fringe is expressed in spiracles, eyes and legs -- all organs in which fringe/Serrate interaction is essential for boundary determination in development (Kim, 1995).


The developing wing disc of Drosophila is divided into distinct lineage-restricted compartments along both the anterior/posterior (A/P) and dorsal/ventral (D/V) axes. At compartment boundaries, morphogenic signals pattern the disc epithelium and direct appropriate outgrowth and differentiation of adult wing structures. However, the mechanisms by which affinity boundaries are established and maintained are not completely understood. Compartment-specific adhesive differences and inter-compartment signaling have both been implicated in this process. The selector gene apterous is expressed in dorsal cells of the wing disc and is essential for D/V compartmentalization, wing margin formation, wing outgrowth and dorsal-specific wing structures. To better understand the mechanisms of Ap function and compartment formation, aspects of the ap mutant phenotype have been rescued with genes known to be downstream of Ap. Fringe, a secreted protein involved in modulation of Notch signaling, is sufficient to rescue D/V compartmentalization, margin formation and wing outgrowth when appropriately expressed in an ap mutant background. When Fng and alphaPS1 (Multiple edematous wings, a dorsally expressed integrin subunit) are co-expressed, a nearly normal-looking wing is generated. However, these wings are entirely of ventral identity. These results demonstrate that a number of wing development features, including D/V compartmentalization and wing vein formation, can occur independently of dorsal identity and that inter-compartmental signaling, refined by Fng, plays the crucial role in maintaining the D/V affinity boundary. In addition, it is clear that key functions of the ap selector gene are mediated by only a small number of downstream effectors (O’Keefe, 2001).

These results suggest that intercompartmental signaling is sufficient to maintain the D/V affinity boundary. In the absence of dorsal identity, compartmental defects associated with ap mutant wing discs can be rescued with the molecule Fng. This argues that signaling between compartments mediated by Fng and Notch, and not the autonomous acquisition of compartment-specific affinity as an aspect of cell identity, plays the crucial role in D/V compartmentalization. Consistent with this are previous findings that both fng and Notch mutant clones generated in the dorsal compartment do not respect the D/V boundary, despite the fact that they likely retain dorsal identity. While the ap alleles used in this study are not molecularly-defined nulls, these allelic combinations clearly reduce Ap function sufficiently to eliminate dorsal identity. Based on both sensory bristle and wing vein morphologies, the Fng and Fng+alphaPS1-rescued wings consist entirely of ventral cell types. The possibility cannot be excluded that these ap allelic combinations might maintain small degrees of dorsal-specific affinity (independent of dorsal identity): the mutant phenotypes indicate that any adhesive differences are clearly not sufficient to maintain D/V compartmentalization (O’Keefe, 2001).

Prospective wing vein cells are identifiable in late third instar wing discs by molecular markers such as rhomboid. Wing disc eversion results in apposition of dorsal and ventral vein components, and interplanar signaling between the dorsal and ventral wing surfaces has been shown to play a crucial role in wing vein differentiation. Clonal analysis has demonstrated that mutations that disrupt or alter vein formation, frequently have non-autonomous effects on the opposite surface, and that these effects are particularly dramatic when the genetic clone lies on the dorsal surface. These results suggest a dorsal-specific signal that induces differentiation of ventral veins. However, when forced to differentiate without interplanar signaling, vein structures are capable of forming on both surfaces, although these veins are defective in terms of refinement and their pattern of corrugation. In the Fng+alphaPS1-rescued wing there is no dorsal identity and, therefore, no dorsal-specific signal directing ventral vein differentiation. Despite this abnormality, vein components on both surfaces differentiate appropriately based on their A/P and proximal/distal position in the wing; these vein components have an entirely ventral identity. This demonstrates that wing vein refinement, alignment and pattern of corrugation can occur independently of dorsal cell types. Although interplanar signaling is certainly essential for proper wing vein differentiation, it is clear that a dorsal-to-ventral signal is not required, and that ventral cell types autonomously contain all the information necessary for wing vein development (O’Keefe, 2001).

An emerging view of selector gene function is that these genes may regulate large numbers of effector genes involved in particular morphogenetic processes. For example, in the differentiation of Drosophila haltere from wing, the transcription factor Ultrabithorax regulates genes at many levels of the wing patterning genetic cascade. So too, the selector homeoproteins Even-skipped and Fushi tarazu (Ftz) have been shown to regulate either directly or indirectly most genes during embryogenesis. However, fusion of the VP16 activation domain to Ftz has suggested that Ftz binds to and regulates only a small number of target genes. The question is therefore unanswered as to whether the number of genes regulated by selectors is large or small. In the absence of normal ap selector gene function, the expression of only two downstream effectors is sufficient to rescue wing structures to a remarkable degree. This result suggests that the compartment-specific selector gene ap regulates only a small number of target genes during wing development. It will be interesting to determine whether selector genes with broader scopes of activities function in a similar manner. Selectors that control the formation of entire structures (such as eyeless) or entire body regions (such as the Hox genes) presumably sit at the top of larger genetic hierarchies than ap, and may control larger sets of target genes to fulfill their developmental roles (O’Keefe, 2001).

Finally, although ap regulates only a small number of downstream effectors to generate the overall morphology of the wing, it may indeed regulate many genes to confer dorsal identity. It is tempting to speculate, however, that Ap may regulate only one additional gene, Dorsal wing (Tiong, 1995), in order to specify dorsal cell fate in the wing. Loss-of-function mutations in the Drosophila Dorsal wing locus result in dorsal-to-ventral transformations in the wing blade, and ventral misexpression of Dorsal wing produces ectopic dorsal structures (Tiong, 1995). While the gene corresponding to this phenotype has yet to be characterized, Dorsal wing likely forms a crucial component of Ap-dependent wing developmental processes (O’Keefe, 2001).

frg was first identified by the expression pattern of ß-galactosidase associated a lacZ-containing P element insertion, 35UZ-1. The P element inserted within a few hundred base pairs of the transcription start site. In the wing disc, ß-galactosidase produced by this insertion is detected exclusively in dorsal regions, and a sharp boundary between cells that either do or do not express ß-galactosidase occurs at or near the presumptive wing margin. Strong expression appears in proximal regions of the ventral wing at the end of larval development, as revealed by cDNA hybridization (Irvine, 1994).

Planar polarization of the denticle field in the Drosophila embryo: roles for Myosin II (zipper) and fringe

Epithelial planar cell polarity (PCP) allows epithelial cells to coordinate their development to that of the tissue in which they reside. The mechanisms that impart PCP as well as effectors that execute the polarizing instructions are being sought in many tissues. The epidermal epithelium of Drosophila embryos exhibits PCP. Cells of the prospective denticle field, but not the adjacent smooth field, align precisely. This requires Myosin II (zipper) function, and it was found that Myosin II is enriched in a bipolar manner, across the parasegment, on both smooth and denticle field cells during denticle field alignment. This implies that actomyosin contractility, in combination with denticle-field-specific effectors, helps execute the cell rearrangements involved. In addition to this parasegment-wide polarity, prospective denticle field cells express an asymmetry, uniquely recognizing one cell edge over others as these cells uniquely position their actin-based protrusions (ABPs; which comprise each denticle) at their posterior edge. Cells of the prospective smooth field appear to be lacking proper effectors to elicit this unipolar response. Lastly, fringe function was identified as a necessary effector for high fidelity placement of ABPs and it was shown that Myosin II (zipper) activity is necessary for ABP placement and shaping as well (Walters, 2006).

Since the prospective denticle field is clearly polarized, it was wondered if smooth field cells were similarly polarized but simply did not express a marker, such as the ABPs, that revealed that polarity. To test this, the formation of ABPs among prospective smooth cells was induced by ectopically expressing the transcription factor svb/ovo in small groups of cells in the ventral epidermis and these cells were marked by co-expression of GFP. Expression of svb/ovo is necessary and sufficient to induce the formation of ABPs, so by expressing svb/ovo in the smooth field, the localization of these protrusions can be visualized within the cell. When svb/ovo is induced in the smooth field, the ectopic ABPs did not preferentially localize to the posterior edge of cells. Instead, they showed a stochastic dispersal around the apical surface of the cell Both anti-phosphotyrosine and phalloidin stains label these misplaced ABPs, indicating that phospho-epitopes as well as actin are present. Out of the thirty-eight ectopic denticles scored, 63% were mis-positioned (nine on an anterior edge, fifteen placed centrally) and only 37% were localized to the posterior edge of the cell (Walters, 2006).

It is also possible that the stochastic ABP localization observed in the svb/ovo-positive cells was not due to the lack of polarity effectors in the smooth field, but instead a simple issue of developmental timing. Since a heat-shock-driven recombinase was used to induce svb/ovo expression, precise control over the timing of the recombination event or svb/ovo induction was not occuring. Attempts were made to remedy this concern by ectopically expressing svb/ovo using Ptc-GAL4. Since patched is expressed well before cell fate specification, any ectopic ABPs that are present should have had ample time to localize to the posterior cell edge. However, even when svb/ovo is expressed at this early time point, ABPs in the smooth field showed no preference for the posterior edge of cells and remained stochastically positioned. These data strongly suggest that smooth cells do not possess a latent ability to place ABPs with the unipolar asymmetry seen among prospective denticle field cells. At the minimum, an effector of unipolar asymmetry must be active only among prospective denticle field cells. Alternatively, unipolar asymmetry is established only late and is restricted to the prospective denticle field. To investigate the unipolar asymmetry further, the phenomena of ABP formation and eventual placement at the posterior edge of denticle field cells was studied (Walters, 2006).

Denticle field cells are aligned and exhibit posterior ABPs at late stages (Stage 14). Since the denticle field is specified between late stage 11 and stage 12, cell alignment and ABP formation were monitored from stage 11 onward. At late stage 12, there was little alignment among cells within each parasegment. However, cell contours evolved through stage 14 (Walters, 2006).

Turning to the development of ABPs, at late stage 12, there was little or no enhancement in actin accumulation at the apical surface of the prospective denticle field cells compared to cells within the smooth field. Early in stage 13, a diffuse actin meshwork appeared at the apical surface of prospective denticle cells. By late stage 13, the apically enriched actin had coalesced into several patches that appear to represent nascent protrusions. Surprisingly, these nascent protrusions were often located away from the posterior edge of a cell. Only later, during stage 14, were ABPs more uniformly at or near each cell's posterior edge. Note that more fully pointed and curved ABPs can be observed at stage 15 (Walters, 2006).

Quantification of actin accumulation within slices across the apical face of a cell throughout these developmental stages supported the notion of a progression to the posterior edge. The intensity of Rhodamine–phalloidin-labeled actin was measured by dividing a prospective denticle field cell into six domains (progressing from A to P) and recording the pixel intensities in each of the domains at the apical face of that cell. The process was repeated for 3 cells in 3 different animals at early stage 13, late stage 13 and stage 14. The results showed that, during early and late stage 13, actin was not biased to any cell edge. At stage 14, actin was enriched dramatically at the posterior edge. It is concluded that actin first accumulates stochastically on the apical face of the cell and then is later positioned at the posterior edge. A core PCP component, frizzled (fz), was tested for its potential role in establishing or maintaining unipolar asymmetry among prospective denticle cells (Walters, 2006).

The frizzled gene is important for polarizing the hairs on the wing and in the abdomen and for ommatidial orientation. Denticle cuticle pattern were examined in the progeny of fzH51/fzP21 mothers crossed with fzH51/TM3 Ubx-LacZ fathers. Most larval cuticles appeared normal, though half of these were expected to be null for fz function. Two of thirty cuticles analyzed did exhibit patterning errors, but these were fusions among segments. This might have been caused by a partially penetrant deficit in canonical Wingless signaling since fz is also used in this pathway (where it is redundant with Dfrizzled2). The ABPs were examined directly in fz-deficient embryos; these were unambiguously identified by the lack of Ubx-LacZ expression. All fz-deficient embryos exhibited normal cell alignment of denticle field cells, and most mutants (six of eight) exhibited normal posterior placement of ABPs. Only two mutants exhibited misplaced protrusions, and in these embryos, the defects were restricted to row 1. Fully penetrant, but similarly restricted defects (to rows 1 and 2) have been reported for the core PCP mutants fz, strabismus, flamingo and even more weakly so for the PCP-specific mutant of disheveled (dsh1. Collectively, the defects observed implicate core PCP genes in denticle field polarization, but the restriction of these defects solely to anterior-most rows suggests a more minor role than expected in executing unipolar asymmetry (Walters, 2006).

