Interactive Fly, Drosophila

Transcriptional regulation (part 3/3)

A new Drosophila Pax gene, sparkling, implicated in eye development, has been isolated and shown to encode the homolog of the vertebrate Pax2, Pax5, and Pax8 proteins. It is expressed in the embryonic nervous system, and in cone, primary pigment, and bristle cells of larval and pupal eye discs. Transcripts are expressed in the posterior portion of the eye disc, with the anterior boundary of expression lagging clearly behind the morphogenetic furrow. In spa(pol) mutants, a deletion of an enhancer abolishes Spa expression in cone and primary pigment cells and results in a severely disturbed development of non-neuronal ommatidial cells. Because Spa is not expressed in R7 cells, its expression in newly recruited cone cells distinguishes their fate from that of R7 cells. Lozenge may be the transcription factor whose synthesis would have to precede that of Spa, which is required for the specification of the R7 equivalence group, including R1/R6, R7 and the cone cells. Lozenge helps define the R7 equivalence group by repressing seven-up (Fu, 1997).

Spa expression is further required for activation of cut in cone cells and of the Bar locus in primary pigment cells. Cut expression is strongly reduced in cone cell of spa(pol) mutants compared to wild type. Interestingly, Cut expression recovers; by 45 hours after pupariation it has risen to levels even above those of wild-type. The lack of Spa protein in cone cells appears to delay the development of the cells, since the shape of their nuclei and the nuclear accumulation of Cut resemble those of earlier stages in wild-type pupal discs. This delay may be caused by a late larval and early pupal requirement of Spa for cut activation, which later becomes independent of Spa. Expression of cut in bristle cells, many of which are mispositioned, appears unaffected during these stages. Expression of both Bar proteins in primary pigment cells is abolished completely in spa(pol) mutants. However, it remains unaffected in the irregularly positioned bristle cells, which continue to express Spa protein. Thus Spa exerts at least part of its control of primary pigment cell development through its regulation of Bar expression. Bar is also expressed in R1 and R6 precuror cells, where Lozenge rather than Spa is one of its activators. It is suggested that close functional analogies exist between Spa and Pax2 in the development of the insect and vertebrate eye. In the absence in Pax2, the optic stalk epithelium develops into pigmented retina and fails to proliferate and differentiate into glial cells, which populate the optic nerve and are essential for guidance of the retinal axons. Thus the cone cell in Drosophila might be considered as a kind of neuronal support, or glial --a cell that may have evolved from a more primitive ancestral glial cell. In favor of such a hypothesis, it is observed that spa is expressed in glial cells in the developing PNS (Fu, 1997).

Cell interactions mediated by Notch-family receptors have been implicated in the specification of tissue boundaries in vertebrate and insect development. Although Notch ligands are often widely expressed, tightly localized activation of Notch is critical for the formation of sharp boundaries. Evidence is presented that the POU domain protein Nubbin contributes to the formation of a sharp dorsoventral (DV) boundary in the Drosophila wing. Nubbin represses Notch-dependent target genes and sets a threshold for Notch activity that defines the spatial domain of boundary-specific gene expression (Neumann, 1998).

Certain features of the abnormal wings in flies mutant for nubbin suggest a possible role for Nubbin protein in spatially limiting Notch activity at the DV boundary of the wing. The row of sensory bristles that makes up the wing margin is disorganized in nubbin wing mutants, suggesting a defect in Wingless or Notch activity. In preparations where the wing margin is viewed edge on, this disorganization reflects a broadening of the region where bristles form. Margin bristles are normally specified in cells very close to the DV boundary, reflecting a requirement for high levels of Wingless signaling activity. The broadening of the margin suggests that Wingless might be ectopically expressed in nubbin mutant wing discs. Wingless is normally expressed in a stripe of two to three cells straddling the DV boundary. In nubbin mutant discs, this stripe is widened considerably. Expression of the Notch targets vestigial and cut are similarly expanded at the DV boundary in nubbin mutants (Neumann, 1998).

During Drosophila wing development growth and patterning are mediated by signaling from the dorsoventral (D/V) organizer. In the metathorax, wing development is essentially suppressed by the homeotic selector gene Ubx to mediate development of a pair of tiny balancing organs, the halteres. Expression of Ubx in the haltere D/V boundary down-regulates the haltere's D/V organizer signaling compared to that of the wing D/V boundary. Somatic loss of Ubx from the haltere D/V boundary thus results in the formation of a wing-type D/V organizer in the haltere field. Long-distance signaling from this organizer was analyzed by assaying the ability of a Ubx minus clone induced in the haltere D/V boundary to effect homeotic transformation of capitellum cells (the capitellum is the main body of the haltere) away from the boundary. The clonally restored wing D/V organizer in mosaic halteres not only enhances the homeotic transformation of Ubx minus cells in the capitellum but also causes homeotic transformation of even Ubx plus cells in a genetic background known to induce excessive cell proliferation in the imaginal discs. In addition to demonstrating a non-cell-autonomous role for Ubx during haltere development, these results reveal distinct spatial roles for Ubx during maintenance of cell fate and patterning in the halteres. Ubx modulates the expression of wingless and cut in the haltere D/V boundary and represses vestigial in the capitellum, thereby suggesting a mechanism for the Ubx mediated down-regulation of the D/V organizer activity in the haltere. While the repression of wingless and cut expression is cell-autonomous, that of the quadrant vestigial-lacZ is non cell-autonomous: pouch cells farther away from the D/V boundary show more severe reduction in lacZ expression. Given the fact that quadrant vestigial-lacZ repression is dependent on the formation of the D/V boundary, the non-cell-autonomy in quadrant vestigial-lacZ repression by ectopic Ubx would not be surprising if Ubx function is to negatively regulate D/V signaling. Thus all the results provide strong evidence for the negative regulation of D/V organizer signaling by Ubx during haltere specification. It is likely that during haltere development, repression of wing patterning signals results in the specification of cell shape and volume that are unique to the haltere (Shashidhara, 1999).

