cubitus interruptus

Promoter Structure

Regulatory sequences responsible for the normal pattern of ci expression have been located. Separate elements regulate ci expression in embryos and imaginal discs. Mutants that delete a portion of an upstream regulatory region express ci ectopically in the posterior compartments of their wing imaginal discs and have wings with malformed posterior compartments. A proximal promoter element drives ci expression in the ventral midline (Schwartz, 1995).

Deformed, ci, and engrailed itself are targets of Engrailed. Engrailed is involved in an auto-regulatory loop in anterior compartments of parasegments, and both deformed and ci are limited to posterior compartments by engrailed function in adjacent anterior compartments. Engrailed binding sites have been found in the promoters of both engrailed and ci (Saenz-Robles, 1995).

Transcriptional Regulation

Evidence that the Engrailed protein normally represses ci in anterior parasegmental compartments (Eaton, 1990) includes the expansion of ci expression into anterior compartment cells that lack Engrailed function, diminution of ci expression upon overexpression of Engrailed protein in anterior compartment cells, and the ability of Engrailed protein to bind to the ci regulatory region in vivo and in vitro (Schwartz, 1995).

A dominant interaction between combgap and engrailed/invected mutations that gives rise to a gap in vein L4 strongly suggests that Cg and En/Inv act together to repress posterior cubitus interruptus transcription. Posterior expression of En represses the transcription of ci resulting in anterior specific expression. En has been shown to interact directly with the ci regulatory elements. In cg mutant wing imaginal discs, weak ectopic expression of ci-lacZ reporter constructs are found in posterior cells, thus Cg may act in concert with En to repress posterior ci. Hypomorphic mutants in either cg or en/inv can give rise to the reduction in vein L4 that is characteristic of ectopic ci expression (Svendsen, 2000).

Many proteins with multiple C2H2 zinc finger motifs like those found in cg have been shown to be transcription factors, DNA-binding proteins or chromatin proteins. The widespread localization of Cg on salivary gland chromosomes is consistent with all of these activities. While the data have not yet established direct action of Cg on the ci regulatory elements, binding of Cg to the ci region of polytene chromosomes suggests that Cg could be a direct regulator of ci transcription. Direct binding of Cg (produced in E. coli) to DNA from the ci regulatory region has not been detected. However, given that the transcriptional regulation of ci is likely to be complex, Cg may not act at the level of direct DNA binding. The involvement of the Pc-group genes in the repression of ci suggests that intricate regulatory modes are necessary to maintain the correct levels and spatial patterns of ci transcription during imaginal disc development. Furthermore, the ci-regulatory regions have been shown to be subject to transvection effects, indicating that interchromosomal interactions also govern ci regulation. Thus Cg may act at any level, from generally influencing the chromosome pairing through to direct binding of ci enhancer elements. Finally, the positive and negative effects of cg mutants on ci transcription and the genetic interaction with en/inv suggest that Cg may be required in conjunction with other transcription factors for the function of ci enhancers and that Cg may not specify activation or repression itself (Svendsen, 2000).

In legs and antennae, overall Ci levels are decreased in the anterior compartment, resulting in circumferential overgrowth of the anterior compartment and ectopic anterior expression of the morphogens wg and dpp. Similar effects on leg morphology have been previously reported when wg and dpp were ectopically activated in anterior cells. The rescue of cg mutant leg defects by additional expression of ci in the anterior compartment using the Gal4/UAS system indicates that the phenotypes result from a reduction of Ci-75 leading to the derepression of wg and dpp. Thus, it is concluded that the Cg protein is critical for the proper levels and spatial patterns of Ci and that the A/P limb patterning defects in cg mutants are due largely, if not completely, to mis-regulation of ci (Svendsen, 2000).

The effects of cg mutants on ci expression are seen only in the anterior compartment of legs but in both anterior and posterior compartments in wings. What is the basis for this difference? While anterior Ci is reduced in both limbs, ectopic posterior Ci is only seen in wings. One possibility is that the alleles that have been studied may have different effects on cg expression in anterior versus posterior compartments and/or legs versus wings. However, little Cg imaginal disc staining was seen in cg²/cg², suggesting little or no Cg protein is produced, and so the phenotype may be near null (there are no deficiencies uncovering the cg locus, so this could not be tested genetically). Cg may not be required for repression of ci in the posterior compartment of leg discs, or alternatively, there may be a much lower threshold for Cg function in legs. The different effects on ci expression in cg mutant leg and wing imaginal discs suggest that while the broad framework is similar, there may be unique aspects to A/P patterning in dorsal versus ventral limbs. The predominance of wing phenotypes in ci^W and similar ci regulatory mutations also suggests a difference in the way ci is regulated in wings versus legs. Another difference was seen in the effects of reduced Ci levels on the expression of dpp. A greater reduction of Ci staining is seen in the anterior compartments of wings compared with leg imaginal discs; paradoxically, ectopic expression of dpp is seen in all cg mutant leg imaginal discs but none is seen in cg²/cg² wing imaginal discs. Although there are limits to how accurately real levels of Ci may be inferred from histochemical staining in different tissues, the simple conclusion is that dpp responds to different thresholds of Ci in legs and wings, and that the effect of cg is indirect (Svendsen, 2000).

