be achieved? There are >300 glycosylenzymes in humans and >100 in Drosophila, but is their enzymatic specificity sufficient to explain the precise modification of all substrates? One possible mechanism that might also contribute to the specific (and diverse) pattern of glycosylation would be the localization/compartmentalization of glycosylenzymes (Yano, 2005).

The Golgi complex, where protein glycosylation takes place, has been regarded as a single functional unit, consisting of cis-, medial-, and transcisternae in mammalian cells. However, the three-dimensional reconstruction of electron microscopic images of the mammalian Golgi structure has suggested the existence of more than one Golgi stack, with the individual stacks being connected into a ribbon by tubules bridging equivalent cisternae. Furthermore, during mitosis, the Golgi cisternae of mammalian cells become fragmented without their disassembly. In Drosophila, Golgi cisternae are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase, although there has been no evidence to date to indicate functional differences among the Golgi fragments (Yano, 2005).

A Drosophila UDP-sugar transporter, Fringe connection (Frc) transports a broad range of UDP-sugars that can be used for the synthesis of various glycans, including N-linked types, GAGs, and mucin types. Interestingly, despite its broad specificity, loss-of-function studies have revealed that Frc is selectively required for Notch glycosylation, but not for GAG synthesis. This observation prompted a study at Frc localization; in this study, it was found that Frc is localized only to a subset of Golgi fragments in Drosophila discs and embryos (Yano, 2005).

Frc, Sfl, a glycosylenzyme of GAGs, and Rho, a processing enzyme of Spi glycoprotein, are localized to distinct Golgi fragments, which are referred to as 'Golgi units,' in Drosophila cells. frc mutants do not exhibit defects in the glycosylation and function of Spi nor do they exhibit defects in glycosylation or function of GAG core proteins. Moreover, biochemically separated distinct Golgi units containing Frc and Rho were isolated by immunoisolation technique. This study clearly shows that there are functionally distinct Golgi units in a Drosophila cell (Yano, 2005).

The Golgi complex is a stack of cis-, medial-, and transcisternae in mammalian cells. In contrast, Golgi markers often do not overlap with each other in Saccharomyces cerevisiae, in which the Golgi cisternae are not stacked but disassembled. The Golgi cisternae of Drosophila are stacked but are not connected to form a ribbon at the embryonic and pupal stages even during interphase. To determine whether Drosophila imaginal disc cells have assembled or disassembled Golgi cisternae, the localizations were compared of the cis-cisternal marker dGM130, the transcisternal marker dSyntaxin16 (dSyx16), and the Golgi-tethered 120-kDa protein, which is commonly used to detect the Golgi complex in Drosophila. The 120-kDa protein was identified by immunoaffinity purification and protein sequencing as a Drosophila homolog of the vertebrate 160-kDa medial Golgi sialoglycoprotein (MG160), which resides uniformly in the medial-cisternae of the Golgi apparatus in vertebrate cells. An antibody specific for the 120-kDa protein also stained numerous Golgi fragments in imaginal disc cells. More than 80% of immunoreactivity for the 120-kDa protein colocalizes with both dGM130 and dSYX16, suggesting that 120-kDa protein-positive fragments of the Golgi complex indeed comprise assembled cisternae; these fragments will be referred to as 'Golgi units'. The distributions of the 120-kDa protein, dGM130, and peanut agglutinin (PNA), another transcisternal marker, also shows that the markers are closely apposed but not identical, suggesting that the Golgi units are polarized. Interestingly, most of the PNA-positive transcisternae are oriented toward the basal side of the cell, within the Golgi complex, whereas most of the GM130-positive cis-cisternae are oriented toward the apical side of the cell. The cis-to-trans polarity of each Golgi unit thus appears to be correlated with the apico-basal polarity of the disc cells (Yano, 2005).

Drosophila mutant larvae defective in the UDP-sugar transporter Frc manifest a highly selective phenotype: the lack of Notch glycosylation in the presence of normal GAG synthesis (Goto, 2001). This limited phenotype was unexpected, given that Frc exhibits a broad specificity for UDP sugars used in the synthesis of various glycans including N-linked types, GAGs, and mucin types. However, given that the frcR29 allele studied previously (Goto, 2001) is hypomorphic, whether the selective glycosylation defect might be a consequence of partial loss of Frc activity was examined. With the use of imprecise excision, a new allele, frcRY34, was generated the presence of which results in the death of most larvae during the second-instar stage, much earlier than the death induced by frcR29. Real-time PCR analysis revealed that the amount of frc transcripts in the second-instar larvae of frcRY34 or frcR29 mutants was 4.2% and 24.4% of that in the wild type, respectively. About 1 kb of the gene, including the transcription initiation site, was deleted in the frcRY34 allele. Together, these observations suggest that frcRY34 is essentially a null allele (Yano, 2005).