A number of wing scalloping mutations were examined to determine their effects on the mutant phenotype of cut mutations and on the expression of the Cut protein. The mutations fall into two broad classes, those which interact synergistically with weak cut wing mutations to produce a more extreme wing phenotype than either mutation alone and those that have a simple additive effect with weak cut wing mutations. The synergistically interacting mutations are alleles of the Notch, Serrate and scalloped genes. These mutations affect development of the wing margin in a manner similar to the cut wing mutations. The mutations inactivate the cut transcriptional enhancer for the wing margin mechanoreceptors and noninnervated bristles and prevent differentiation of the organs (Jack, 1992).

Armadillo, dishevelled and shaggy/zeste white 3 encode elements of a unique wingless signaling pathway used several times throughout development. cut and achaete are targets of shaggy signalling in the wing margin region, reflecting the activity of wg and probably mediating its function. Thus an important aspect of wingless function is the enhancement of neurogenesis, acting through Shaggy to regulate transcription of cut and achaete. The functional relationship between these genes and wg is the same as that which exists during the patterning of the larval epidermis (Couso, 1994).

cut and wingless as possible Notch targets are suggested by the results of Notch overexpression in the wing disc. Notch gain-of-function alleles in which Notch activity is not restricted to the dorsoventral boundary cause misexpression of cut and wingless and overgrowth of the disc, illustrating the importance of localised Notch activation for wing development (de Celis, 1996).

Notch function is required at the dorsoventral boundary of the developing Drosophila wing for its normal growth and patterning. Clones of cells expressing either Notch or its ligands Delta and Serrate in the wing mimic Notch activation at the dorsoventral boundary, producing non-autonomous effects on proliferation and activating expression of the target genes E(spl), wingless and cut. The analysis of these clones reveals several mechanisms important for maintaining and delimiting Notch function at the dorsoventral boundary:

Notch activation in the wing leads to increased production of Delta and Serrate generating a positive feedback loop that maintains signaling.
wingless expression is upregulated by Notch signaling in both dorsal and ventral cells during the early stage of boundary formation. It is proposed that during normal development, Wingless co-operates with Notch to reinforce this positive feedback.
Cut, which is activated by Notch at late stages, acts antagonistically to prevent Delta and Serrate expression in the central D/V cells at the margin. High levels of Delta and Serrate have a dominant negative effect on Notch; at late stages, Notch can only be activated in cells next to the ligand-producing cells that are adjacent to cells of the margin.

Thus the combined effects of Notch and its target genes cut and wingless regulate the expression of Notch ligands, which restricts Notch activity to the dorsoventral boundary (de Celis, 1997).

Notch-dependent activation of wg, cut and vestigial at the wing margin depends on the activity of Suppressor of Hairless. Su(H)-mutant cells lose expression of the vestigial early enhancer, of wingless and of cut in a cell autonomous manner. Clones of Su(H)-mutant cells cause loss of wing tissue and scalloping of the wing, but only in Notch mutant clones at the D/V boundary. vestigial expression at the D/V boundary does not depend on wingless, since misexpression of wild-type wg cDNA, which results in wing margin bristles, does not cause an expansion of vestigial expression. Likewise, wingless expression does not depend on an early function of vestigial. Both Notch and wingless cooperate to activate cut at the D/V boundary. Later expression of vestigial in the wing pouch is, however, wingless dependent. vestigial is expressed in a broad domain throughout the wing. Removing of Wg activity in late second instar larvae leads to almost complete loss of the secondary expression of vestigial in the wing pouch without affecting expression at the D/V boundary. Taken together with the observation that clones of cells lacking shaggy activity show a cell-autonomous increase of Vestigial expression, these results suggest the vestigial is a direct target of the Wg pathway (Neumann, 1996).

Strawberry notch is a nuclear protein that functions downstream of Notch. Subjecting temperature sensitive strawberry notch to heat shock results in a down regulation of wg at the wing margin. Expression of wg in other regions of the wing disc as well as in other imaginal discs is unaffected by the loss of sno function. Likewise sno is required for the expression of vestigial, cut and E(spl)-m8 at the wing margin (Majumdar, 1997).

The role of the Notch and Wingless signaling pathways has been investigated in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified in the area of the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells is flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. Cut acts to maintain margin wingless expression, providing a potential explanation for the cut mutant phenotype. Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; the results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. It is proposed that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression (Micchelli, 1997).