Denticle field cells elaborate ABPs, and this has allowed it to be established that these cells are polarized in the plane of the epithelium. Forcing smooth field cells to elaborate ABPs failed to reveal any latent unipolarity within this portion of the parasegment. Nevertheless, the evidence supports the proposition that both fields are polarized, albeit in different ways. This study establishes bipolar enrichment of Myosin II on prospective smooth and denticle field cells. This suggests that the whole ventral epithelium exhibits bipolar PCP, and all cells can discriminate their A/P from their D/V edges. Since this bipolar enrichment emerges during stage 12, it is possible that this reflects de novo establishment of polarity within the epithelium. However, at earlier stages, cells of this epithelium exhibit a strikingly similar bipolar distribution of Myosin II then used for convergence extension. Thus, the bipolar redeployment of Myosin II might reflect a memory of that earlier polarization. This possibility is particularly compelling given that Myosin II orchestrates the rearrangements of cell junctions necessary for convergence extension. Perhaps Myosin II is being engaged similarly at the later stages, and the re-emergence of a bipolar preference is a precondition to accomplish the necessary junctional re-organization for cell alignments. What signals direct this re-emergence are not yet known. In addition, since Myosin II is bipolar among both prospective denticle and smooth cells, though the latter do not align, there must either be cues or effectors specific to the denticle field that initiate the cell alignment process (Walters, 2006).

Note also that the bipolar enrichment of Myosin II yields no clues as to how the denticle field cells uniquely identify their 'P' cell edge and faithfully position the ABPs to this edge. One possibility is that only the prospective denticle field cells have proper effectors to transmute the global bipolarity into asymmetric unipolarity. Since it is difficult to imagine how this might occur, a second possibility is that unipolarity is imparted locally, only across the prospective denticle field. The failure to observe proper ABP placement after ectopic expression of svb/ovo in the prospective smooth field supports this possibility. If unipolarity is imparted locally, then this also places constraints on the timing of the signals for unipolarity. The epithelium is sorted into smooth and denticle fields only late in development as a consequence of the antagonism between Wingless signaling and EGF receptor activation. This results in the establishment of the domain of expression for Svb. Thus, the denticle field is only established after this time, and the idea is favored that unipolarity is assigned after this stage. It is believed that analysis of Fringe supports this contention: fringe comes to be expressed within denticle field cells only after this stage and mutation of fringe interferes with unipolarity across the whole denticle field. This identifies fringe as, at the minimum, an effector of denticle field unipolarity (Walters, 2006).

Among a set of core genes involved in the establishment and maintenance of polarity in other tissues is frizzled, and surpriskingly it did not play a major role in denticle field polarization. Only two of eight embryos exhibited any defects, and in these, only row 1 cells appeared affected. Yet, a role for frizzled signaling in denticle field unipolarity cannot be ruled out due to possible redundancy with DFrizzled 2. In fact, recent work has implicated several core PCP genes in denticle field unipolarity. Mutation of frizzled, dsh, flamingo or strabismus lead to mild defects restricted to rows 1 and 2 reminiscent of what are report here for the minority of fz embryos. In addition, enrichment for Flamingo, Fz and Dsh occurs on the edges of prospective denticle field cells. In aggregate, these data are very suggestive for a role of core PCP genes in ABP polarity. However, the restriction of the phenotype to the very anterior rows of the denticle field leaves open the possibility that these genes do not play as major of a role as they do in, for example, the wing. In addition, while it has been shown that Fz and Dsh are enriched on certain cell edges, it will be of interest to know whether the enrichment is uniquely to one edge of cells, as it is in wing cells. For example, the data show that Zipper and Squash-GFP are enriched to cell edges but that these are likely both A and P edges of each cell. In denticle field cells, if Fz and Dsh exhibit such bipolar enrichment, rather than the unipolar asymmetry observed in wing cells, then their role in the denticle field would be quite distinct from that currently proposed for wing cells (Walters, 2006). It will be difficult to establish definitively whether Fz or Dsh play more extensive roles in denticle field polarity, especially since this data strongly suggest that effectors for this polarity are likely established late, after the epithelium is sorted into smooth versus denticle field cells. It would not be possible to easily interpret the removal of all function for dsh and frizzled (which likely would entail the removal of both frizzled and Dfrizzled2) because both proteins play earlier and essential roles in Wingless signal transduction. Removing both maternal and zygotic dsh (or fz Dfz2) function might well lead to polarity phenotypes, but these may be secondary to earlier deficits in Wg signaling. This is especially the case as Wg (and Hedgehog as well) plays a major role in establishing the denticle versus smooth field and subdividing parasegments into smaller signaling territories as well as establishing the responses of the cells to those signals (Alexandre, 1999, Gritzan, 1999, Hatini and DiNardo, 2001b and Wiellette and McGinnis, 1999). These considerations also raise a caution in drawing the conclusion from Wg or Hh null mutants that these pathways play any direct role in polarization (Walters, 2006).

Effects of Mutation or Deletion

Flies homozygous for strong fng mutations die by early larval stages, making it impossible to directly observe effects on formation of the adult wing. Viable fng mutations result in the loss of tissue from the wing margin. Clones of cells homozygous for strong fng mutations in the ventral wing have no apparent affect on wing development. Clones in the dorsal wing form ectopic wing margins along clone borders.

These ectopic wing margins are composed of both wild type and mutant cells, with the clone boundary always splitting the middle of the ectopic margin. This indicates that juxtaposition of cells possessing and cells lacking fng expression can induce wing margin formation, and implies the existence of a signaling process between such cells (Irvine, 1994).

The normal wing margin forms at the boundary between dorsal and ventral cells. Along the anterior of the normal wing margin, dorsal and ventral cells form distinct types of bristles. However, the ectopic wing margins induced along fng clone boundaries are composed of mirror-image duplications of dorsal-type bristles. This suggests that dorsal identity is determined independently of fng. When ectopic margins are induced at apterous clone boundaries, the wild-type cells make dorsal bristles, but the apterous mutant cells make ventral bristles. Thus apterous or the absence of apterous determines dorsal or ventral identity respectively, while fng function is restricted to boundary formation (Irvine, 1994).

Clone boundaries near normal wing margins consistently have ectopic wing margins, but clone boundaries distant from the normal margin often fail to induce ectopic margins. This suggests that an additional, spatially restricted factor is required for effective wing margin induction at fng expression boundaries. The distribution of this factor defines a "competence region" within the disc in which wing margins can form at fng clone borders (Irvine, 1994).

Dorsal surface clones can be associated with an expansion of the dorsal wing surface. In certain cases, rather than simply expanding the dorsal surface, the growth associated with fng clones can effect a second proximal-distal axis. Distal outgrowths appear to form only from positions near the anterior-posterior compartment boundary. This suggests that interactions between cells at fng expression boundaries and the anterior-posterior compartment boundary promote distal wing outgrowth. The size of distal outgrowths varies with their location within the wing; outgrowths from the tip are small and outgrowths from the base are large (Irvine, 1994).

Notch signalling mediates segmentation of the Drosophila leg

The legs of Drosophila are cylindrical appendages divided into segments along the proximodistal axis by flexible structures called joints, with each leg having 9 segments. The separation between segments is already visible in the imaginal disc because folds of the epithelium and cells at segment boundaries have a different morphology during pupal development. The joints form at precise positions along the proximodistal axis of the leg; both the expression patterns of several genes in the leg and the results of regeneration experiments suggest that different positions along the proximodistal axis have different identities. Two signaling molecules, wingless (wg) and decapentaplegic (dpp) play a central role in patterning the leg discs. These genes are activated in complementary anterior dorsal (dpp) and anterior ventral (wg) sectors in response to the secreted protein Hedgehog, which is only expressed in posterior cells. The asymmetry of dpp and wg expression is maintained by mutual repression: dpp and wg act antagonistically to regulate several genes involved in generating differences along the dorsoventral axis. It is therefore likely that the proximodistal patterning system initiated by wg and dpp determines the localization of presumptive joints in developing leg discs, but the identity of the gene products mediating this process is unknown (de Celis, 1998 and references).

Although the mechanism underlying joint formation is not understood, the fusion of segments caused by some Notch alleles indicates a requirement for Notch signaling. In the leg imaginal disc most segments form concentric rings, with the most distal in the center of the disc. The exceptions are the distal femur and proximal tibia, which are indistinguishable in the larval imaginal disc and only separate during pupariation. This separation occurs through the formation of lateral invaginations that fuse creating two epithelial tubes constricted at the femur/tibia joint. When Notch activity is compromised in Nts1 larvae during early and late third instar stage, the legs that develop are misshapen, with some fusion between femur/tibia (early) and tarsal (late) segments (de Celis, 1998).

To distinguish which elements of the Notch pathway are required during leg development, clones of homozygous mutant cells were generated, using lethal alleles in fng, Dl and Su(H) as well as a deficiency of the E(spl) complex. Lethal Ser alleles can survive into adults and they have a low frequency of joint fusions. The phenotype of Dl and Su(H) mosaics are similar to each other and, like Notch, result in a failure to make joints when mutant cells are in the position where a joint should have formed. Again, the wild-type cells near the clones can still form joints, but the length of the leg is reduced when the mutant clones are large and span more than one segment. In contrast, mutant cells homozygous for a deficiency that removes the E(spl)bHLH genes form normal joints even when they span more than one segment and are characterized by the differentiation of a vast array of ectopic sensory organs. These develop without intervening epidermal cells, indicating that E(spl) is required for the lateral inhibition mechanism that allows the spacing between sensory organs. The larger clones cause a slight reduction in the overall size of the leg (12% in area and 8% in length), but it is likely that these effects are due to the differentiation of ectopic sensory organs rather than direct effects on growth. Cells mutant for fng also result in fusions between segments. However, these effects are position dependent. Thus, with clones spanning the boundary between the femur and tibia the phenotypes are indistinguishable from those of Notch and Su(H), resulting in a fusion of these two segments and shortening of the leg, whereas in more distal segments defects in the joint can only be detected between the proximal two tarsal segments. The fact that fng is important in leg segmentation suggests that boundaries similar to the wing dorsal-ventral boundary are being created in at least some of the presumptive joints (de Celis, 1998).

In the developing wing the localized activation of Notch can be detected by the activation of certain target genes such as E(spl) and vestigial. Furthermore, the domains of expression of Dl and Ser are important in creating this localized activation of Notch. The expression of Ser, Dl, fng, Notch and E(spl)m beta were therefore examined during leg development. Heterogeneities in the expression of all these genes are detected in the third instar imaginal disc, where Dl and E(spl)m beta RNA are expressed in narrow concentric rings. In evaginating leg discs (0-4 hours APF) and in pupal legs, when the separation between leg segments becomes more evident, E(spl)m beta expression is localized to a ring of distal cells in each leg segment, suggesting that larval expression of E(spl)m beta also defines the distal end of each segment. The expression of fng is also restricted, and is only detected in several broad rings localized to the presumptive tibia and first tarsal segment, and in two groups of distal cells in the fifth tarsal segment that could correspond to the presumptive claws. At this stage, no heterogeneity could be detected in the expression of Notch RNA, but by 24 hours after puparium formation the levels are higher in the places where the joints are being formed, which appear to be the same cells where E(spl)m beta is expressed. At these later stages, Dl also accumulates in rings of cells located at the distal end of each segment and at the separation between the femur and tibia, as well as in many clusters of cells that correspond to developing sensory organs. Expression of E(spl) genes is dependent on Notch activity and hence the localization of E(spl)m beta mRNA to rings of cells in the imaginal and pupal leg disc indicates that there are high levels of Notch activation in the distal-most set of cells in each segment. To determine more precisely the relationship between the E(spl)m beta-expressing cells and the expression of other components of the Notch pathway, a reporter gene was generated in which 1.5 kb of genomic DNA upstream of E(spl)m beta was used to drive expression of a rat cell surface protein, CD2. As a landmark for the segment boundaries an enhancer trap in the bib gene, bib lacZ was used, which is expressed at higher levels in single-cell wide rings at the distal end of each leg segment during both larval and pupal development. The expression of E(spl)m beta-CD2 is localized to a narrow ring, 1-2 cells wide, which coincides with the cells expressing bib lacZ and with cells that have higher levels of lacZ expression in the N lacZ1 enhancer trap line. The expression of N lacZ1 at the dorsoventral boundary and at vein-intervein boundaries is dependent on Notch activity itself. Thus the coincident Notch, E(spl)m beta and bib expression indicates that high levels of Notch activation during imaginal leg development are restricted to the most distal cells of each segment. The accumulation of Notch ligands is also localized within the developing leg segments, with the highest levels of Dl and Ser detected in a narrow stripe of cells localized proximally to those expressing bib lacZ both in the larval imaginal disc and at pupal stages (de Celis, 1998).