Clonal cells of the frcRY34 mutant exhibit normal levels of GAGs, as detected by immunostaining with the 3G10 antibody, whereas the amount of GAGs was reduced in clones of tout-velu (ttv) mutant cells. Given that GAGs are required for signaling by Hedgehog (Hh), Wingless (Wg), and Decapentaplegic (Dpp),the expression was examined of corresponding target genes [patched (ptc) for Hh signaling and Dll for Wg and Dpp signaling] in the wing discs of the frcRY34 mutant. Expression of ptc and that of Dll in the ventral compartment of the wing discs were unaffected in the mutant clones, suggestive of normal GAG function (Yano, 2005).

Given that Notch glycosylation by Fringe (Fng), a fucose-specific ß1,3-N-acetylglucosaminyltransferase, requires Frc activity, Notch glycosylation was examined in the frcRY34 mutant. The frcRY34 mutant clones in the dorsal compartment, but not those in the ventral compartment, of the wing discs induce wg expression at their borders, as has been observed with fng mutant clones, suggesting that Notch glycosylation is impaired in the frcRY34 mutant. The ectopic expression of Wg induced by the frcRY34 mutant clones is likely responsible for the observed induction of Dll expression in the dorsal compartment (Yano, 2005).

To determine why the loss of a UDP-sugar transporter with a broad specificity selectively affects Notch glycosylation, the subcellular localization of Frc was examined. Frc tagged with the Myc epitope was expressed in imaginal discs under the control of the arm-Gal4 driver. The Gal4-induced expression of Frc-Myc rescues the frc mutant phenotype, suggesting that Frc-Myc is functional and properly localized. Immunostaining of imaginal discs of wild-type larvae expressing Frc-Myc with antibodies to Myc and to the 120-kDa protein revealed that Frc localizes to only a small subset of Golgi units. Thus, it is hypothesized that the Golgi units might be functionally heterogeneous, and that those containing Frc might modify some proteins, including Notch, but not others (Yano, 2005).

To test this hypothesis, the localizations of various molecules involved in protein modification in the Golgi complex were compared with that of Frc. It was found that Sfl is also restricted to a subset of Golgi units, but that its distribution does not overlap with that of Frc. This differential localization of Sfl and Frc might thus explain the observation that frc mutant clones in wing discs do not show any defect in GAG synthesis by Sfl (Yano, 2005).

The Spi-processing enzyme Rho was also localized to a subset of Golgi units distinct from those containing Frc, in addition to its presence in other compartments. This result indicates the existence of at least two types of Golgi units, those containing Rho and those containing Frc. To determine whether these two types of Golgi units differ functionally, the glycosylation state and function of Spi were examined in frc mutants (Yano, 2005).

Given that the extent of Notch glycosylation, as detected by wheat germ agglutinin (WGA), is markedly reduced in frc mutants compared with that in the wild-type background (Goto, 2001), whether the WGA-reactive glycan of Spi is also affected by frc mutation was examined. Myc epitope-tagged Spi was expressed in the wild type or the frcRY34 mutant. Spi-Myc was then precipitated from larval homogenates with antibodies to Myc and was examined for its glycosylation by SDS/PAGE and subsequent blot analysis with WGA. The reactivity of the Spi glycan with WGA was similar in the frc mutant and in the wild type. Whether the frcRY34 mutation affects the Spi glycan was examined by mobility shift analysis. The electrophoretic mobility of glycosylated Spi from the wild type was also similar to that from the frc mutant. Deglycosylation of Spi by neuraminidase, peptide-N-glycosidase (PNGase) F, and O-glycanases also increased its mobility to the same extent in wild-type and frc mutant larvae, suggesting that the core protein is not affected by the frc mutation. Together, these results indicate that the function of Frc is not necessary for formation of the Spi glycan (Yano, 2005).