E(spl) proteins activate cut expression

A common consequence of Notch signaling in Drosophila is the transcriptional activation of seven Enhancer of split [E(spl)] genes, which encode a family of closely related basic-helix-loop-helix transcriptional repressors. Different E(spl) proteins can functionally substitute for each other, hampering loss-of-function genetic analysis and raising the question of whether any specialization exists within the family. Each individual E(spl) gene was expressed using the GAL4-UAS system in order to analyse each gene's effect in a number of cell fate decisions taking place in the wing imaginal disk. A focus was placed on sensory organ precursor determination, wing vein determination and wing pattern formation. All of the E(spl) proteins affect the first two processes in the same way: they antagonize neural precursor and vein fates. Yet the efficacy of this antagonism is quite distinct: E(spl)mbeta, which is normally expressed in intervein regions, has the strongest vein suppression effect, whereas E(spl)m8 and E(spl)m7 are the most active bristle suppressors. While E(spl)m8 is more effective in abolishing the notum microchaeta fate, E(spl)m7 is most active against wing margin bristles (Ligoxygakis, 1999).

During wing patterning, Notch activity orchestrates a complex sequence of events that define the dorsoventral boundary of the wing. Two phases within this process have been discerned, based on the sensitivity of N loss-of-function phenotypes to concomitant expression of E(spl) genes. E(spl) proteins are initially involved in repression of the vg quadrant enhancer, whereas later they appear to relay the Notch signal that triggers activation of cut expression. Of the seven proteins, E(spl)mgamma is most active in both of these processes (Ligoxygakis, 1999).

How do E(spl) proteins, implicated in gene processing, come to activate cut expression? The present work suggests that cut expression requires Gro and partly also depends on E(spl)bHLH factors. One possibility is that E(spl) [at least E(spl)mgamma and E(spl)mdelta], like a number of other transcription factors, might have a dual function as either a transcriptional repressor or an activator, depending on context. Such an activation role has never been suggested before for either E(spl) or Gro. Alternatively, two models can be envisaged that reconcile a repressor activity of E(spl) proteins with their role in cut activation. In one, E(spl) can act by repressing a negative regulator of cut transcription. In the other, E(spl) can repress a negative regulator of Notch signaling. In the latter case, E(spl) expression would promote a positive feedback loop to enhance Notch signaling, thus increasing the signaling output from the severely compromised N^ts1 receptor at the restrictive temperature. The fact that no restoration is observed in the expression of two other Notch targets, namely wg and E(spl)m8-lacZ , argues against this hypothesis. A direct role of E(spl)mgamma and E(spl)mdelta in cut expression is favored, either as activators or as repressors of a repressor, but not as general positive regulators of Notch signaling (Ligoxygakis, 1999).

Ectopic expression of E(spl)mgamma/E(spl)mdelta is not sufficient for cut expression in a wild-type background. Rather, it appears that the ability of ectopic E(spl)mgamma/E(spl)mdelta to induce cut is spatially restricted to the normal domain of cut expression. Since activated Notch is sufficient to ectopically turn on cut, it follows that some other Notch-responsive event, other than E(spl) expression, must also contribute to cut expression. This is consistent with findings that early reduction of Notch activity abolishes cut expression despite concomitant ectopic expression of E(spl)mdelta. Molecular analysis has shown that cut expression requires the transcription factor Scalloped (Sd). sd is a candidate target gene of Vg, which in turn is initially activated by Notch independent of E(spl). It is possible that expression of vg and sd at the wing margin during early L3 could make these cells competent for cut expression. This would only be initiated later, when a second pulse of Notch signaling during mid-L3 activates (or relieves the repression of) cut via E(spl)mgamma or another Gro-interacting protein. In conclusion, E(spl) proteins have partially redundant functions, yet they have evolved distinct preferences in implementing different cell fate decisions, which closely match their individual normal expression patterns (Ligoxygakis, 1999).

Notch regulations cut expression along the presumptive wing margin

In the Drosophila wing the cut gene is activated by Notch signaling along the dorso-ventral boundary but not in other cell types. Additional regulatory components, scalloped and strawberry notch, that are targets of the Notch pathway, are expressed specifically within the wing anlagen. As suggested by physical interactions, these proteins could be co-factors of Vestigial the cut trans-regulator. Additional regulatory input comes from the Wingless pathway. These data support a model whereby context specific involvement of distinct co-regulators modulates Notch target gene activation (Nagel, 2001).

These data show that the complex regulation of ct along the D/V boundary is based on a bifurcation of the Notch signaling pathway. Most signals from the Notch pathway are mediated by Su(H), which seems to act as a repressor on its own that is converted to an activator by N^act. Since Su(H) has the capacity to bind directly to the ct wing margin enhancer, the repression of ct by Su(H) and the activation by N^act/Su(H) might be direct. However, although sufficient for the activation of ct along the D/V boundary, a number of additional factors downstream of N^act are required. These include the products of wing fate selector genes vg and sd, that seem to be, together with Sno, part of a multi-factor trans-activation complex that binds to the ct wing margin enhancer. Thereby, Sd binds directly to the ct promoter, presumably recruiting the other factors by protein-protein interactions. In agreement with this hypothesis, respective physical interactions are observed between Vg and Sd or Sno. However, all three genes are targets of the Notch signaling pathway and are activated upon the overexpression of Su(H) specifically within presumptive wing tissue. Activation of Vg is also observed also within the wing pouch, although Su(H) acts as a repressor on the vg quadrant enhancer, indicating that the isolated enhancer elements reveal only a subset of the normal pattern and might contribute differently in a wild type context (Nagel, 2001).