The posterior wing venation defect in cg hypomorphs is very similar to that found in ci mutants and this phenotype is enhanced in cg/+;ci/+ transheterozygotes. These ci mutants, however, are gain-of-function mutants; they show ectopic expression of ci in the posterior. In fact, direct misexpression of ci in the posterior using the UAS/Gal4 system can also produce the same vein defects as seen in these mutants and in cg mutants. Analysis of cg mutant discs reveals ectopic ci expression in the posterior, indicating that the cg posterior phenotype is almost certainly the direct result of deregulation of ci expression in this compartment (Campbell, 2000).

Ci expression is also abnormal in the anterior of cg mutant discs, being found at much lower levels than in wild-type discs. Loss of ci expression in the wing results in hedgehog gain-of-function phenotypes, including overgrowth and misexpression of dpp. Reduced Ci levels in the leg also result in the characteristic overgrowth phenotype, with ectopic expression of wg and dpp, found following ubiquitous expression of Hh -- i.e., the same phenotype as that found in cg mutant leg discs. Support for the proposal that the anterior combgap phenotype in the leg is also the direct result of deregulation of ci expression, in this case lowered levels of expression, comes from the observation that raising ci levels in cg mutant leg discs using the UAS/Gal4 system can suppress the overgrowth and ectopic dpp expression (Campbell, 2000).

One difference between ci and cg mutants is that wing discs from the former have a hedgehog gain-of-function phenotype with overgrowth and ectopic dpp in the anterior, while the latter do not show overgrowth and only very weak ectopic dpp. It is possible that the leg and wing are differentially sensitive to Ci levels and the Ci levels are still high enough in the wing in cg mutants to repress most dpp expression. Protein levels detected with antibody staining in ci hypomorphs and cg mutants are too low to detect significant differences with confidence, so the reason for the difference between ci and cg wings remains to be determined. Ci is also required during embryogenesis, but the putative null cg mutant survives to the early pupal stage. This suggests either that lower levels of Ci are sufficient for embryonic but not larval development or that cg RNA is maternally supplied. The first possibility is supported by the observation that hypomorphic ci mutants are not embryonic lethal and survive to the early pupal stage. However, in situ analysis reveals that CG RNA is maternally supplied so that the question of whether cg is required during embryogenesis will require the generation of germline clones (Campbell, 2000).

Full-length Ci acts as a transcriptional activator and there is evidence that the lowered levels of Ci in cg mutants also compromises Ci function as an activator. Although, dpp is misexpressed in cg discs, the level of expression, even at the compartment border, is lower than that found in wild-type discs. A similar phenomenon has been demonstrated for loss of ci in the wing and it appears that the high levels of dpp in wild-type discs require activation by Ci-155, as well as the absence of Ci-75. Thus, the lower levels of dpp in cg discs are presumably due to lower levels of Ci-155. Another gene directly activated by Ci is en in late third instar wing discs. Ci-dependent en activation in the anterior compartment does not occur in cg mutant cells, again presumably because the level of the Ci-155 activator form is too low (Campbell, 2000).

Thus Cg is required to activate ci expression to its normal levels in the anterior compartment and to repress ci expression in the posterior. The Cg protein contains multiple zinc fingers and is most probably a DNA-binding protein that would be expected to bind to elements at the ci locus. However, understanding the mechanism by which it regulates ci expression requires further studies. It is possible that Cg functions as a standard transcription factor and activates ci transcription in the anterior and represses it in the posterior. If this is the case, its activity must be modified in either the anterior or posterior compartments. Analysis of the Cg protein outside of the zinc fingers does not reveal any classical activator or repressor domains, but as these are often not well defined it is impossible to determine whether the protein has these activities without more-detailed studies (Campbell, 2000).

An argument against such a direct involvement of Cg in transcription is the well-documented role of En in regulating ci expression. En is a transcription factor that represses expression of several genes including ci, dpp and wg, and has been shown to bind to elements at the ci locus. It would appear likely that En is the primary factor that represses transcription of ci in the posterior. If this is the case, the function of Cg in regulating transcription may be indirect and may be to assist the binding of other transcription factors to the ci gene. If so, the misexpression of Ci in the posterior of cg mutant discs would be due to a lowered ability of En to bind in the absence of Cg protein, while the lowered Ci levels in the anterior would be due to a lowered ability to bind a currently unidentified transcriptional activator of ci. There are several possible mechanisms by which Cg might affect the binding of other factors. For example, there may be direct physical interactions between Cg and these other factors. Alternatively, Cg action could be more indirect, for example, it could modify chromatin structure at the ci locus producing a more open conformation. Further studies are required to test these possibilities (Campbell, 2000).