Overall the effects produced by ectopic Dl and Ser are similar: the altered morphology of the resulting legs includes both fusion of segments and ectopic joints. However there are positional differences in the way the ligands exert their effects. Thus, the strongest effects of mis-expressing Dl are observed in the tarsal segments, where joint formation is perturbed resulting in foreshortened fused tarsi. This resembles Notch loss-of-function phenotypes suggesting that the levels or position of Dl expression are interfering with normal Notch activity. In addition, an abnormal structure forms at the junction between the first and second tarsal segments, which seems to consist of a partial perpendicular joint. The strongest effects of Ser mis-expression are suggestive of dominant negative effects, since the tibia is foreshortened and forms abnormal joints with the femur and tarsi. In addition, incomplete ectopic joints can be observed at low frequency in distal tarsal segments. Thus, the phenotypes indicate that both activation and repression of Notch occurs when high levels of Notch ligands are expressed. It is likely that the differential effects of misexpression of Dl and Ser are related to the distribution of fng, because the strongest dominant negative effects of Ser occur in the tibia, where fng expression is maximal, and those of Dl occur in distal tarsal segments, where fng is absent or expressed at low levels. Similar effects occur when the ligands are expressed in the wing using the GAL4 system, where the outcome is in part determined by interactions between Notch and Fng (de Celis, 1998).

Notch-mediated segmentation and growth control of the Drosophila leg

The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, the Notch signaling pathway has been shown to play a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream of the action of Wingless and Decapentaplegic. While Dl expression overlaps fngand Ser, in some cases, it appears to extend into regions of the disc where neither fng nor Ser is expressed (Rauskolb, 1999).

fng mutant clones also result in fused joints and shortened legs. fng is required with the formation of all joints except the tibia-tarsal (ta1: basitarsus) joint. In most cases, the formation of the joints appears to be an autonomous property of wild type cells, while the failure to form joint structures is an autonomous property of cells mutant for Notch, Dl, Ser or fng. However, some exceptions have been observed in which joint formation is inhibited within wild type cells that border mutant clones or mutant cells appear to contribute to joint structure (Rauskolb, 1999).

When fng is ectopically expressed, Nub expression can be induced along the inside of clone borders. Similarly, joint structures in the adult can be induced along fng-expression borders and can be inhibited within patches of fng-expressing cells. These observations are consistent with prior studies of fng action during Drosophila wing and eye development, in that Notch activation is positioned along the borders of fng expression (Rauskolb, 1999).

A re-evaluation of the contributions of Apterous and Notch to the dorsoventral lineage restriction boundary in the Drosophila wing

The Drosophila limb primordia are subdivided into compartments -- cell populations that do not mix during development. The wing is subdivided into dorsal (D) and ventral (V) compartments by the activity of the selector gene apterous in D cells. Apterous causes segregation of D and V cell populations by at least two distinct mechanisms. The LRR transmembrane proteins Capricious and Tartan are transiently expressed in D cells and contribute to initial segregation of D and V cells. Signaling between D and V cells mediated by Notch and Fringe contributes to the maintenance of the DV affinity boundary. Given that Notch is activated symmetrically, in D and V cells adjacent to the boundary, its role in boundary formation remains somewhat unclear. The roles of Apterous and Fringe activities in DV boundary formation have been re-examined and evidence is presented that Fringe cannot, by itself, generate an affinity difference between D and V cells. Although not sufficient, Fringe is required via Notch activation for expression of an Apterous-dependent affinity difference. It is proposed that Apterous controls expression of surface proteins that confer an affinity difference in conjunction with activated Notch. Thus, Apterous is viewed as instructive and Notch activity as essential, but permissive (Milán, 2003).

The LRR transmembrane proteins Capricious and Tartan contribute to DV boundary formation, but their role is transient. Maintenance of the boundary requires an additional mechanism. Notch activity has been implicated in this process, but its role has been questioned. Models for maintenance of the DV boundary must take into account the fact that Notch is activated symmetrically in cells on either side of the DV boundary. Therefore, an Ap-dependent process must be invoked to confer a DV difference. One proposal is that Fringe mediates the required Ap-dependent activity by acting in a Notch-independent manner, in addition to its role in Notch signaling. According to this view, confrontation of Fringe-expressing and non-expressing cells should induce a cell affinity difference. Increasing or decreasing Fringe activity has some effect, but does not produce affinity differences comparable with those produced by manipulating Apterous activity. Furthermore, the effects of restoring Fringe in D cells that lack Apterous activity can be reproduced independently by blocking Notch activation using Necd. Thus, it is unlikely that Fringe has a Notch-independent role in DV cell interactions (Milán, 2003).

A second, very different, model proposes that Notch activation confers a boundary-specific affinity state and that this is modulated into D and V states by Apterous expression. According to this model, there should be an affinity difference between boundary cells and internal cells within a compartment but not between D and V cells in the absence of Notch activity. This model proposes that Notch activity is sufficient to produce an affinity difference and hence smooth clone borders. However, clones of cells expressing the activated Notch receptor do not exhibit this property. This model is also difficult to reconcile with the observation that the borders of fringe mutant clones in the D compartment are highly irregular. It is also incompatible with the finding that restoring Notch activity in the absence of Apterous function is not sufficient to generate a smooth DV boundary and prevent mixing of D and V cells (Milán, 2003).

The results reported here support the view that Notch activity is needed for cell affinity differences between D and V cells, but indicate that Notch activation is not sufficient to cause these differences. A new model is proposed that differs in one crucial respect from the model discussed above. The role of Notch activation is considered to be permissive rather than instructive, and it is suggested that Apterous controls expression of surface proteins in D and V cells. It is envisaged that Notch activity is an essential co-factor in allowing cells to convert this into an affinity state. In molecular terms, one possibility is that D and V surface proteins form complexes with activated Notch (N*). In this scenario D+N* and V+N* are the active components, D and V are needed and instructive but have no activity alone. Interestingly, it has been observed that loss of Notch activation only in one compartment does not alter the DV affinity boundary. Thus, production of either the dorsal (D+N*) or the ventral (V+N*) boundary-specific cell state is sufficient to induce an affinity difference with cells of the opposite compartment. Another plausible molecular scenario is that Notch activity might control the subcellular localization of the predicted D and V proteins (Milán, 2003).

These examples are presented to illustrate how Notch activity can be seen as a permissive co-factor rather than as an instructive principle defining cell affinity. Many other molecular explanations are possible. This model provides a satisfactory explanation for how Notch can be required, but not sufficient for boundary maintenance. The essential difference between the permissive and instructive models for Notch function lies in the observation that Notch activation leads to an affinity difference only in the context of juxtaposition of cells with opposite DV identity. Notch activation per se does not induce a robust affinity boundary, whereas clones expressing dLMO and Necd do so only when Notch is not blocked in the cells outside the clone. Comparable results have been obtained with clones expressing Apterous and Necd (Milán, 2003).

Are the transmembrane proteins Serrate and Delta the D and V proteins, respectively? Early in development, Serrate is expressed in D cells and Delta in V cells. Late in development, both genes are regulated by Wg and are expressed in cells adjacent to the Wg-expressing cells at the DV boundary. Given that the Serrate- and Delta-expressing cells are offset from the DV boundary, it is considered unlikely that they confer the D* and V* activities. However, the possibility that they might contribute to the establishment of the DV affinity boundary in collaboration with Caps and Tartan cannot be excluded (Milán, 2003).

The interface between D and V cells behaves as an affinity boundary and as a signaling center where Notch activation is required for the growth of the wing disc. Clones of cells can be induced to sort into the opposite compartment by manipulating Apterous or Fringe activities. A distinction can be made between crossing and pushing the DV boundary as possible mechanisms. Cells with altered Apterous activity also have altered Fringe activity. It is suggested that these clones can cross the boundary and mix freely with cells in the opposite compartment because they change both their affinity state and signaling properties. Clones in which only Fringe activity is altered adopt signaling properties of the opposite compartment and displace the signaling center relative to the endogenous compartment boundary. In wild-type discs, symmetric activation of Notch and its targets leads to symmetric growth of D and V compartments. If growth is symmetric with respect to the displaced signaling center, the clone could be pushed into the opposite compartment by growth of the surrounding tissue (Milán, 2003).

At first glance, differential growth might explain how cells could be pushed to the interface between compartments. Can the model presented in the preceding section explain why some dorsal fringe mutant clones become able to mix with cells of the opposite compartment? Notch is not activated in V cells adjacent to fringe mutant clones abutting the boundary. The model presented here suggests that these cells would become V instead of V+N*; hence, there would not be a sustained affinity difference between fringe mutant D cell and the adjacent V cells. This may explain why fringe mutant D cells can sometimes mix with V cells when they are pushed into the V compartment. A similar case can be made to explain how V cells expressing Fringe can be pushed into the D compartment and mix with D cells. In both situations, it is noted that these clones form smooth borders with the cells of the compartment of origin, suggesting symmetric growth induced by Notch may contribute to the smoothness of the affinity boundary. This type of 'pushing' mechanism provides a useful explanation for the behavior of clones of cells that contact the DV boundary. It is noted that the behavior of cells expressing Apterous and Fringe was not the same when the entire P compartment was involved. P cells of ventral origin expressing Apterous were able to sort into the dorsal posterior quadrant, but cells expressing Fringe were not. It is suggested that this reflects an underlying difference between cells that have acquired a fully dorsal affinity state from those in which only the signaling properties have been altered. Fringe activity clearly plays an important role in the maintaining the segregation of D and V cells, but it is not the sole mediator of Apterous activity in this process (Milán, 2003).

A Notch/Delta-dependent relay mechanism establishes anterior-posterior polarity in Drosophila

The anterior-posterior axis of Drosophila becomes polarized early in oogenesis, when the oocyte moves to the posterior of the germline cyst because it preferentially adheres to posterior follicle cells. The source of this asymmetry is unclear, however, since anterior and posterior follicle cells are equivalent until midoogenesis, when Gurken signaling from the oocyte induces posterior fate. Asymmetry is shown to arise because each cyst polarizes the next cyst through a series of posterior to anterior inductions. Delta signaling from the older cyst induces the anterior polar follicle cells, the anterior polar cells signal through the JAK/STAT pathway to induce the formation of the stalk between adjacent cysts, and the stalk polarizes the younger anterior cyst by inducing the shape change and preferential adhesion that positions the oocyte at the posterior. The anterior-posterior axis is therefore established by a relay mechanism, which propagates polarity from one cyst to the next (Torres, 2003).