Spi function was evaluated by examining developmental processes such as photoreceptor recruitment and bract formation, both of which require Spi activation. During eye development, although Spi is not necessary for the primary induction of the photoreceptor R8, it is required for the subsequent recruitment of R1 to R7. Given that photoreceptors R1 to R8 express ELAV and that R1 and R6 express Bar, the expression of these proteins was examined in frc mutants. In mutants harboring the hypomorphic allele frcR29, all photoreceptors are normally induced, although their direction is irregular as seen in fringe or Notch mutants. Similar results were obtained by clonal analysis of frcRY34 mutants. Spi function in photoreceptor recruitment thus did not appear to be impaired in the frc mutants. The frcR29 mutant also formed normal bracts on malformed legs. Tests were performed for genetic interaction between rho and frc mutations in wing vein formation. The rhove1 mutant is viable but shows partial loss of L3-5 veins. This phenotype is also apparent in rhove1, frcRY34/rhove1, frc+ flies, suggesting that Frc does not affect Rho function. From these results, it is concluded that the function of the Rho-Spi pathway is not affected by frc mutation (Yano, 2005).

To confirm that the Golgi units containing Frc and those containing Rho are distinct, whether these Golgi units could be selectively isolated was tested by using antibodies to Myc (for Myc-tagged Frc) or HA (for HA-tagged Rho). Because it is difficult to collect enough of the imaginal discs, the starting material was switched to embryos, and whether Frc and Rho are also localized to distinct Golgi units in embryos was examined. Frc-Myc and Rho-HA were coexpressed in the embryos by the arm-Gal4 driver; immunostaining with antibodies to Myc and to HA revealed that the Golgi units containing Frc-Myc (45.4% of total Golgi units) and those containing Rho-HA (43.0% of total Golgi units) are largely distinct: only 11.6% of total Golgi units were positive for both Frc-Myc and Rho-HA. Immunoisolation was attempted from embryonic lysates by using either antibody to Myc or HA and how much Frc-Myc and Rho-HA were coisolated in each immunoisolate was examined. When Frc-Myc was immunoisolated with an antibody to Myc, the recovery of Frc-Myc was 5.7 times greater than that of Rho-HA. Moreover, when Rho-HA was immunoisolated with an antibody to HA, the recovery of Rho-HA was 18.3 times greater than that of Frc-Myc. The immunoblot analysis of these immunoisolates with the anti-120-kDa antibody confirmed that the Golgi units were concentrated in these immunoisolates. These results support the notion that Frc-Myc-containing fraction is distinct and can be separated from Rho-HA-containing fraction (Yano, 2005).

Whether these distinct Golgi units contain different constituents was examined. Fringe (Fng) is one of the candidate molecules that may be colocalized with Frc. Therefore, expression of ectopically expressed Fng was examined in Rho- and Frc-containing immunoisolates. It was found that expression of Fng in Frc-containing immunoisolates was 26 times greater than in Rho-containing immunoisolates, supporting the idea that Fng is localized in the Frc-positive Golgi units rather than the Rho-positive Golgi units. It was also confirmed by immunostaining analysis that Fng colocalizes mostly with Frc (88.1% of the FNG-positive Golgi units), but not with Rho (16.6% of the Fng-positive Golgi units), by immunostaining analysis (Yano, 2005).

The data suggest that different Golgi units perform different functions, a notion that is also supported by the observation that Tn antigen (O-linked N-acetylgalactosamine) was detected in only a subset of Golgi units in imaginal eye disc cells. In addition, it was found that most of these Tn antigen-positive Golgi units are distributed in the basal region of the disc cells, suggesting that the differential distribution of Golgi units might contribute to the apicobasal polarity of glycan distribution (Yano, 2005).

In contrast to the larval stage, Frc is required for GAG synthesis at the early embryonic stage (Goto, 2001; Selva, 2001). To determine why the Frc requirement for GAG synthesis differs between the embryonic and larval stages, embryos expressing Frc-Myc were stained with antibodies to Sfl and to Myc. Sfl was found to be colocalized with Frc, likely explaining the importance of Frc for GAG synthesis at the embryonic stage. In addition, this embryonic requirement of Frc for GAG synthesis excludes the possibility that the selective defects in Notch and not in GAG synthesis observed in frc mutant larvae are caused by the selective Frc-dependent transport of a subset of UDP-sugars used only for glycosylation of Notch but not for GAGs synthesis (Yano, 2005).

It summary, these results provide evidence for the existence of functionally distinct Golgi units in Drosophila cells. Such functional heterogeneity of Golgi units is likely responsible for the diversity of protein glycosylation. At least two types of Golgi units containing either Frc or Sfl are present in larval disc cells. Two distinct sets of proteins, exemplified by Notch and GAG core proteins, might thus be selectively transported to Frc- or Sfl-containing Golgi units, respectively, where they undergo glycosylation by different sets of molecules (Yano, 2005).