The differentiation of cells in the Drosophila eye is precisely coordinated in time and space. Each ommatidium is founded by a photoreceptor (R8) cell. These R8 founder cells are added in consecutive rows. Within a row, the nascent R8 cells appear in precise locations that lie out of register with the R8 cells in the previous row. The bHLH protein Atonal determines the development of the R8 cells. The expression of atonal is induced shortly before the selection of a new row of R8 cells and is initially detected in a stripe. Subsequently, atonal expression resolves into regularly spaced clusters (proneural clusters) that prefigure the positions of the future R8 cells. The serial induction of atonal expression, and hence the increase in the number of rows of R8 cells, requires Hedgehog function. In addition to this role, Hedgehog signaling is also required to repress atonal expression between the nascent proneural clusters. This repression has not been previously described and appears to be critical for the positioning of Atonal proneural clusters and, therefore, the position of R8 cells. The two temporal responses to Hedgehog are due to direct stimulation of the responding cells by Hedgehog itself (Dominguez, 1999).

The initial expression of ato in the eye discs occurs in a strip of cells anterior to the morphogenetic furrow. The levels of Ato within this stripe vary, with enhanced Ato expression corresponding to the approximate position of proneural clusters. Behind the furrow, the only cells that express ato are the future R8 cells. In mature R8 cells, the expression of ato is repressed. When ato and hh expressions are compared, it appears that the refinement of ato expression occurs in cells close to the hh-expressing cells, whereas the continuous stripe of ato, which is believed to be induced by Hh, is 5-7 ommatidial rows in front of the first row of hh-expressing cells. This observation suggests that Hh acts at a distance to induce ato. Such a long-range action of Hh could either be direct or indirect (relay by a secondary signal) (Dominguez, 1999).

In the eye disc, the Ci protein is expressed dynamically, with the highest levels of Ci protein overlapping with Ato expression. Accordingly, misexpression of high levels of Ci in clones of cells showed that Ci is able to induce Ato. The Ci accumulation in cells ahead of the furrow depends on Hh, because cells lacking smo activity have low uniform levels of Ci. Loss of Hh reception in more posterior regions results in the failure to downregulate Ci levels and consequently mutant cells have inappropriately high Ci protein levels when compared to wild-type neighbors. This indicates that Hh stimulates (at long-range) and inhibits (at short-range) Ci accumulation (Dominguez, 1999).

The regulation of ato by the Hh-signaling pathway was studied further by generating clones of marked cells expressing a membrane-tethered Hh protein tagged with CD2 (Hh-CD2). ato expression in cells that have gained hh was examined. Misexpression of hh-CD2 can either activate (when clones are lying anteriorly) or repress (when they lie adjacent to the furrow) the expression of ato. Repression of ato is autonomous in the hh-CD2 cells, suggesting that Hh may repress ato directly. These observations suggest that Hh is secreted near the advancing furrow: close to the source ato expression is inhibited, further away it is induced. If hh-CD2 is misexpressed, naive cells begin to express ato prematurely and this ectopic ato initiates precocious ommatidial formation. However, slightly later (and within the region of influence of the endogenous hh), misexpression of hh-CD2 results in the premature repression of ato. Thus, cells experiencing the extra Hh exhibit no ato expression while the wild-type neighbors just begin to express ato. This model has been tested by manipulating the reception of the Hh signal using in vivo assays. Genetic evidence shows that Hh is required for both promoting and inhibiting ato expression (Dominguez, 1999).

In the proposed model, the induction of Hh has two effects in the responding cells: (1) as an ato inducing signal, through the activation (by upregulation) of the Zn-finger transcription factor Ci, and (2) as an inhibitory signal, through activation of Rough, to inhibit ato expression in the cells in and behind the furrow. The two responses occur in a cell sequentially, as monitored by ato and rough expression in the wild-type pattern and by analysis of their expression in marked clones. The expression domains of ato, Ci protein and rough and their relationship with Hh supports the model. Ci and rough are activated and expressed, respectively, by Hh in restricted spatial domains across the furrow and their expression either overlaps (in the case of Ci) or is complementary (in the case of rough) with ato, consistent with their respective roles in promoting or inhibiting ato expression (Dominguez, 1999).