The follicle stem cells reside in region 2b of the germarium and give rise to two distinct lineages: the epithelial follicle cell precursors, which proliferate until stage 6 to generate most of the cells that surround each cyst, and the polar/stalk precursors. The latter exit mitosis at stage 1 to 2 of oogenesis and give rise to the symmetric pairs of polar cells at the anterior and posterior poles of the cyst and to the stalk that separates each cyst from the adjacent one. Delta mutant germline clones and Notch follicle cell clones fail to form polar cells, indicating that Delta signals from the germline to activate the Notch receptor in the polar/stalk precursors to induce them to adopt the polar cell fate. This induction requires fringe, which is upregulated in the polar/stalk precursors and renders these precursors competent to respond to the Delta signal. Once the polar cells are specified, they express Unpaired, the ligand for the JAK/STAT pathway, and the resultant activation of JAK/STAT signaling plays two key roles in patterning the rest of the follicle cells. (1) The polar cells induce uncommitted polar/stalk cell precursors to become stalk cells. Overexpression of Unpaired causes all polar/stalk cell precursors to differentiate as stalk, whereas loss-of-function mutations in hopscotch (JAK) or STAT92E cause a loss of the stalk. (2) Unpaired signaling from the polar cells induces the adjacent epithelial follicle cells at each pole of the egg chamber to adopt a terminal fate. This induction is essential for axis formation because only the terminal cells are competent to respond to Gurken by becoming posterior. Unpaired also acts as a morphogen to specify three distinct terminal cell types at the anterior: the border cells, the stretched follicle cells, and the centripetal cells. In the absence of Gurken signaling, all three cell types also form at the posterior of the egg chamber, indicating that the graded activity of JAK/STAT pathway creates a symmetric prepattern at both poles (Torres, 2003 and references therein).

Establishment of a distinct boundary of cells that express fng and those that do not is not necessary in lgd mutant wing discs

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule Fringe (Fng). The activity domain will be established symmetrically at each side of the boundary between Fng-expressing and non-expressing cells. Evidence is presented that the Drosophila tumor-suppressor gene lethal giant discs (lgd), a gene whose coding region has yet to be identified, is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development (Klein, 2003).

It has also been observed that wingless (wg) is expressed ectopically in the pouch of lgd mutants during wing development. Similar phenotypes are observed, if the Notch pathway is ectopically activated during wing development, raising the possibility that the lgd mutant phenotype could stem from the ectopic activation of the Notch pathway. The Notch pathway is indeed ectopically active in lgd mutants, and hyperactivation as well as ectopic activation of the pathway accounts for the lgd phenotype during wing development. In lgd mutants, the expression of Notch target genes along the D/V boundary is expanded, indicating that Lgd is required for the restriction of Notch activity to the D/V boundary. Furthermore, the mutant phenotype of lgd is suppressed by concomitant loss of Presenilin or Suppressor of Hairless function, indicating that the mutant phenotype is caused by the activation of the Notch pathway. Evidence is provided that the activity of fng and Serrate seem to be dispensable in lgd mutant wing disc and that Delta can activate Notch efficiently enough to maintain its activity during wing development. The presented results indicate that the negative regulation of Notch by Lgd is not restricted to wing development and occurs during several other developmental processes, such as vein, eye, and bristle development, suggesting that Lgd suppresses the activity of the Notch pathway in a variety of developmental processes (Klein, 2003).

Loss of lgd function leads to an overgrowth of the imaginal discs, clearly noticeable in the wing region of the wing disc, which becomes enlarged and flat (Bryant, 1971). wg expression is normally restricted to the D/V boundary of the wing pouch. In lgd mutants, wg is activated ectopically in a much broader domain that extends into the wing pouch. In addition, lgd mutant wing discs often develop a second wing pouch in the region of the anlage of the scutellum. Similar phenotypes are caused by gain-of-function alleles of N (for example, Abruptex) and are also observed upon expression of the activated intracellular form of Notch, Nintra, or expression of Notch ligands, such as Dl. The ectopic activation of wg can already be observed in early third instar wing discs and precedes the visible morphological changes that occur at later stages. The deficiency Df(2L) FCK-20 deletes the lgd locus, allowing the classification of the relative strength of the available alleles. The phenotype is always variable, but the overall phenotype of lgdd7 and lgdd10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating that these two alleles are strong, probably amorphic alleles. lgdd4 and lgdd1 are weaker alleles. All alleles display a qualitatively similar phenotype over the deficiency as in homozygotes, indicating that the observed phenotype is probably caused by the loss-of-function of the lgd gene (Klein, 2003).

The similarity between the loss of lgd function and ectopic N activation suggests that the phenotype of lgd could be caused by ectopic activation of the Notch pathway. To examine this possibility, the expression of E(spl)m8, cut, Dl, and Ser was monitored as well as the activity of the vg-boundary enhancer (vgBE) in mutant wing discs. The expression of all these markers is initiated in cells at the D/V boundary in a Notch-dependent manner. The vgBE is initially expressed along the D/V boundary of the wing, but late in the third instar, it is activated in an additional stripe along the anteroposterior compartment boundary (A/P boundary), which is also dependent on Notch activity. Both domains depend on the presence of a single Su(H) binding site in the enhancer. Similarly, the expression of cut and E(spl)m8 is initiated in cells at the boundary by the Notch-pathway, and E(spl)m8 is also dependent on the presence of Su(H) binding sites in its promoter. As described above, the expression of Dl and Ser is more complex but always dependent on the activity of Notch in cells at the D/V boundary. In lgd mutant wing discs, the vgBE as well as cut, Dl, Ser, and E(spl)m8 are activated ectopically within the wing pouch. The activation of the vgBE is dependent on the presence of the Su(H) binding site in the enhancer, since a version lacking it shows no ectopic activity in the mutants. As in the case of wg, the expression of the vgBE is already expanded in early third larval wing discs. Altogether, these results show that the loss of lgd function leads to the ectopic expression of Notch target genes. This suggests that the Notch pathway is ectopically activated in lgd mutants (Klein, 2003).

All tested Notch-target genes are ectopically activated in lgd mutant wing discs or lgd mutant cell clones. The ectopic activation of Notch target genes as well as the observed overproliferation of lgd mutants is abolished in lgd;Psn double mutants. In addition, Notch target gene expression is also abolished in Psn or Su(H) mutant clones generated in lgd mutant wing imaginal discs. These data suggest that the Notch pathway becomes ectopically active in the absence of lgd function. Furthermore, the fact that Delta alone seems to provide sufficient Notch activity to sustain wing development in lgd mutants indicates that the pathway can be activated more efficiently in the mutant background. The activation of Notch is a consequence of loss of lgd function also in other developmental processes, such as bristle, leg, and wing vein development. Thus, the presented data make lgd a good candidate gene that regulates activity of the Notch pathway during adult development of Drosophila (Klein, 2003).

The activation of the Notch pathway in the wing along the D/V boundary depends on the presence of a boundary between cells that express and cells that do not express the Fng protein. Consistent with this model, expression of UASfng with ptcGal4 interrupts the expression of Notch-dependent genes along the D/V boundary and induces a new domain of expression along the posterior end of the ptc domain, where cells expressing high levels of Fng are juxtaposed to nonexpressing cells. In contrast, performing the same experiment in lgd mutant discs, Fng does not interrupt the expression of wg at the D/V boundary. This raises the possibility that establishment of a distinct boundary of cells that express fng and those that do not is not necessary in lgd mutant wing discs. To further confirm this conclusion, UAS fng was expressed throughout the wing blade with sdGal4 to remove a sharp expression boundary of fng throughout wing development. Expression of UASfng in this way during normal development results in the loss of the wing blade and distal hinge. However, in lgd mutant discs, the expression of UAS fng by sdGal4 has little effect on wing development, and the disc develops a wing blade similar to that of lgd mutants. This result supports the conclusion that a sharp boundary between fng-expressing and nonexpressing cells is not required in lgd mutant wing discs for wing development. To find more evidence for this conclusion, fng13 mutant clones were induced in lgd mutant wing discs. Dorsal clones induced by sdGal4 UAS FLP in wild type wing discs led to the ectopic activation of the Notch pathway and the activation of wg expression at the clone boundaries. Mutant clones located in the ventral half of the pouch have no effect since fng is not expressed there during early development, and hence no ectopic boundary of fng-expressing and nonexpressing cells is generated. In lgd mutant wing discs, fng mutant clones, which do not include the D/V boundary, behave like the clones in wild type discs and wg expression is activated at the clonal boundaries in the dorsal half of the blade. However, unlike in the wild type, dorsal clones that are located within the expanded expression domain lead only to a weakening of wg expression in the center of the clone but do not result in a loss of wg expression, as in the wild type. This result suggests that, in lgd mutant wing pouches, wg expression can be induced by Notch in the absence of Fng. Furthermore, clones that cross the D/V boundary do not lead to an interruption of wg expression at the D/V boundary within the mutant area, and clones that include parts of the ventral half of the expanded domain do not affect Wg expression at all, indicating that Fng has no function in the regulation of the ventral half of the expanded domain of Notch target genes. Altogether, the clonal analysis of fng13 confirms that, in the absence of lgd, a boundary of fng-expressing and nonexpressing cells is not necessary for activation of Notch. Nevertheless, an ectopic boundary of Fng-expressing and nonexpressing cells can activate Notch (Klein, 2003).

Fringe is required for polar cell fate during oogenesis: Pole cells act as an organizers that pattern surrounding follicle cells and establish the A/P axis of the oocyte

fringe encodes a glycosyltransferase that modulates the ability of the Notch receptor to be activated by its ligands. fringe functions during early stages of Drosophila oogenesis. Animals mutant for hypomorphic alleles of fringe contain follicles with an incorrect number of germline cells, which are separated by abnormally long and disorganized stalks. Analysis of clones of somatic cells mutant for a null allele of fringe has localized the requirement for fringe in follicle formation to the polar cells, and demonstrates that fringe is required for polar cell fate. Clones of cells mutant for Notch also lack polar cells and the requirement for Notch in follicle formation appears to map to the polar cells. Ectopic expression of fringe or of an activated form of Notch can generate an extra polar cell. These results indicate that fringe plays a key role in positioning Notch activation during early oogenesis, and establish a function for the polar cells in separating germline cysts into individual follicles (Grammont, 2001).

In wild-type ovaries, each follicle contains 16 germline cells. In stronger fng mutants, follicles often contain an abnormal number of germline cells. In most cases (31/36), these abnormal follicles include more than 16 germline cells, although occasional examples (5/36) of follicles containing less than 16 germline cells were found. Follicles with multiples of 16 germline cells contain extra oocytes. Follicles that contain less than 16 germline cells are always adjacent to follicles that contain more than 16 germline cells, with the total number of germline cells in adjacent follicles being a multiple of 16. Along the length of the entire ovariole, the oocyte:nurse cell ratio is normal (1:15). Phalloidin staining reveals that all of the germline cells are connected by normal ring canals. Together, these observations indicate that the presence of abnormal numbers of germline cells derives from defects in the enclosure of germline cysts by somatic cells (Grammont, 2001).

In wild-type ovaries, a single column of four to six cells separates adjacent follicles. In fng mutant ovaries, long stalks can form that contain, on average, 10 cells. Thirteen of 58 stalks examined exhibit an additional defect, in which the stalk cells are not correctly organized. Instead of forming a single column of cells, they form clusters. Thus, fng influences both the number and the organization of stalk cells. In wild type, follicle cells form an even, single-layered columnar epithelium around the germline cells. By contrast, in fng mutant ovaries the posterior follicular cells can form extra cell layers. This defect is most obvious in stage 9 and later follicles, but can also be detected earlier. Thus, fng is required to maintain a monolayer of epithelial cells around the oocyte (Grammont, 2001).

Although some aspects of fng expression during oogenesis have been described previously, a more detailed description is presented here. fng expression is first detected in the germarium. This staining appears in some germaria as a solid block in somatic cells throughout region II, while in others it appears as a broad stripe throughout region IIa, absence of staining in region IIb, and a thin stripe of staining between regions IIb and III. Oogenesis is a dynamic process in which germline cysts move towards the posterior of the germarium as they mature. The variability in fng expression presumably reflects this dynamism, and derives from individual germaria having been frozen by fixation at different points of follicle development. fng expression along the IIb-III border corresponds to the somatic cells that invaginate between and separate adjacent cysts. At the posterior end of a stage 1 follicle (region III of the germarium), fng expression appears to be restricted to the polar cells and stalk cells. Between stage 2 and 3 follicles, fng expression is detected in the polar and stalk cells. From stages 3 to 6 of oogenesis, fng expression is restricted to the polar cells (Grammont, 2001).