A combination of Sd and Su(H) binding sites is sufficient to drive expression along the D/V boundary within the wing anlagen. This synthetic enhancer is too simplified to faithfully model ct regulation. Since the overexpression of Su(H) affects the accumulation of all the important trans-activator components, Vg, Sd, Sno and Su(H) itself, ct expression would be expected. Instead, repression of ct is observed: this might be due to a lack of N^actas co-activator of Su(H). However, repression can be overcome by concurrent expression of Wg resulting in strong ct activation. It is concluded that factors downstream of the Wg signaling cascade are able to convert Su(H) from a repressor to an activator, maybe by supplying a respective co-activator or by a cooperative combinatorial activity, e.g. together with the Wg signaling mediator dTCF, in accordance with a presumptive dTCF binding site within the ct wing margin enhancer (Nagel, 2001).

These signaling events appear to be unique to the activation of ct along the D/V boundary of the wing disc. Another important role of ct is the specification of external sensory organ cells during embryogenesis and imaginal development alike. Although Notch signaling is essential for setting up the correct number of neuronal cells in the peripheral nervous system by lateral specification, it appears not to be involved in the transcriptional activation of ct within these cells. The complex mechanism of ct trans-activation from the wing margin enhancer is, therefore, not a general paradigm for ct gene regulation. Moreover, neither wg, sd nor sno are under the direct regulatory influence of the Notch pathway in various embryonic tissues suggesting that this remarkably complex control is strictly tissue specific (Nagel, 2001).

These data confirm and extend the model of context dependent activity of Notch signaling towards the regulation of ct expression along the presumptive wing margin. The regulation of ct requires the combined input of components downstream of Su(H) and Wg, including Vg, Sd and Sno. The latter three components have the potential to form a multi-protein complex which seems to be a pre-requisite for the trans-activation of the ct wing margin enhancer. Whether Su(H) is part of this specific complex or other, similar complexes has to be elucidated in the future. Although there are no indications for direct interactions between Su(H) and Sd, Vg or Sno, Su(H) has the capacity to bind to the ct wing margin enhancer and act in a combinatorial manner together with the Sd/Vg/Sno transactivation complex and components of the Wg pathway. Presumably, in many instances of Notch signaling, where Su(H) acts as a DNA-binding molecule and signal transducer, a number of additional positive or negative co-regulators confers tissue and cell specificity. Therefore, the identification of corresponding factors should help to further the understanding of the context dependent outcome of Notch signaling events (Nagel, 2001).

Control of a genetic regulatory network by a selector gene: Reduction of sd function reduces or eliminates the expression of the Cut and Wingless (Wg) proteins

The formation of many complex structures is controlled by a special class of transcription factors encoded by selector genes. It has been shown that Scalloped, the DNA binding component of the selector protein complex for the Drosophila wing field, binds to and directly regulates the cis-regulatory elements of many individual target genes within the genetic regulatory network controlling wing development. Furthermore, combinations of binding sites for Scalloped and transcriptional effectors of signaling pathways are necessary and sufficient to specify wing-specific responses to different signaling pathways. The obligate integration of selector and signaling protein inputs on cis-regulatory DNA may be a general mechanism by which selector proteins control extensive genetic regulatory networks during development (Guss, 2001).

The discovery of genes whose products control the formation and identity of various fields, dubbed 'selector genes', has enabled the recognition and redefinition of fields as discrete territories of selector gene activity. Although the term has been used somewhat liberally, two kinds of selector genes have been of central interest to understanding the development of embryonic fields. These include the Hox genes, whose products differentiate the identity of homologous fields, and field-specific selector genes such as eyeless, Distal-less, and vestigial-Scalloped (vg-sd) whose products have the unique property of directing the formation of entire complex structures. The mechanisms by which field-specific selector proteins direct the development of these structures are not well understood. In principle, selector proteins could directly regulate the expression of only a few genes, thus exerting much of their effect indirectly, or they may regulate the transcription of many genes distributed throughout genetic regulatory networks (Guss, 2001).

In the Drosophila wing imaginal disc, the Vg-Sd selector protein complex regulates wing formation and identity. Sd is a TEA-domain protein that binds to DNA in a sequence-specific manner, whereas Vg, a novel nuclear protein, functions as a trans-activator. To determine whether direct regulation by Sd is widely required for gene expression in the wing field, the regulation of several genes that represent different nodes in the wing genetic regulatory network and that control the development of different wing pattern elements were analyzed. Focus was placed in particular on genes for which cis-regulatory elements that control expression in the wing imaginal disc have been isolated, including cut, spalt (sal), and vg (Guss, 2001).