ato expression is controlled by two enhancer elements located 5' or 3' to the coding sequences (Sun, 1998). A 3' enhancer directs initial expression in a stripe anterior to the furrow and a distinct 5' enhancer drives expression in the proneural clusters and R8 cells within and posterior to the furrow. The 5' enhancer, but not the 3' enhancer, depends on endogenous ato function. The identification of the factors that activate the 5' enhancer element will require refining the ato regulatory sequences followed by binding studies in vitro and in vivo. One of the factors binding to these ato promoters might be Ci. Preliminary results for the loss of ci in mitotic clones are consistent with Ci acting as a positive transcriptional regulator of ato (M. D. and E. Hafen, unpublished, cited in Dominguez, 1999). During furrow progression, Ci is upregulated in the cells anterior to the furrow and in groups of cells in the furrow that coincide with cells expressing ato. These high levels of Ci are then later downregulated to a low level behind the furrow. Ci is thought to act as a transcriptional factor activating or repressing target genes in a concentration-dependent manner. The transcriptional activator form of Ci is thought to correlate with high levels of full-length Ci protein induced by Hh. This upregulation of Ci proteins by Hh is a conserved feature of Hh signaling in all systems. Therefore it is surprising that in the eye Ci is not upregulated near to the Hh source but only in cells far away. The analysis of Ci distribution in smo³, hh ^AC and viable fused alleles (where the reception and transduction of the Hh signal is blocked or very reduced) suggests that high levels of Hh protein may inhibit Ci protein levels. Probably this regulation is required to restrict the domain of Ci activation and therefore, the cells that are competent to express ato. Thus, by combining a positive long-range inductive signal with short-range inhibition of Ci, Hh may act to pattern ato expression along the anteroposterior axis and refine the array of R8 cells (Dominguez, 1999 and references).

The localized expression of Hedgehog (Hh) at the extreme anterior of Drosophila ovarioles suggests that it might provide an asymmetric cue that patterns developing egg chambers along the anteroposterior axis. Ectopic or excessive Hh signaling disrupts egg chamber patterning dramatically through primary effects at two developmental stages. (1) Excess Hh signaling in somatic stem cells stimulates somatic cell over-proliferation. This likely disrupts the earliest interactions between somatic and germline cells and may account for the frequent mis-positioning of oocytes within egg chambers. (2) The initiation of the developmental programs of follicle cell lineages appears to be delayed by ectopic Hh signaling. This may account for the formation of ectopic polar cells, the extended proliferation of follicle cells and the defective differentiation of posterior follicle cells, which, in turn, disrupts polarity within the oocyte. Somatic cells in the ovary cannot proliferate normally in the absence of Hh or Smoothened activity. Loss of protein kinase A activity restores the proliferation of somatic cells in the absence of Hh activity and allows the formation of normally patterned ovarioles. Hence, localized Hh is not essential to direct egg chamber patterning (Zhang, 2000).

Hh signaling in Drosophila generally regulates the abundance and activity of Ci proteins without altering CI mRNA levels. By contrast, vertebrate Hh homologs frequently regulate transcription of the Ci-related GLI family of transcriptional effectors. The induction of CI RNA in ptc mutant follicle cells provides the first evidence that this circuitry can also be found in Drosophila. Other consequences of altering the activity of Hh signaling components in ovarian somatic cells substantiate the hypothesis that Hh signaling activates at least two distinct intracellular pathways. One pathway, involving protection of Ci-155 from proteolysis and perhaps also release from cytoplasmic anchoring, is phenocopied by PKA and cos2 mutations. In the ovary, cos2 mutations elicit stronger phenotypes than PKA mutations, perhaps because cos2 mutations preferentially disrupt cytoplasmic anchoring of Ci-155. The second pathway increases the specific activity of Ci-155 in opposition to the inhibitory effects of Su(fu). This pathway is elicited by ptc, but not by PKA mutations and requires Fu kinase activity. In accordance with this model, PKA Su(fu) double mutant cells produce phenotypes almost as strong as for ptc mutants in ovaries, whereas ptc fu double mutant cells exhibit minimal phenotypes and PKA mutant phenotypes are not greatly altered by additional loss of Fu kinase activity. In imaginal discs high level Hh signaling to nearby cells is phenocopied by ptc mutations and requires Fu kinase activity, whereas only low level Hh signaling to more distant cells can be phenocopied by PKA mutations and does not require Fu kinase activity. PKA mutations in somatic ovarian cells can effectively substitute for Hh activity: Fu kinase activity is not essential for somatic cell proliferation and ptc mutations engender excessive Hh signaling phenotypes even in the absence of Hh activity. Hence, it is surmised that ovarian somatic cells normally undergo only low levels of Hh signaling, in keeping with the observation that the source of Hh in the germarium is separated from its target cells by several cell diameters (Zhang, 2000).

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).