Cell-lineage studies have revealed that somatic cells in the germarium become subdivided into two groups: the precursors of the polar and stalk cells, and the precursors of the main-body follicle cells. The molecular basis for this subdivision is unknown, and it has not been possible to distinguish the two groups until after follicles exit the germarium. However, the studies presented here reveal that fng is expressed in a group of cells along the border between regions IIb and III of the germarium. As this corresponds to a group of cells that separate cysts, and fng is expressed exclusively in the polar and stalk cells from the earliest time at which they are morphologically recognizable (between stage 1 and 2 follicles), it is suggested that this stripe of fng expression in the germarium includes the progeny of polar-stalk precursors. Although this would make fng the earliest known gene whose expression is largely restricted to polar-stalk cells, the observation that fng- cells can be recovered in stalks at later stages of development indicates that fng is not actually required to specify this lineage (Grammont, 2001).

However, fng is required autonomously to specify polar cell fate. The observations that fng expression becomes restricted to the polar cells, and that overexpression of fng can generate an extra polar cell, further imply that fng is a key player in polar cell specification. Nonetheless, other factors must also contribute to polar cell specification, because fng is expressed in a larger group of precursors within the germarium, and because overexpression of fng in most cases can generate only a single extra polar cell (Grammont, 2001).

In genetic mosaics, the requirement for fng in follicle formation maps to the polar cells. Analysis of fng mutant clones thus identifies key requirements for the polar cells in enclosing and separating germline cysts. However, the phenotypes observed depend not just on the presence of polar cells in a single follicle, but also on the presence of polar cells in the adjacent follicle. The main-body follicle cells of each egg chamber are separated from the next by a polar-stalk unit consisting of a pair of polar cells, a stalk, and another pair of polar cells. Since more than one polar-stalk precursor participates in the formation of each unit, a polar-stalk unit could be mosaic for fng function in these experiments. When only one pair of polar cells forms, adjacent follicles are still separated. However, one of two morphological defects occurs. In some cases, the egg chamber without polar cells appears open, with a gap in the epithelium. In other cases, stalkless follicles form, separated by a bilayered epithelium. Notably, the observation that normal egg chamber formation actually depends on the specification of two pairs of polar cells per polar-stalk unit implies that polar and stalk cells acquire distinct fates prior to follicle formation (Grammont, 2001).

More severe phenotypes are observed when all of the cells in a polar-stalk unit are fng-, and no polar cells can form. In most cases, this results in the formation of a compound follicle. The compound follicle ends whenever polar cells form. Moreover, normal follicles appear to form as long as fng+ polar cells form, even if all other cells are fng-. These observations imply that the polar cells have the capacity to recognize distinct cysts, to migrate in between them, and to separate them. Although in some cases adjacent cysts can be separated even without any polar cells, this separation is composed of only a monolayer of epithelial cells. Thus, the occasional ability of the main-body follicular cells to migrate between cysts is not sufficient to generate independent follicles (Grammont, 2001).

Several other genes, including brainiac, egghead, toucan, daughterless, hedgehog and cut, have been identified as playing important roles in the packaging of cysts into separate follicles. hedgehog is required for the normal proliferation of somatic cells in the germarium, so it is possible that hedgehog mutation leads to the formation of compound follicles because of a decrease in the number of polar-stalk precursors. Three of these genes, brainiac, egghead and toucan, are required in the germline. Determination of their roles may provide new insights into the interactions between germline and somatic cells required to organize a follicle (Grammont, 2001).

fng is also required for stalk formation, and this requirement maps in genetic mosaics to the polar-stalk lineage. However, the requirement is complex in two respects. (1) The influence of fng on stalk formation is non-autonomous, and appears to map largely to the polar cells. In the complete absence of polar cells, no stalk forms, while stalks that are composed almost entirely of fng mutant cells can form as long as wild-type polar cells exist at each end. Moreover, stalks exhibit more severe defects whenever polar cells at one end are missing. Even in cases where abnormally long stalks have fng+ polar cells at each end, it is conceivable that the long stalk phenotype relates to a requirement for fng in the polar cells. That is, it is hypothesized that fng- cells that would ordinarily have been fated to be polar cells may instead be incorporated into the stalk, and that polar-stalk precursors may continue to join the stalk until fng+ polar cells arrive (Grammont, 2001).

(2) In some cases fng mutant clones result in stalkless follicles, but in other cases they result in long stalks. This observation, together with the analysis of Notch mutant clones, indicates that there is no direct correlation between stalk length and Notch activity. Instead, it is suggested that the number, timing and/or location of polar cells within the polar-stalk lineage at the time of stalk formation is crucial to stalk organization (Grammont, 2001).

The phenotypes observed in fng mutant clones are also observed in Notch mutant clones. Moreover, as for fng, the requirements for Notch appear to map to the polar cells. While these results confirm that Notch plays important roles in separating follicles and organizing stalks, they overturn previous conclusions about the nature of this requirement. Most notably, it had been suggested that loss of Notch function leads to extra polar cells. Although after stage 8 Fas III becomes expressed in Notch mutant cells, this occurs regardless of their location and in the absence of other aspects of polar cell fate. Instead, the results described here imply that Notch is required for polar cell specification, and that Notch is required only in these cells for the separation of germline cysts into distinct follicles. Furthermore, the specific expression of E(spl)mß-lacZ in the polar cells confirms that Notch is activated within these cells, and the induction of extra polar cells by activated Notch demonstrates that the activation of Notch can be sufficient to specify polar cells within a competent subpopulation of somatic cells (Grammont, 2001).

In the Drosophila wing and eye, as well as in cultured mammalian cells, fng has been shown to potentiate the activation of Notch by Delta and to inhibit the activation of Notch by Serrate. Notch, Serrate and Delta all appear to be expressed ubiquitously in follicle cells throughout the germarium. In many circumstances, expression of Notch ligands can exert an autonomous inhibition of Notch activation. It has been hypothesized that fng may potentiate Delta signaling by allowing Notch activation within ligand-expressing cells. Similarly, it is proposed that in the germarium, fng expression overrides autonomous inhibition to allow Notch activation by Delta within ligand-expressing cells, thereby positioning Notch activation to a subset of cells (the polar cells) within a broad domain of Notch and ligand expression. This is consistent with the observation that Notch and fng are both positively required within polar cells for them to adopt their fate, and that fng expression soon becomes restricted to these cells (Grammont, 2001).

Although fng is expressed in a discrete pattern that correlates with its genetic requirements, ectopic expression of fng during oogenesis has surprisingly little effect. This contrasts with the very dramatic effects on Notch activation associated with fng mis-expression during imaginal development. The limited consequences of fng mis-expression are also surprising in light of the observation that mirror (mirr) mutant ovaries have compound follicles. It has been hypothesized that the mirr phenotype derives from de-repression of fng transcription. mirr expression is complementary to fng, and ectopic expression of mirr represses fng expression and generates phenotypes similar to those observed in fng mutants. It is suggested that the simplest resolution to this discrepancy would be the existence of co-factors that are required for fng function and are co-regulated by mirr during oogenesis. Since Fng is involved in the synthesis of an O-linked tetrasaccharide, the enzymes that are responsible for catalyzing other steps in its synthesis are candidate Fng co-factors, and it will be interesting to determine whether they too are expressed in discrete patterns (Grammont, 2001).

Patterning of the Drosophila egg requires the establishment of several distinct types of somatic follicle cells, as well as interactions between these follicle cells and the oocyte. The polar cells occupy the termini of the follicle and are specified by the activation of Notch. Their role in follicle patterning has been investigated by creating clones of cells mutant for the Notch modulator fringe. In the absence of fng or Notch function, polar cells do not form, and the requirement for these genes in polar cell fate is strictly cell autonomous. This genetic ablation of polar cells results in cell fate defects within surrounding follicle cells. At the anterior, the border cells (the immediately adjacent follicle cell fate) are absent, as are the more distant stretched and centripetal follicle cells. Conversely, increasing the number of polar cells by expressing an activated form of the Notch receptor increases the number of border cells. At the posterior, elimination of polar cells results in abnormal oocyte localization. Moreover, when polar cells are mislocalized laterally, the surrounding follicle cells adopt a posterior fate, the oocyte is located adjacent to them, and the anteroposterior axis of the oocyte is re-oriented with respect to the ectopic polar cells. These observations demonstrate that the polar cells act as an organizer that patterns surrounding follicle cells and establishes the anteroposterior axis of the oocyte. The origin of asymmetry during Drosophila development can thus be traced back to the specification of the polar cells during early oogenesis. Only one gene, upd, is known that encodes for a signaling molecule that is expressed by polar cells. Although loss of upd, or other components of the JAK-STAT pathway, reduces the number of border cells, this contrasts markedly with the complete elimination of border cells observed in the absence of polar cells. Moreover, loss of upd does not have obvious effects on any of the other terminal cell fates that are polar-cell dependent. Thus, the existence of additional signaling molecules must be invoked to account for the organizing activity of the polar cells (Grammont, 2002).

Posterior terminal cells have two crucial functions during oogenesis: to localize the oocyte to the posterior of the follicle at stage 1, and to establish the developmental axes of the oocyte at stage 6. The posterior localization of the oocyte is achieved through differential adhesion: the oocyte expresses higher levels of E-cadherin than do other germline cells, and the posterior follicular cells express higher levels of E-cadherin than do central follicular cells. Homophilic adhesion mediated by E-cadherin then maintains the oocyte at the posterior of the follicle. Mosaic analysis has demonstrated that the requirement for E-cadherin maps to posterior terminal cells, but not specifically to the polar cells. To examine the role of the polar cells in oocyte localization, fng mosaic follicles that lacked posterior polar cells were sought (Grammont, 2002).

Four fng mosaic follicles without posterior polar cells were identified, and in all cases the oocyte was abnormally localized to the anterior of the follicle, in contact with the anterior polar cells. The anterior terminal cells also expressed elevated levels of E-cadherin, and posterior clones mutant for E-cadherin similarly resulted in mislocalization of the oocyte to the anterior. Importantly, fng mosaics in which only the two polar cells were fng+, and all other posterior cells were mutant, formed wild-type follicles with a posteriorly located oocyte (four examples). Together, these observations localize the requirement for fng in oocyte positioning to the polar cells, and demonstrate that posterior follicle cells are unable to localize the oocyte in the absence of polar cells (Grammont, 2002).

To determine whether a polar cell-dependent terminal cell fate is required for the initial asymmetric localization of the oocyte within the follicle, or only for its maintenance, fng mosaics were examined at stage 1, when the oocyte first becomes localized to the posterior. Since no markers are available for polar cells at this stage, stage 1 follicles surrounded entirely by fng- cells, which can not form polar cells, were sought. Ten fng- stage 1 follicles were found, and the oocyte was mislocalized in all ten. Thus, the dependence of oocyte positioning on fng begins during follicle formation and not later, during follicle maturation. Because Fng acts cell-autonomously to specify polar cell fate, and is later not required in any other follicle cells for oocyte localization, these observations imply that the polar cells have an essential role during early oogenesis in oocyte localization, well before they become recognizable through their expression of specific molecular markers (Grammont, 2002).

In 33 cases, fng mosaic follicles were recovered that lacked posterior polar cells, but instead had two polar cells along the lateral side of the follicle. Although it is not understood how these abnormally constructed follicles arise, they presented an opportunity to investigate the ability of ectopic polar cells to control the position of the oocyte. In all cases, the oocyte was in contact with these mislocalized polar cells rather than at the posterior or anterior termini of the follicle. Since prior studies have ruled out any role for the oocyte in the induction of polar cell fate and for the polar cells in the specification of the oocyte, it is concluded from the co-localization of the oocyte and the polar cells that the polar cells are not only necessary but also sufficient to direct the localization of the oocyte within the follicle (Grammont, 2002).