First it was tested whether sd gene function is required for the expression of various genes in the wing field. Mitotic clones of cells homozygous for a strong hypomorphic allele of sd were generated and the expression of gene products or reporter genes was assessed within these clones. Reduction of sd function reduces or eliminates the expression of the Cut and Wingless (Wg) proteins and of reporter genes under the control of the sal 10.2-kb and the vg quadrant enhancers, demonstrating a cell-autonomous requirement for selector gene function for the expression of these genes in the wing field (Guss, 2001).

These results, however, do not distinguish between the direct and indirect regulation of target gene expression by Vg-Sd. To differentiate between these possibilities, whether the DNA binding domain of Sd could bind to specific sequences in cut, sal, and vg wing-specific cis-regulatory elements were tested. Using DNase I footprinting, Sd-binding sites were identified in all of the elements assayed. Thus, Sd may control the expression of these genes by binding to their cis-regulatory elements (Guss, 2001).

To determine whether Sd binding to these sites is necessary for the function of these cis-regulatory elements in vivo, specific Sd-binding sites within each of the elements were mutated such that they reduced or abolished Sd binding in gel mobility-shift assays. The mutation of tandem Sd-binding sites in the cut and sal elements results in complete loss of reporter gene expression in vivo. Similarly, mutation of the four single Sd-binding sites identified in the vg quadrant enhancer eliminated or dramatically reduced reporter gene expression. These results show that Sd binds to and directly regulates the expression of four genes (cut, sal, vg, and DSRF) in the wing genetic regulatory network. This molecular analysis and the genetic requirement for Sd function for the expression of other genes suggest a widespread requirement for direct Vg-Sd regulation of genes expressed in the wing field (Guss, 2001).

Each of the Sd targets analyzed is activated in only a portion of the wing field, in patterns controlled by specific signaling pathways. For instance, cut is a target of Notch signaling along the dorsoventral boundary, and the sal and vg quadrant enhancers are targets of Dpp signaling along the anteroposterior axis. Binding sites for the transcriptional effectors of the Notch- and Dpp-signaling pathways, Suppressor of Hairless [Su(H)], and Mothers Against Dpp (Mad), and Medea (Med), respectively, have been shown to be necessary for the activity of a number of wing-specific cis-regulatory elements, and occur in these elements. This observation, coupled with the data demonstrating a direct requirement for Sd binding, suggests that gene expression in the wing field requires two discrete inputs on the cis-regulatory DNA: one from the selector proteins that define the field, and one from the signaling pathway that patterns the field (Guss, 2001).

These findings also raised the possibility that the combination of selector and signal inputs may be sufficient to drive field-specific, patterned gene expression. To test this, there were built a number of synthetic regulatory elements comprised of combinations of Sd binding sites with binding sites for Su(H) or Mad/Med. The activity of these elements was compared with those composed of tandem arrays of just selector- or signal effector-binding sites, or combinations of different signal effector sites. Each of the binding sites used in these constructs was selected from sequences found in native Drosophila cis-regulatory elements that have been demonstrated to function in vivo (Guss, 2001).

Elements containing only single classes of binding sites for the selector or signal effectors were unable to drive reporter gene expression in the wing. In contrast, the synthetic elements in which binding sites for both selector and signal effector were combined drove field-specific expression restricted to the wing and haltere discs in patterns predicted by the specific signaling inputs to each element. That is, the [Sd]₂ [Su(H)]₂ element drove wing-specific expression along the dorsoventral margin, consistent with Notch activation along this boundary, and the [Sd]₂ [Mad/Med] element drove expression in a broad domain oriented with respect to the anteroposterior axis of the disc, consistent with Dpp-signaling activity along this boundary. These patterns of expression are similar to those of the native cut and vg quadrant cis-regulatory elements that also respond to Notch- and Dpp-signaling inputs, respectively. However, regulatory elements containing a combination of Su(H) and Mad/Med sites were not active in vivo, demonstrating that combinatorial input in the absence of selector input is not sufficient to drive gene expression. These results suggest that the Vg-Sd complex provides a qualitatively distinct function required to generate a wing-specific response to signaling pathways (Guss, 2001).

Notch-dependent downregulation of the homeodomain gene cut is required for the mitotic cycle/endocycle switch and cell differentiation in Drosophila follicle cells

During Drosophila mid-oogenesis, follicular epithelial cells switch from the mitotic cycle to the specialized endocycle in which the M phase is skipped. The switch, along with cell differentiation in follicle cells, is induced by Notch signaling. The homeodomain gene cut functions as a linker between Notch and genes that are involved in cell-cycle progression. Cut is expressed in proliferating follicle cells but not in cells in the endocycle. Downregulation of Cut expression is controlled by the Notch pathway and is essential for follicle cells to differentiate and to enter the endocycle properly. cut-mutant follicle cells enter the endocycle and differentiate prematurely in a cell-autonomous manner. By contrast, prolonged expression of Cut causes defects in the mitotic cycle/endocycle switch. These cells continue to express an essential mitotic cyclin, Cyclin A, which is normally degraded by the Fizzy-related-APC/C ubiquitin proteosome system during the endocycle. Cut promotes Cyclin A expression by negatively regulating Fizzy-related. These data suggest that Cut functions in regulating both cell differentiation and the cell cycle, and that downregulation of Cut by Notch contributes to the mitotic cycle/endocycle switch and cell differentiation in follicle cells (Sun, 2005).