The variety of Golgi units might be established by separate transport of secretory proteins and glycosylenzymes from the endoplasmic reticulum (ER) to the distinct Golgi units. In yeast, glycosylphosphatidylinositol (GPI)-anchored proteins exit the ER in vesicles distinct from those containing other secretory protein. Given that the GAG core protein Dally in Drosophila is anchored to the membrane by GPI, it is possible that Dally and Notch are loaded into distinct vesicles as they exit the ER (Yano, 2005).

Combinations of glycosylenzymes and transporters, such as Sfl and Frc, contained in Golgi units of Drosophila differ not only between embryos and larval disc cells but also among cell types. For example, Frc is localized to all Golgi units in salivary gland cells at the larval stage. It has also been shown that all of the Golgi complexes dispersed in oocytes may have the ability to process the Gurken precursor protein, which is usually cleaved in a subset of the Golgi complexes residing in the dorso-anterior region. The Golgi units may thus be altered in a manner dependent on development, cell type, and signaling processes (Yano, 2005).

The functional diversity of Golgi units also might contribute to the polarized distribution of glycans along the apicobasal axis of cells. It was found that Tn antigen is synthesized in the basal Golgi units of larval disc cells. Furthermore, certain types of glycans are distributed along the apicobasal axis of pupal ommatidia. These glycans might thus be synthesized differentially in the Golgi units that are asymmetrically distributed along the apicobasal axis and then be secreted at either the apical or basal cell surface (Yano, 2005).

Whereas Golgi units are dispersed throughout Drosophila cells, the Golgi complex in mammalian cells is thought to be a single entity that is located in the pericentriolar region through its association with the microtubule-organizing center in interphase and which is fragmented at the onset of mitosis. The Golgi fragments apparent in mammalian cells during mitosis are highly similar to the Golgi units of Drosophila cells in both electron and confocal microscopic images. The mammalian Golgi complex during interphase may therefore be comprised of functionally distinct units that are associated with the microtubule-organizing center and connected with each other (Yano, 2005).

The glycosyltransferase Fringe promotes Delta-Notch signaling between neurons and glia, and is required for subtype-specific glial gene expression

The development, organization and function of central nervous systems depend on interactions between neurons and glial cells. However, the molecular signals that regulate neuron-glial communication remain elusive. In the ventral nerve cord of Drosophila, the close association of the longitudinal glia (LG) with the neuropil provides an excellent opportunity to identify and characterize neuron-glial signals in vivo. This study found that the activity and restricted expression of the glycosyltransferase Fringe (Fng) renders a subset of LG sensitive to activation of signaling through the Notch (N) receptor. This is the first report showing that modulation of N signaling by Fng is important for CNS development in any organism. In each hemisegment of the nerve cord the transcription factor Prospero (Pros) is selectively expressed in the six most anterior LG. Pros expression is specifically reduced in fng mutants, and is blocked by antagonism of the N pathway. The N ligand Delta (Dl), which is expressed by a subset of neurons, cooperates with Fng for N signaling in the anterior LG, leading to subtype-specific expression of Pros. Furthermore, ectopic Pros expression in posterior LG can be triggered by Fng, and by Dl derived from neurons but not glia. This effect can be mimicked by direct activation of the N pathway within glia. These genetic studies suggest that Fng sensitizes N on glia to axon-derived Dl and that enhanced neuron-glial communication through this ligand-receptor pair is required for the proper molecular diversity of glial cell subtypes in the developing nervous system (Thomas, 2007).

This study identified Fng as a means by which a specific subtype of glia, the anterior LG, are made sensitive to N activation, evidence was provided that Dl, expressed on axons, activates N signaling in these glia leading to subtype-specific gene expression. Fng is required for maintenance of Pros expression in the anterior LG, which can also be blocked by antagonism of the N pathway with no effect on their survival or positioning. This is in contrast with studies of pros mutants, which found a role for Pros earlier in CNS development in establishing glial cell number. The role of Pros in mature LG is poorly understood, but it has been proposed to retain mitotic potential in these cells for use in repair or remodeling of the nervous system in subsequent larval or adult stages. It will be important to determine the consequences of lost Pros expression from mature anterior LG, and whether additional features and functions of the anterior LG are controlled by N signaling from axons (Thomas, 2007).