When follicles lack normal anterior as well as posterior polar cells, they tend to be round, or even elongated perpendicular to the axis of the ovariole. This phenotype is reminiscent of that of Leucocyte antigen related (Lar) mutations. Lar encodes for a receptor-like tyrosine phosphatase, which is required for epithelial planar polarity during oogenesis. These observations are thus consistent with a hypothesized role for the polar cells in a Lar-dependent reorganization of actin filaments that influences follicle elongation (Grammont, 2002).

Posterior terminal cells also have a second crucial function during oogenesis, in establishing the anteroposterior and dorsoventral axes of the egg through reciprocal signaling with the oocyte. Prior to stage 6, a microtubule organizing center (MTOC) is located at the posterior of the oocyte, resulting in a network of microtubules with their minus ends at the posterior of the oocyte and their plus ends at the anterior of the follicle. At stage 6, the oocyte signals to the follicle cells through the EGFR ligand Gurken. This signal represses anterior terminal follicle fate and establishes posterior terminal follicle fate. The posterior follicular cells then send back an unknown signal to the oocyte that inactivates the existing MTOC. In parallel, a new microtubule network is established with the minus ends of the microtubules at the anterior of the oocyte and the plus ends at the posterior. This new microtubule network is essential for the correct localization of the anterior, posterior and dorsal determinants within the oocyte, and consequently for the later establishment of embryo polarity. The posterior terminal cells also express specific genes or markers of posterior identity, such as the pointed gene (Grammont, 2002).

Prior studies have established that only posterior terminal cells, and not central follicle cells, are competent to express these genes and to signal back and inactivate the first MTOC in response to the Gurken signal. These studies also show that this competence does not depend on signaling from the oocyte. Although this competence maps to posterior terminal cells and not specifically to polar cells, it is hypothesized that the distinct behavior of the posterior terminal cells could nonetheless be established by signaling from the polar cells. It is not possible to examine the fate of posterior cells or the microtubule network in oocytes without posterior polar cells, because in such mosaics the oocyte simply relocalizes to the anterior. Thus, to address the question of whether the polar cells can induce a terminal fate in neighboring follicular cells, fng mosaic follicles were analyzed that lacked posterior polar cells and instead possessed two lateral polar cells. The oocyte localizes to the side of the follicle in these cases (Grammont, 2002).

In 7/7 fng follicles with lateral polar cells that carried an enhancer trap in the pointed gene (pnt-lacZS99812), ß-galactosidase staining was observed in follicle cells surrounding the ectopic polar cells. Thus, ectopic polar cells induce a competence in neighboring cells to respond to the Gurken signal by expressing a posterior follicle marker. If these cells are fully functional posterior cells, they should also be able to signal back to the oocyte to inactivate the first MTOC. To examine the polarity of the microtubule cytoskeleton, advantage was taken of nod:lacZ and kin:lacZ reporter constructs. These constructs fuse the minus end directed motor Nod or the plus-end-directed motor kinesin to ß-galactosidase, and consequently they serve as reporters of the minus and plus ends of microtubules. In seven out of seven fng follicles with lateral polar cells that carried the kin:lacZ marker, ß-galactosidase staining was observed where the oocyte contacts follicle, near the polar cells. In seven out of seven fng follicles with lateral polar cells that carried the nod:lacZ marker, ß-galactosidase staining was observed where the oocyte contacts the nurse cells, far from the polar cells. Thus, in these lateral oocytes the microtubule cytoskeleton is oriented perpendicular to the normal AP axis of the follicle, but is nonetheless correctly established with respect to the polar cells. It is concluded from this that the follicle cells surrounding these lateral polar cells have been instructed by the polar cells to adopt a terminal follicular fate that renders them competent to adopt a posterior fate in response to Gurken signaling from the oocyte (Grammont, 2002).

The possibility that the polar cells influence the fate of surrounding cells has been suggested previously, however, it was not possible to establish a definitive link between the function of the polar cells and the fates of surrounding cells, because it was not possible to eliminate or specifically manipulate the polar cells. At the anterior of the follicle, each of the three distinct cell types that surround the polar cells (border cells, stretched cells, and centripetal cells) fails to form in the absence of polar cells. Instead, these cells appear to adopt the fate of central follicle cells; that is, to migrate over the nurse cells towards and around the oocyte, leaving the anterior germ cells uncovered. The conclusion that the polar cells serve as organizers of follicle patterning is also supported by the ability of additional or mispositioned polar cells to redirect the fates of neighboring cells. When the number of anterior polar cells is increased by expression of an activated form of the Notch receptor, the number of border cells is increased, and this increase occurs in proportion to the number of extra polar cells. Similarly, activation of the HH pathway can result in both extra polar cells and a corresponding increase in border cells (Grammont, 2002).

In contrast to the anterior terminal cells, the posterior terminal cells do not exhibit obvious differences in morphology or behavior from central follicle cells. Nor are distinct molecular markers available, because all of the known posterior-specific genes are also targets of EGFR signaling from the oocyte; EGFR signaling cannot occur in the absence of polar cells due to the mislocalization of the oocyte. However, fng mosaic follicles are sometimes abnormally constructed such that polar cells are formed along the sides of the follicle, rather than at the posterior. These ectopic polar cells are sufficient to confer to the neighboring cells a posterior identity, which then directs the reorganization of the oocyte cytoskeleton (Grammont, 2002).

Thus, it is concluded that polar cells are both necessary and sufficient to direct the fates of surrounding cells. The polar cells exhibit the hallmarks of an organizer because they not only influence the fates of surrounding cells, but they establish distinct cell fates at different distances, and they can redirect the fates of surrounding cells at ectopic locations. Although the induction of distinct fates at different distances normally only occurs at the anterior of the follicle, the capacity to establish these distinct fates also exists at the posterior, but is suppressed there by EGFR signaling. The observations that distinct cell fates arise in rings at discrete distances from the polar cells at both poles in the absence of EGFR signaling points to the polar cells as a potential source of a signal that directs terminal fate. The current analysis confirms this hypothesis and leads to an understanding of terminal patterning as a two-step process in which cells at each end of the follicle first receive identical polar-cell signals that distinguish terminal from central follicle cell fates, and then later GRK signaling from the oocyte represses anterior fates and promotes posterior fate (Grammont, 2002).

Although more complex models are possible, two basic mechanisms for organizer activity are a morphogen or a signal relay. In the morphogen model, a signal would be produced by polar cells and then spread to all of the cells whose fate is polar-cell dependent. The signal would exist in a concentration gradient, with different amounts of the signal being required to induce the distinct border cell, stretched cell, and centripetal cell fates. In the signal relay model, the polar cells would produce a short range signal that induces border cells, which would then produce a second signal that induces stretched cells, which would, in turn, then express a third signal that induces centripetal cells. Although these models can not be definitively distinguished until the polar cell signal(s) are identified, several observations together suggest that a combination of the morphogen and signal relay mechanisms are actually employed (Grammont, 2002).

In support of a single long-range signal, reduction in the number of border cells by mutation of components of the JAK-STAT pathway does not result in any obvious reduction of the number of stretched or centripetal cells. Nor does ablation of the border cells by expression of a toxic protein exert obvious effects on stretched or centripetal cell fate. Thus, the establishment of more distant polar-cell dependent cell fates does not require the establishment of intervening cell fates. Conversely, however, the determination that the specification of the centripetal cells depends in part upon DPP signaling from the stretched cells supports the idea of a signal relay from the stretched to the centripetal cells (Grammont, 2002).

Further insight into the nature of the polar cell signal may be gleaned from the time and distance over which it acts. Polar cell signaling must occur at the anterior and at the posterior of the follicle prior to stage six, when terminal cells are required to confer a distinct responsiveness to EGF-R signaling from the oocyte. Additionally, recent observations suggest that the polar cells behave as polarization centers with respect to the organization of F-actin within the follicular cells; F-actin gradually aligns from the poles to the center of the follicle during stages 5-7. This phenomenon suggests that a signal should exist from the polar cells even before the end of follicle cell proliferation at stage 5 (Grammont, 2002).

The anteroposterior and dorsoventral axes of Drosophila are established during oogenesis by localized determinants. These consist of mRNAs for bcd and nanos localized, at the anterior and the posterior pole for the AP axis, respectively, and mRNAs for grk around the oocyte nucleus for the DV axis. The localization of these mRNAs is dependent upon the establishment of the correct polarity of the microtubule cytoskeleton. Thus, prior work has made it possible to trace the establishment of both the AP and the DV axes of Drosophila back to the signaling process between oocyte and terminal follicle cells that regulates the oocyte cytoskeleton. This signaling further requires that the oocyte be correctly localized to the end of the follicle, which is dependent upon differential cadherin expression in the germarium (Grammont, 2002).

The observations made in this study now trace the origin of asymmetry back further, to the specification of the polar cells in the germarium, and their initial contact with the oocyte. As the germline cysts move from region IIb to region III of the germarium, the oocyte localizes to the posterior of the cyst. This localization is dependent upon the polar cells, presumably because of their ability to upregulate the expression of E-cadherin. Although the possibilities that the autonomous upregulation of E-cadherin effects oocyte localization, or that the non-autonomous upregulation induced by polar cell signaling also contributes to oocyte localization cannot be distinguished, in either case, oocyte localization and hence the initial AP asymmetry of the follicle, is established by the polar cells. All available evidence indicates that the anterior and posterior polar cells are equivalent, and the posterior localization of the oocyte is likely a consequence of the more advanced development of the posterior follicle cells surrounding region IIb cysts, which thus have the first opportunity to localize the oocyte (Grammont, 2002).

The localization of the oocyte at the posterior is then a necessary precondition for the second essential role of the polar cells in establishing oocyte polarity -- the promotion of terminal follicle cell fate. Terminal follicle cell fate then confers a distinct responsiveness to EGFR signaling from the oocyte; this is manifest in follicle cell ability to signal back to the oocyte to destroy the initial posterior MTOC. Destruction of the initial MTOC in turn allows establishment of the correctly polarized microtubule cytoskeleton that is necessary for the ultimate establishment of the AP and DV axes. Notably, the dual roles of the polar cells in initiating the establishment of the axes of the oocyte thus work in concert, since the localization of the oocyte to the posterior of the follicle by the polar cells places the oocyte in the correct position to participate in the later reciprocal signaling process with terminal follicle cells (Grammont, 2002).

Throughout Drosophila oogenesis, specialized somatic follicle cells perform crucial functions in egg chamber formation and in signaling between somatic and germline cells. In the ovary, at least three types of somatic follicle cells, polar cells, stalk cells and main body epithelial follicle cells, can be distinguished when egg chambers bud from the germarium. Although specification of these three somatic cell types is important for normal oogenesis and subsequent embryogenesis, the molecular basis for establishment of their cell fates is not completely understood. Studies reveal the gene eyes absent (eya) to be a key repressor of polar cell fate. Eya is a nuclear protein that is normally excluded from polar and stalk cells, and the absence of Eya is sufficient to cause epithelial follicle cells to develop as polar cells. Furthermore, ectopic expression of Eya is capable of suppressing normal polar cell fate and compromising the normal functions of polar cells, such as promotion of border cell migration. Finally, it has been shown that ectopic Hedgehog signaling, which is known to cause ectopic polar cell formation, does so by repressing eya expression in epithelial follicle cells (Bai, 2002).

One germline signal that is known to play a role in polar cell specification is Delta, which signals from the germline to Notch in the soma to control the differentiation of polar cells. Epithelial follicle cells do not respond to Delta in the same way, presumably because, unlike polar cells, they do not express fringe. fringe encodes a glucosyltransferase that potentiates the ability of the Notch receptor to be activated by its ligand, Delta. Mutation of either Notch or fringe leads to the disappearance of polar cells. As a result, Eya-negative cells are not found in the follicles. Mis-expression of either Fng or activated Notch produces ectopic polar cells only at the poles of the egg chamber, whereas loss of Eya can cause polar cells to form throughout the follicle epithelium. Thus Notch signaling appears to be necessary, but not sufficient to repress Eya expression and leads to polar cell formation. Surprisingly, activated Notch also can produce ectopic polar cells cell-nonautonomously at the poles of the egg chamber. The reason for this could be that activated Notch signaling might activate the expression of Delta, which, in turn, can activate Notch signaling in the adjacent cells (Bai, 2002).