The switch of cell-cycle programs in Drosophila follicle cells provides an excellent opportunity to study how developmental signals control the intrinsic cell-cycle machinery. A switch from the mitotic cycle to the endocycle in follicle cells is induced by the Notch pathway. Cell-cycle regulators such as CycA, CycB, Stg and Fzr are regulated by Notch during this process. The homeodomain gene cut acts between the Notch pathway and some of these cell cycle regulators. Its expression is downregulated by the Notch pathway in main-body follicle cells during the mitotic cycle/endocycle switch. Cut function was required cell-autonomously for maintenance of the mitotic cycle and an immature cell fate in main-body follicle cells. Cut downregulation by Notch signaling is a key step allowing proper entry into the endocycle and cell differentiation. Fzr, an adaptor of the APC/C that promotes endocycle, is negatively regulated by Cut, but Stg is not regulated by Cut (Sun, 2005).

Several roles for cut during Drosophila oogenesis have been described. (1) cut negatively interacts with Notch for partitioning of individual germline cysts into egg chambers. (2) cut defines a signaling pathway from the follicle cells to the oocyte to maintain the germline integrity. In addition, the rarely produced ct^C145 mutant follicle-cell clones have fewer, but larger, cells. This last phenotype agrees with findings that (3) cut is required to maintain the follicle cells in mitotic cycle and that ct^db7 mutation results in premature entry into the endocycle. Study of the involvement of cut in this process required generation of clones after the egg chamber exited the germarium, so that defects caused by germarium requirements of cut would not interfere. Interestingly, during both egg-chamber encapsulation and cell-cycle switch, cut function is related to the Notch pathway. Cut expression is downregulated by Notch signaling during the cell-cycle switch at stages 6-7. In the germarium, cut negatively interacts with Notch; heterozygous cut mutation suppresses the Notch phenotype. Whether Cut acts as a target of Notch signaling at this stage is unclear. Gain- or loss-of-function clones of Notch prior to stage 6 were studied and no obvious change of Cut expression was detected; this argues that Cut is not a downstream target of Notch during early oogenesis (Sun, 2005).

The interaction between Notch and cut is not restricted to the follicle cells during Drosophila development. In wing imaginal discs, cut also interacts with Notch, but the Notch pathway positively regulates Cut expression in DV boundary formation. Clones of Notch eliminate Cut expression cell-autonomously at the DV border, a pattern opposite that of the follicle-cell-cycle switch. Ectopic expression of constitutively active Notch causes Cut to be ectopically activated in the disc. Notch downstream genes, such as strawberry notch and Enhancer of split (E(spl)), also positively regulate Cut expression in the disc, suggesting Notch-regulated Cut expression is indirect. In follicle cells, downregulation of Cut expression by Notch signaling seems to be indirect as well, because NICD/Su(H) usually functions as a transcriptional activator. Downregulation of Cut is probably achieved by a transcriptional repressor activated by NICD/Su(H). E(spl) is unlikely to be the mediator in this process, because loss of E(spl) has no effect on follicle-cell cycle transition and follicle-cell differentiation. Two other nuclear proteins, bHLH transcription factor dMyc (Dm, encoded by diminutive - FlyBase) and bHLH protein Emc (encoded by extra macrochaetae), are both required in follicle cells for proper entry into the endocycle. Removal of the function of dMyc results in lack of endocycle in follicle cells, but whether dMyc expression is regulated by Notch is uncertain, because the expression of dMyc is uniform throughout oogenesis. Emc expression is detected in main-body follicle cells from the germarium to stage 8 of oogenesis; its expression after stage 6 depends on Notch signaling. Loss of emc function results in upregulated CycB and FasIII in follicle cells after stage 6, a phenotype similar to that of loss of Notch. Emc could function as a transcriptional repressor, and it is known to be involved in promoting differentiation, so it is a good candidate for mediating Notch-dependent downregulation of Cut during the mitotic cycle/endocycle switch. No change of Cut expression however, was found in emc loss-of-function clones generated by a null allele, emc^AP6, thus excluding the possibility that cut is repressed by emc during the mitotic/endocycle switch. Another transcription factor may therefore be involved (Sun, 2005).

The known role of Cut in Drosophila is mostly related to cell differentiation. During neurogenesis, Cut is involved in cell-fate determination in sensory-organ cells. Loss of cut function causes transformation of the external sensory organ into the chordotonal sensory organ, whereas overexpression of Cut has the opposite effect. In main-body follicle cells, overexpression of Cut maintains expression of immature cell-fate markers FasIII and Eya, whereas loss of cut function in early stages represses their expression. Although Cut is involved in cell differentiation in these two developmental processes, its roles in the two are significantly different. In sensory organs, the role of Cut is post-mitotic, whereas in main-body follicle-cell differentiation, it appears to be correlated with Cut function in the mitotic cycle. The requirement for Cut in main-body follicle-cell differentiation may be related to its function in cell-cycle regulation (Sun, 2005).