The importance of glycosylation for N function has been demonstrated in vivo. The addition of O-linked fucose to EGF repeats in the N extracellular domain is essential for all N activities and is mediated by O-fucosyltransferase-1 (O-fut1). By contrast, Fng is selectively used in specific developmental contexts, and has been best studied in the formation of borders among cells in developing imaginal tissues. Fng catalyzes the addition of GlcNac to O-linked fucose, to which galactose is then added. The resulting trisaccharide is the minimal O-fucose glycan to support Fng modulation of Notch signaling. Fng activity reduces the sensitivity of N for the ligand Ser but increases its sensitivity for Dl. By contrast with imaginal discs, in which modulation of N sensitivity to both ligands appears to be important, loss of Fng in LG resulted in reduced N activation only, consistent with reduced response to Dl. Expression of Pros in LG can be triggered by Dl derived from neurons but not glia, and this effect can be mimicked by direct activation of the N pathway within glia. Genetic experiments implicate neuron-derived Dl as the relevant N ligand for Pros expression in anterior LG, consistent with the ability of Fng to sensitize N to signaling by Dl. Enriched Fng expression in the anterior LG probably renders them differentially sensitive to sustained N signaling from Dl-expressing axons (Thomas, 2007).

The final divisions of the six LG precursors that give rise to 12 LG are thought to be symmetric, with low levels of Pros first distributed evenly between sibling cells after division. However, Pros is maintained and in fact upregulated in the anterior LG, and downregulated in sibling LG that migrate posteriorly. fng transcripts first appear to be expressed in all LG, then become enriched in the anterior LG and reduced in the posterior LG. It is speculated that refinement of fng expression may involve a positive feedback mechanism to consolidate and enhance N signaling in the anterior LG, since preliminary evidence suggests that N signaling can positively influence fng expression in the LG (Thomas, 2007).

Like Pros, Glutamine synthetase 2 (Gs2) is specifically expressed in the anterior LG but not posterior LG, indicating that these are functionally distinct glial subtypes with respect to their ability to recycle the neurotransmitter glutamate. The specificity of N signaling for Pros but not Gs2 indicates that N signaling is unlikely to influence cell fate decisions in the LG lineage and that Fng is unlikely to be the primary determinant of anterior versus posterior LG identity. Rather, Fng probably serves to consolidate this distinction through sustained N signaling (Thomas, 2007).

NICD is a potent activator of Pros expression in the posterior LG. This leads to a consideration of what factors limit Pros expression to the anterior LG in wild-type animals, since posterior LG are indeed capable of expressing Pros in response to constitutive N activity. (1) Based on analysis of fng mutants and Fng misexpression, it is proposed that Fng is a major determinant. The finding that misexpression of Fng causes ectopic Pros in posterior LG supports the argument that Dl-expressing axons do not contact the anterior LG only. It is likely that they make contact with at least some of the posterior LG. Therefore, in wild-type animals, in which Fng is reduced on posterior LG, contact from the subset of Dl axons is alone not sufficient to drive Pros expression. (2) Misexpression of Dl in all postmitotic neurons led to ectopic expression of Pros in posterior LG, indicating that the restricted expression of Dl on a subset of neurons also limits N activation. (3) N appears to be expressed in most or all LG, though it was also found that overexpression of full-length N caused ectopic expression of Pros. From these data a threshold model is proposed for N activation in LG that invokes a combination of factors, including Fng-regulated N sensitivity, exposure of N to ligand, N expression levels, and perhaps others. Increasing any of these factors can provide sufficient signaling for ectopic Pros induction in posterior LG. In wild-type embryos, these factors are also likely to combine with one another in the anterior LG to achieve supra-threshold N signaling and sustained Pros expression during normal development (Thomas, 2007).

Signaling through N is important for glial cell development in Drosophila, although it is context-dependent. Both an embryonic sensory lineage and the subperineurial CNS glial lineage utilize N activation to promote Gcm expression and glial fate. By contrast, in the sensory organ of adult flies, antagonism of N leads to Gcm expression in the glial precursor cell. In vertebrates, signaling through Notch receptors promotes the differentiation of peripheral glia, Müller glia, radial glia and mature oligodendrocytes. A Fng ortholog, lunatic fringe, is expressed in the developing mouse brain in a pattern consistent with glial progenitors. It will be interesting to determine whether Fng-related proteins in vertebrates have a role in glial cell differentiation, and whether they too can modulate N sensitivity and the context of N signaling between neurons and glia (Thomas, 2007).

fringe: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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