Genetic interaction of Lobe with its modifiers in dorsoventral patterning and growth of the Drosophila eye

Dorsoventral (DV) patterning is essential for growth of the Drosophila eye. Recent studies suggest that ventral is the default state of the early eye, which depends on Lobe (L) function, and that the dorsal fate is established later by the expression of the dorsal selector gene pannier (pnr). However, the mechanisms of regulatory interactions between L and dorsal genes are not well understood. For studying the mechanisms of DV patterning in the early eye disc, a dominant modifier screen was performed to identify additional genes that interact with L. The criterion of the dominant interaction was either enhancement or suppression of the L ventral eye loss phenotype. Forty-eight modifiers were identified that corresponded to 16 genes, which included fringe (fng), a gene involved in ventral eye patterning, and members of both Hedgehog (Hh) and Decapentaplegic (Dpp) signaling pathways, which promote L function in the ventral eye. Interestingly, 29% of the modifiers (6 enhancers and 9 suppressors) identified either were known to interact genetically with pnr or were members of the Wingless (Wg) pathway, which acts downstream from pnr. The detailed analysis of genetic interactions revealed that pnr and L mutually antagonize each other during second instar of larval development to restrict their functional domains in the eye. This time window coincides with the emergence of pnr expression in the eye. These results suggest that L function is regulated by multiple signaling pathways and that the mutual antagonism between L and dorsal genes is crucial for balanced eye growth (Singh, 2005).

Axial patterning plays a crucial role in organizing growth and in differentiating developing fields. To understand how the DV pattern is established in the Drosophila eye, the genetic relationships between dorsal and ventral eye genes were analyzed. A group of new genes was identified that modifies the L mutant eye phenotype not only by misexpression but also by reduced gene function (Singh, 2005).

In the early eye disc, fng is preferentially expressed in the ventral eye. The DV domain specification by Fng is also important for growth of the eye disc as its ubiquitous overexpression in the eye disc blocks eye development. Even though L and fng play important roles during ventral eye growth and patterning, the developmental interaction between the two has been unknown. This study showed that overexpression of fng can partially compensate for the loss of L gene function in the eye. This suggests that fng works either downstream or parallel to L in the growth of the ventral eye. It is possible that L and fng interact through the induction of a common target, Ser, in the eye (Singh, 2005).

Like several other pathways, Wg signaling has multiple functions during eye development. This study identified Sgg, a serine/threonine kinase, as a modifier that suppresses the L mutant phenotype upon overexpression. Sgg is known to inhibit the Wg signaling pathway by downregulating Armadillo (Arm) via ubiquitin-mediated proteosomal degradation. Other components of the Wg signaling pathway such as pygo and dally were also identified as modifiers, which, upon overexpression in the eye, enhanced the L mutant phenotype. These results suggest that Wg signaling acts antagonistically to L function in the ventral eye. The genetic interaction of these EP lines with the L mutations represents specific enhancement rather than additive effects, since antagonists of Wg signaling were identified as suppressors, whereas members required for Wg signaling were identified as enhancers of the L mutant phenotype in the EP screen (Singh, 2005).

In this screen, it was found that the overexpression of Daughters against Dpp (Dad), an antagonist of Dpp signaling, enhances the L mutant phenotype, whereas EP insertions at hh and its receptor gene smo were identified as suppressors of the L mutant phenotype. The members of these two signaling pathways are known to be involved in eye growth and differentiation. These results raise another interesting possibility of the possible role of Hh and Dpp signaling pathways in early eye growth and patterning (Singh, 2005).

During Drosophila eye development, Hh controls progression of the furrow by inducing the expression of dpp and atonal (ato), a proneural gene responsible for R8 photoreceptor formation. In the eye, hh and dpp are involved in a positive feedback loop for the initiation and movement of the MF whereas Wg signaling acts antagonistically to Dpp signaling to block MF movement and progression. This antagonistic relation may be present even during early eye development since wg and dpp are localized to opposing regions of the undifferentiated younger eye primordia: dpp along the posterior margin and wg across the dorsal anterior region. These results suggest that the early function of Hh and Dpp signaling is to promote L-mediated ventral eye growth whereas Wg signaling acts as an antagonist (Singh, 2005).

BarH1 and BarH2 were identified as enhancers of the L mutant phenotype. BarH1 and BarH2 are a pair of homeobox proteins that express in a subset of photoreceptors and in the basal undifferentiated cells of the eye disc. B is required for the negative regulation of eye development by repressing the expression of the proneural gene ato. However, it is not known whether B plays a role in early eye growth, prior to retinal differentiation. Clonal analysis has not yet revealed evidence for B function in DV asymmetric eye patterning. However, the current data showed genetic interactions of L mutants with GOF and LOF mutants of B. The suppression of the L2/+ eye phenotype by a LOF mutation of B, which by itself has no defects in the eye, raises the possibility that B itself may not have DV asymmetric function but needs to be downregulated by L for normal growth of the early eye disc. This is also consistent with the dramatic eye reduction observed when ey-GAL4 drive B is overexpressed during early eye development. It has been shown that Wg and B have both positive and negative regulatory relationships in prepatterning of the notum. B expression is activated by Wg in the scutum whereas B represses Wg expression in the most anterior part of the notum. L mutants respond to GOF and LOF of both Wg signaling and B in a similar fashion, suggesting that Wg and B may be regulating each other positively during early eye development (Singh, 2005).

L is known to act downstream of N. In the eye, emc and h, the repressors of ato, are downregulated by N. During eye development, emc acts in collaboration with hairy (h) as the negative regulator of the morphogenetic furrow by repressing ato. Therefore, identification of emc as an antagonist of L-mediated early ventral eye growth seems possible. Interestingly, both emc and B have also been identified as modifiers of pnr (Singh, 2005).

Some of the genes that were identified as L modifiers, such as B, emc, and smo, have been well characterized, but their roles in early eye disc growth and/or DV asymmetric function have not been studied. Genes were identified involved in cell survival and growth such as disc over grown (dco), a member of the serine/threonine protein kinases family, and genes involved in vesicular trafficking, including RhoGAP68F, an ion transport such as nrv 1, and the acetyl transferase nej. It is possible that potential DV asymmetric function of these genes might have been missed by LOF analysis because of functional redundancy or these genes may be modifying the early growth function of L in the eye. More in-depth studies will be necessary to explore these possibilities. However, it is important to note that both GOF and LOF of these genes exhibit specific genetic interactions with L mutant backgrounds. In addition to the well-characterized genes, a few novel genes like EP1229 and EP1595 were identified whose functions are not known. These genes were not listed in this study as the specificity of their genetic interaction with L was not tested by using LOF mutations (Singh, 2005).

The results demonstrate that the level of pnr gene function is a crucial factor for DV patterning of the eye as increased levels of pnr gene function enhance the L mutant phenotype of ventral eye loss to no eye, whereas reduction of pnr gene function rescued the loss of the ventral eye phenotype of the L mutant. Further, the phenotypes of LOF clones of L where only the ventral cells are lost can be rescued by reducing the levels of pnr gene function. These results suggest that pnr acts antagonistically to the ventral eye growth function of L. However, it was also found that the antagonism of pnr and L is mutual. This conclusion is based on the fact that the gain-of-function phenotype of pnr in the eye is significantly enhanced when L function is reduced. These conclusions were also validated by showing that the dorsal eye enlargements associated with LOF clones of pnr can be prevented by reducing the levels of L gene function. These results suggest that optimal levels of pnr and L are necessary for DV patterning and growth of the eye. It was also found that the downstream dorsal eye selectors, Iro-C members (ara, caup, mirr) are involved in a mutually antagonistic relationship with L. These studies demonstrate that the antagonism of L holds true for key components involved in dorsal fate selection during early eye development (Singh, 2005).

The time window of the second instar of larval development was identified as the periord during which mutual antagonistic interaction of L and pnr is required for DV patterning and growth in the eye. Previously, it was shown that the pnr function in eye development is critically required during the second instar larval stage. This time window is coincident with the one that is required for the antagonism of pnr and L as shown in this study, suggesting that a major function of L in early eye development is to establish the DV domains by negatively regulating the dorsal selectors. These studies also support the physiological relevance of this mutually antagonistic interaction in DV patterning (Singh, 2005).

It is not known how L antagonizes Pnr function. One possibility is that L may be required for restricting the pnr expression domain to the dorsal margin of the eye disc. It was difficult to check whether L is cell-autonomously required for pnr repression because LOF clones of L result in the elimination of the entire or ventral eye, depending on the time when the clones are generated. Alternatively, the effect of a L mutation on pnr expression was studied. Interestingly, pnr expression, which is restricted to the dorsal eye margin in wild type eye discs, shows a nearly twofold expansion in L2/+ mutant discs. It remains to be studied whether L is required for the repression of pnr expression or for the inhibition of growth of pnr-expressing cells. On the basis of these data it is suggested that during early DV patterning, the onset of pnr expression might restrict the functional domain of L and Ser to the ventral eye. It is possible that pnr may also suppress L gene function via the Wg signaling pathway (Singh, 2005).

The results support the view that various developmental pathways cross-talk with each other to define the final form of a developing eye field. Such genes are likely to interact with both pnr and L. It is interesting to note that several pnr-interacting genes were identied as L modifiers in the screen. This illustrates the importance of the interaction of L and pnr pathways and also the efficacy of the screen. Further study of new modifiers of L may provide important clues to the mechanism of pnr-L interactions in the control of growth and/or DV patterning of the eye. Since the compound eye of Drosophila shares some similarities with the vertebrate eye and genetic machinery is highly conserved, it would be interesting to see if these antagonistic interactions between the dorsal eye selectors and the ventral eye genes play roles in the DV patterning and growth of vertebrate eyes (Singh, 2005).

Sequential Notch signalling at the boundary of fringe expressing and non-expressing cells

Wing development in Drosophila requires the activation of Wingless (Wg) in a small stripe along the boundary of Fringe (Fng) expressing and non-expressing cells (FB), which coincides with the dorso-ventral (D/V) boundary of the wing imaginal disc. The expression of Wg is induced by interactions between dorsal and ventral cells mediated by the Notch signalling pathway. It appears that mutual signalling from dorsal to ventral and ventral to dorsal cells by the Notch ligands Serrate (Ser) and Delta (Dl) respectively establishes a symmetric domain of Wg that straddles the D/V boundary. The directional signalling of these ligands requires the modification of Notch in dorsal cells by the glycosyltransferase Fng and is based on the restricted expression of the ligands with Ser expression to the dorsal and that of Dl to the ventral side of the wing anlage. In order to further investigate the mechanism of Notch signalling at the FB, the function of Fng, Ser and Dl was analyzed during wing development at an ectopic FB and at the D/V boundary. Notch signalling was found to be initiated in an asymmetric fashion on only one side of the FB. During this initial asymmetric phase, only one ligand is required, with Ser initiating Notch-signalling at the D/V and Dl at the ectopic FB. Furthermore, the analysis suggests that Fng has also a positive effect on Ser signalling. Because of these additional properties, differential expression of the ligands, which has been a prerequisite to restrict Notch activation to the FB in the current model, is not required to restrict Notch signalling to the FB (Troost, 2012).

During wing development, the activity of the Notch pathway is required to establish a stripe-like domain of expression of several genes along the D/V boundary that control wing growth and patterning, chief among them are wg and vg. The D/V boundary is a FB, which provides an interface that is crucial for the activation of Notch and establishment of this organising centre. The current understanding is that Fng promotes Dl signalling and prevents Ser signalling through the modification of Notch. As a result, Dl signals strongly from ventral to dorsal and Ser from dorsal to ventral cells boundary cells. The simultaneous signalling of the ligands in opposite direction establishes the expression of Notch target genes at both sides of the FB. It is essential for this model to work at the D/V boundary that induction of expression of Ser is restricted to dorsal and that of Dl to ventral cells. If e.g. Dl expression could also be induced in dorsal cells by the Notch pathway, the activity of Notch would immediately spread throughout the dorsal half of the wing anlage. In agreement with this requirement, it has been observed that expression of Ser is restricted to dorsal cells upon expression of activated Notch in dorsal and ventral cells. The combination of spatially restricted expression of the ligands and the Dl/Ser loop restricts the activation of Notch to the D/V boundary during early stages of wing development. At the middle of the third larval instar stage the Dl/Ser/Wg loop takes over to maintain the activity of Notch. Thus, a critical step is the establishment of expression of Wg in boundary cells. Once this is achieved the second feedback-loop assures expression of Wg and Notch signalling throughout wing development (Troost, 2012).