The role of cut in polar-cell differentiation is intriguing. Cut expression is normally retained in these specialized cells while its expression in main-body cells is decreased. Consistent expression of Cut leads follicle cells to take the immature main-body cell fate, but these cells eventually take the polar-cell fate. Main-body cell fate and polar/stalk-cell fate are separated in the germarium, which requires Notch activity. Continuous Cut activity seems able to reverse this differentiation process (Sun, 2005).

In contrast to the role of Drosophila Cut in cell differentiation, mammalian Cut has mainly been shown to be involved in regulating cell-cycle progression in some cell types. CDP, the mammalian homolog of Cut, has been shown to be physically associated with the complex regulating the G1/S progression. Cut can functionally replace E2F in forming a complex with RB in regulating cell-cycle progression. The requirement for Cut in maintaining the mitotic cell cycle in Drosophila follicle cells echoes its role in mammalian systems. Whether Drosophila E2F has a function in follicle cell proliferation is not known: weak alleles of E2F1 and E2F2 affect gene amplification, whereas no defect appears in the mitotic cycle. Cut may functionally replace E2F for cell-cycle progression in proliferating follicle cells, but it is not an essential regulator of the cell cycle machinery because the mitotic cycle did not seem to be affected in cut germline clones. In addition, cut function has been extensively studied during embryogenesis and in imaginal discs, but no reported function is related to cell-cycle regulation in these developmental stages. The requirement for Cut in cell-cycle regulation is therefore probably specific to follicle cells in Drosophila (Sun, 2005).

Hindsight mediates the role of Notch in suppressing Hedgehog signaling and cell proliferation

Temporal and spatial regulation of proliferation and differentiation by signaling pathways is essential for animal development. Drosophila follicular epithelial cells provide an excellent model system for the study of temporal regulation of cell proliferation. In follicle cells, the Notch pathway stops proliferation and promotes a switch from the mitotic cycle to the endocycle (M/E switch). This study shows that zinc-finger transcription factor Hindsight mediates the role of Notch in regulating cell differentiation and the switch of cell-cycle programs. Hindsight is required and sufficient to stop proliferation and induce the transition to the endocycle. To do so, it represses string, Cut, and Hedgehog signaling, which promote proliferation during early oogenesis. Hindsight, along with another zinc-finger protein, Tramtrack, downregulates Hedgehog signaling through transcriptional repression of cubitus interruptus. These studies suggest that Hindsight bridges the two antagonistic pathways, Notch and Hedgehog, in the temporal regulation of follicle-cell proliferation and differentiation (Sun, 2007).

How developmental signals coordinate to control cell proliferation and differentiation remains largely unknown. These data reveal a molecular mechanism that links signal-transduction pathways and the cell-cycle machinery. Hnt is induced by Notch signaling and mediates most, if not all, Notch functions in the downregulation of Hh signaling and the M/E switch in follicle cells during midoogenesis. Loss of hnt function in follicle cells results in an extra round of the mitotic cycle after stage 6 and a delayed entry into the endocycle. In contrast, misexpression of Hnt at an earlier stage causes the follicle cells to differentiate prematurely and enter the endocycle. Hnt suppresses both stg and Cut, whose expression must be downregulated to ensure the M/E switch. In addition, Notch signaling appears to act through Hnt to downregulate Hh signaling by suppressing ci transcription, so Hnt links the two antagonistic signaling pathways in follicle-cell development. The transcriptional repression of ci is probably not mediated by Hnt alone, because ttk exhibited a similar defect in transcriptional regulation of ci and stg (Sun, 2007).

Studies have shown that downregulation of Cut mediates part of Notch function during the M/E switch. Specifically, Cut promotes cell proliferation and maintains an immature-cell fate, but Stg, the Cdc25 homolog, is not regulated by Cut. To induce the mitotic division ectopically during midoogenesis in follicle cells, both Cut and Stg must be misexpressed. The current study suggests that both Cut and Stg are suppressed by Hnt. Without Stg activity, a major regulator of G2/M transition, follicle cells are arrested before they enter the M phase, and downregulation of Cut allows accumulation of Fzr, causing degradation of CycA and CycB by the UPS, thus lowering CDK activity. This process allows endocycling follicle cells to by-pass the M phase and enter the next S phase. Repeated gap phases and S phases constitute the endocycle (Sun, 2007).

The finding that hnt follicle cells enter the endocycle after one additional round of the mitotic cycle suggests that hnt mutation causes a delay in the M/E switch. Mutations of the Notch pathway may also result in only a delay in entering the endocycle. In Notch mosaics, the cell number in mutant clones is approximately twice that of the twin spots, suggesting that an additional cell cycle also takes place. Further testing of this hypothesis requires a detailed analysis of the DNA content and clone size in Notch pathway mutants. Alternatively, Hnt may not be the sole mediator of the Notch effect; for example, Su(H)-independent Notch signaling may also be required in the M/E switch. Although hnt mutant cells can enter the endocycle late, they could not enter the chorion-gene-amplification program even much later, suggesting that Hnt function is also required for chorion-gene amplification (Sun, 2007).

The removal of negative components of the Hh pathway such as ptc causes overproliferation in follicle cells. Loss-of-function analyses of fu, a positive regulator of the pathway, revealed fewer cells in the mutant clones than in twin spots. The nuclear sizes of fu mutant cells were similar to those of the wild-type at the same developmental stage, and no fragmentation of the chromosomes was observed. Hh signaling therefore promotes cell proliferation in follicle cells during early oogenesis. Thus, Hnt-mediated downregulation of Hh signaling through suppression of ci transcription plays an important role in the M/E switch. Hh signaling is probably not involved in regulating Cut or Stg expression, because ectopic expression of Ci-155 in follicle cells during midoogenesis did not extend Stg-lacZ or Cut expression beyond stage 6, and fu mutant follicle cells showed normal Cut expression during early oogenesis. Other factors may therefore mediate the role of Hh signaling to modulate proliferation of follicle cells (Sun, 2007).

Hnt is not only required to mediate the role of Notch in regulating the M/E switch in follicle cells, but it is also sufficient to drive premature entry into the endocycle. Only a few cells misexpressing Hnt at the early stages of oogenesis were recovered, consistent with the role of Hnt in terminating the mitotic phase. In an extreme case, a stage-4 egg chamber contained only ~20 follicle cells, most of which misexpressed Hnt. Hnt misexpression suppresses Cut and stg-lacZ expression, suggesting that Hnt acts as a transcriptional repressor. Consistent with this interpretation, the mammalian homolog of Hnt, RREB1, also acts as a transcriptional repressor in several cellular contexts (Sun, 2007).

An interesting observation from these studies is that ttk clones have a phenotype similar to that of hnt clones. As in Notch regulation of Hnt, ttk is possibly downstream of Notch, but the current analysis of Notch mutants in stage-1 to stage-10 egg chambers showed no obvious change in Ttk expression. It was also found that Hnt has no role in regulating ttk expression. The findings that ttk expression is not regulated by Hnt or Notch during midoogenesis is perhaps not surprising given that Ttk69 is evenly expressed throughout early and midoogenesis. The phenotypic similarity between hnt and ttk mutants suggests that ttk and hnt act cooperatively to suppress gene expression at the M/E transition. Ttk may act as a permissive signal for Hnt to regulate Ci expression and the M/E switch. In the absence of either one, the M/E switch cannot take place properly. Consistent with this hypothesis, Ttk is known to act as a transcriptional repressor in the Drosophila eye. Whether Hnt and Ttk bind directly to the regulatory sequence of the cell-cycle genes and/or ci remains unclear (Sun, 2007).

Several lines of evidence suggest that the role of Hnt in promoting the M/E switch is not universal. First, during embryogenesis, a hnt-deficiency line enters the G1 arrest normally after cycle 16 in epidermal cells and undergoes normal M/E switch in the salivary gland, although Fzr is required for this process. Second, nurse-cell endoreplication does not require Hnt; no obvious defect was detected in hnt germline clones. The specific role of Hnt in follicle-cell-cycle regulation may stem from its role in regulating cell differentiation. For example, Hnt expression may cause upregulation of Fzr through the downregulation of Cut. This indirect role of Hnt suggests that the cell-cycle regulation may be a by-product of cell differentiation (Sun, 2007).

Both Notch and Hh signaling pathways are implicated in the regulation of differentiation and proliferation, but precisely how the two interact in regulating cellular processes is poorly understood. Depending on the cellular environment, their effects on proliferation and differentiation differ. In Drosophila eye imaginal discs, Notch triggers the onset of proliferation during the second mitotic wave (SMW), the opposite of its role in follicle-cell development. In the SMW, Notch positively affects dE2F1 and CycA expression and promotes S phase entry. In these cells, Hh signaling, along with Dpp, activates Dl expression, thereby activating the Notch pathway. Hh and Notch therefore act sequentially and positively during the SMW, whereas, in follicle cells, they act antagonistically. Hh signaling is active in the mitotic follicle cells in early oogenesis, but it is downregulated during the M/E switch when Notch signaling is activated. Notch appears to be superimposable on Hh signaling; mutation of the negative regulator of the Hh pathway, ptc, in follicle cells cannot interfere with the activation of Notch signaling as long as these cells are in direct contact with the germline cells. These ptc mutant cells show no accumulation of Ci-155, consistent with the finding that Notch signaling suppresses ci transcription through Hnt. The ptc^S2 cells that were out of contact with germline cells remained in the mitotic cycle because they could not receive Dl signaling from them, suggesting that Hh signaling is sufficient to keep these cells in the undifferentiated and mitotically active state (Sun, 2007).

Notch-dependent activation of Hnt and downregulation of Ci may be involved in another follicle-cell process, the migration of a specialized group of anterior follicle cells toward the border between the nurse cells and the oocyte at stage 9. These so-called border cells showed downregulation of ci during migration. When slbo-Gal4 was used to drive Ci overexpression in border cells, ~66% of egg chambers showed defects in border-cell migration. Notch signaling, as well as ttk, has been reported to be required for border-cell migration. Hnt was found to be expressed in the border cells and depended on Notch signaling. The occasional hnt border-cell clones observed also showed defects in border-cell migration, so the crosstalk between Hh and Notch through Hnt may go beyond the regulation of the M/E switch in follicle cells (Sun, 2007).

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