While this model can explain the events at the D/V boundary, it cannot explain the events at an ectopic FB, since differential expression of the ligands is unlikely to occur there. Nevertheless, the expression of Wg is also restricted at the ectopic FB. The presented work provides further evidence for the current model of Fng action, but adds new details that enable it to explain also the events at an ectopic FB. One addition is the initial sequential establishment of the expression domain of Wg through asymmetric Notch signalling during early stages of wing development. Asymmetric expression of Wg was observed only in ventral boundary cells (VBCs) at the D/V boundary, indicating that these cells achieve sufficiently high activation of Notch to initiate expression of Wg. The existence of the early asymmetric phase of Notch activation was surprising given the fact that activation of Notch results in the activation of the expression of Dl and Ser. Consequently, the activation of Notch in ventral cells should immediately lead to up-regulation of expression of Dl in ventral cells and back-signalling to dorsal cells. It was therefore expected that if an asymmetric phase exists, it would be too short in time to be detected. Importantly, the initial asymmetric phase appears to be a general property for Notch signalling at a FB, since it was observed on both analysed FBs. The existence of the asymmetric phase also indicates that a FB can be used to generate activity domains of the Notch pathway where the feedback-loop that regulates the expression of the ligands through Notch activation does not occur. So far the loop has been found only in the wing pouch. In the absence of the loop the asymmetric state would remain and thus, a defined stripe of high Notch activation would be generated in a field of cells that uniformly express Dl even in the absence of Ser (Troost, 2012).

At the ectopic FB, it was found that eliminating the activity of the Notch pathway in PBCs does not result in the loss of expression of Wg in ABCs. It only prevents the late symmetric phase. This indicates that establishment of a Dl/Ser feedback loop in A/P boundary cells is not essential for reaching sufficient high levels of Notch signalling to induce Wg expression during the asymmetric phase. The same was observed for the D/V boundary: Depletion of Su(H) function or over-expression of H causes a restriction of expression of Wg to VBCs, but not its abolishment. Thus, the Dl/Ser loop is probably mainly required for the later occurring patterning of the future wing margin, but not for the establishment of the wing primordium (Troost, 2012).

A further addition is that the basic expression of Dl is independent of Ser signalling. This is indicated by the observation that 1) Dl is expressed throughout the wing anlage in early discs and 2) Dl signals to DBCs at the D/V boundary in absence of Ser function. This holds true also for the ectopic FB: here Dl signals to the anterior boundary cells (ABCs) in the absence of Ser. However, in both cases Dl signalling is not strong enough to initiate Wg expression, which is a crucial event for wing development (Troost, 2012).

The results also reveal an unanticipated requirement of Ser in ABCs for the expression of Wg. This requirement could be explained through Ser signalling from the ABCs to PBCs to up-regulate Dl there, which in turn signals back to ABCs (Ser/Dl loop). This explanation would imply that the levels of Notch activation required for the induction of Dl expression are lower than that for Wg. Otherwise, the expression of Wg would not stay asymmetric as it is observed. However, it was found that Notch signalling is not required in PBCs during the early asymmetric phase. This excludes the mentioned explanation and suggests that Ser must activate Notch in the Fng expressing ABCs. This assumedly weak activation contributes to the total activity of Notch in these cells and guarantees levels of Notch signalling above the threshold required for expression of Wg. In the absence of Ser the activation by Dl from PBCs appears to fail to reach the threshold level in a fraction of discs. In agreement with this notion it has been shown that expression of Ser is broadly induced upon ectopic expression of Fng (Troost, 2012).

It was further observed that concomitant loss of Ser and Dl function in ABCs always abolishes the expression of Wg at the ectopic FB. Hence, Dl must also signal in Fng expressing ABCs and contribute to Notch signalling in these cells. The results suggest that the total amount of Notch activity in ABCs is the sum of signalling by Dl and Ser in the Fng domain and Dl signalling from PBCs to ABCs. Whereby the signal from posteriors to anterior is the more important one, since its loss always abolishes expression of Wg. A similar requirement at on both sides of the boundary for Dl had been described for the D/V boundary. Interestingly, the data indicate that Dl plays a similar role there as Ser at the ectopic FB (Troost, 2012).

Another addition to the current model is that Fng has two antagonistic effects on each ligand. The modification of Notch by Fringe is known to polarise signalling at the D/V-boundary by enhancing Dl- and suppressing Ser-signalling. Loss of function of fng during early stages of wing development abolishes expression of Wg at a time where it is solely dependent on Ser signalling. This observation indicates that Fng has a positive effect on Ser signalling in addition to its known negative one: it enhances Ser-signalling from Fng expressing dorsal to non-expressing ventral cells. This enhancement is required to activate expression of Wg in cells across the boundary. This finding is in good agreement with previous work that reports that Fng can enhance the ability of Ser to induce ectopic wing margins upon their co-overexpression. It is a possibility that this positive influence on Ser is indirect: The modification of Notch mediated by Fng results in a decrease of binding Ser to Notch. As a consequence high levels of free Ser are available in Fng expressing boundary cells at the FB that can bind in trans to the unmodified Notch on the adjacent Fng non-expressing boundary cells. At the analysed ectopic FB, it was found that Dl is required in Fng non-expressing PBCs to raise the activity of Notch signalling to a level that is sufficient for expression of Wg in Fng expressing anterior boundary cells, although Dl is expressed ubiquitously in early discs and thus, present in both cell populations at the same levels. This finding indicates that although Dl can induce activity of Notch in the Fng domain, this activity is insufficient to initiate the expression of Wg. The activity rises beyond the threshold only if the cells receive an additional Fng-enhanced Dl signal from non-expressing. This suggests that Fng has a suppressing effect on Dl signalling within its domain. The opposing effects on the activity of each ligand have important implications for restricted Notch-signalling at the FB. Restricted expression of the ligands on opposite sides of the FB is not required to restrict Notch activation to the FB. Only Dl outside the Fng domain is sufficiently active to induce Wg expression in Fng expressing cells. In turn only Ser in Fng expressing cells is sufficiently active to induce Wg expression in Fng non-expressing cells. These conditions are only met at the FB. These properties assure that expression of Wg is restricted to a FB even in a tissue where Dl or Ser are initially expressed uniformly, as it is the case for the ectopic FB (Troost, 2012).

A difference between the ectopic FB and the D/V boundary is that the roles of the ligands are reversed: at the ectopic FB, Dl signalling is required for the establishment of the initial asymmetric phase and Ser to establish the symmetric phase and to maintain expression of Notch activity at a high level. In contrast, Ser signalling is required for the initial asymmetric phase at the D/V boundary and Dl for the establishment of the later symmetric phase and probably maintenance during later stages. Thus, activation of Notch-signalling at a FB can be initiated by both ligands. Another difference is the location of the asymmetric stripe of wg expression, which is located in Fng expressing cells at the ectopic FB, but in non-expressing cells at the DV boundary. It is believed that the events observed at the ectopic FB represents the more general mode of interactions, since the interactions occur solely in the ventral pouch where the cells differ mainly with respect to the expression of Fng. In contrast, at the D/V boundary dorsal cells differ from ventral cells by expression the selector Ap, which might modify the outcome of the interactions (Troost, 2012).

On the basis of these results, the events at the ectopic boundary can be summarized. During early stages of wing development, Dl is ubiquitously expressed throughout the wing anlage. Ectopic expression of fng with ptcGal4, creates a band-like Fng domain in the ventral pouch. During early stages Dl and Ser (probably induced by Dl) activate Notch signalling throughout the Fng domain at low levels that are not sufficient for activation of Wg. At the sharp posterior FB, Dl signalling from PBCs to ABCs, enhanced by Fng, raises the levels of Notch in ABCs beyond the threshold required for expression of Wg. The asymmetric phase is established. Dl signalling also up-regulates expression of Ser in ABCs. Over time Ser signalling from ABCs to PBCs (enhanced by Fng), induces Wg expression in PBCs. After the solid induction of symmetric expression of Wg, the Dl/Ser/Wg loop takes over and maintains Notch signalling at the D/V boundary. It was observed that the expression of Gbe+Su(H)-lacZ is broader upon ectopic expression of Fng in Ser mutant early third instar discs. Moreover, Wg is ectopically expressed throughout the ventral ptc domain upon ectopic expression of Fng by with ptcGal4 in Ser H, but not in H mutant discs. Thus, Ser probably contributes to keeping the Notch activity in the Fng domain at low level. It is known that the expression of Ser can contribute to the suppression of Notch activity in a cell-autonomous manner through cis-inhibition. Cis-inhibition has been discovered during analysis of wing development and appears to be involved in regulation and directional Notch signalling in several processes. This mechanism causes strong Ser signalling only from Ser expressing to non-expressing cells. Since Ser expression is induced in the Fng domain, strong signalling occurs from ABCs to PBCs. Thus, it is likely that cis-inhibition contributes to the directional signalling of from ABCs to PBCs to induce the later symmetric phase (Troost, 2012).

At the D/V boundary signalling is initiated differently. This study has shown that loss of the activity of the Notch pathway in dorsal cells does not prevent the establishment of expression of Wg along the D/V boundary, but restricts it to VBCs and allows wing development to proceed. Thus, the dorsal to ventral signal, which is mediated by Ser is most important. This notion is also in agreement with the null phenotype of Ser. Ser and Fng are initially expressed in all dorsal cells. Fng enhances Ser signalling to VBCs, but suppresses signalling among dorsal cells. This strong polarised signalling results in the activation of Wg expression and up-regulation of Dl expression in VBCs. It is likely, that the cis-inhibitory effect of Ser contributes to the suppression of the activation of Notch in dorsal cells through the initial phase of wing development, since the expansion of the expression of Gbe+Su(H)–lacZ over the whole dorsal wing anlage in early Ser mutant discs. This is probably induced by the weak ubiquitous expression of Dl that was observed in early wing discs. The analysis of the Ser null and Ser H double mutants indicates that Dl can activate Notch signalling in absence of Ser function, but not strong enough to induce Wg expression, despite the presence of the FB. It appears that at the D/V boundary, Ser has to up-regulate the expression of Dl in VBCs over time beyond the threshold that is required to induce Wg expression in DBCs. Fng contributes to induction of Wg expression by enhancing Dl signalling from Fng non-expressing VBCs to expressing DBCs. The requirement for accumulation of Dl could contribute to the observed delay of the establishment of the symmetric phase of expression. After the initial asymmetric phase, the Ser/Dl/Wg loop is established to maintain Notch signalling and symmetric expression of Wg. These results indicate that the initial Fng enhanced Ser signal is sufficiently strong to induce Wg in VBCs in a manner that enables also the establishment of the Ser/Dl/Wg loop. This is indicated by the observation that suppression of Notch activity in all dorsal cells throughout wing development does not prevent maintenance of expression of Wg in late stages of the third instar and even allows the development of adult wings with only minor patterning problems. Thus, the later signal from VBCs to DBCs is mainly required for positioning and patterning of the wing margin (Troost, 2012).

Asymmetric signalling through the Hh pathway has been shown to establish the organising centre for the A/P axis at the anterior side of the boundary. It has been assumed that one difference in the establishment of the D/V organising centre is its symmetric placement on the D/V boundary. The current results suggest that at least during initial phases, the signalling at the D/V boundary is also asymmetric and that the established organising centre can also work if it is displaced ventrally. Thus, it appears that the signalling events at both boundaries are more similar than previously anticipated (Troost, 2012).


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

date revised: 10 April 2017

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