Delta

Effects of Mutation or Deletion

Table of contents

Neurogenetic and other effects of Delta mutation

All three germ cell layers (ectoderm, mesoderm and endoderm) are affected by mutations in Delta and Notch, as well as the process of oogenesis (Hartenstein, 1992). Larval stages are also affected, including wing, eye and leg imaginal discs, sensory bristle development, larval midgut (Parody, 1993) and epidermal (Notum) development (Schweisguth, 1995).

Two extreme models can be envisioned for lateral inhibition. The first implicates the Notch pathway in the choice of a single precursor via negative a feedback loop. This process could be random in some cases. The second model postulates that the precursor is pre-determined by some mechanism other than Notch signaling, and that Notch signaling then serves only to mediate mutual, uniform repression of other cells and ensure development of a single precursor. Studies concerning the physical spacing of precursors for the microchaetes of the peripheral nervous system suggest the existence of a regulatory loop under transcriptional control between Notch and its ligand Delta. Activation of Notch leads to repression of the achaete-scute genes, which are themselves known to regulate transcription of Delta; this regulation may perhaps be direct (Seugnet, 1997).

Neuroblast segregation was studied in embryos lacking both the maternal and the zygotic forms of either Notch or Delta. A seven-up-LacZ marker was used to follow neuralization of 5-2 and 7-4 neuroblast groups. In the absence of Notch signaling, the cells with an equivalence group do not enter the neural differentiation pathway simultaneously. Neuralization within a group is progressive with two or three cells segregating early and several more later. This suggests that neural potential is not evenly distributed among these cells. A requirement for transcriptional regulation of Notch and/or Delta during neuroblast segregation in embryos was tested by providing Notch and Delta ubiquitously at uniform levels. Neuroblast segregation occurs normally under conditions of uniform Notch expression, suggesting that transcriptional regulation of Notch is not necessary for many aspects of development of the larval CNS and PNS. In particular, it is dispensable both before and after neuroblast segregation, implying that it is not a necessary component of neuroblast segregation, per se. Under conditions of uniform Delta expression, a single neuroblast segregates from each proneural group in 80% of the cases; in the remaining 20%, more than one neuroblast segregates from a single group of cells. Thus transcriptional regulation of Delta is largely dispensable, with only a small percentage of multiple neurons segregating in each cluster. The possibility is discussed that segregation of single precursors in the central nervous system may rely on a heterogeneous distribution of neural potential between different cells of the proneural group. Genes such as achaete, scute, extramacrochaete, and wingless could be responsible for local differences in proneural activity. Notch signaling would enable all cells to mutually repress one another; only a cell with an elevated neural potential could overcome this repression (Seugnet, 1997).

Sanpodo regulates Notch-mediated sibling cell fate decisions but is not involved in Notch-mediated lateral inhibition. Notch functions in the neurogenic ectoderm to limit the number of cells adopting a neural fate. spdo mutation does not alter the number of neuroblasts that delaminate from the ectoderm, but instead is involved only in regulating sibling cell fate in the progeny of neuroblasts. Although the spdo sibling neuron phenotype is identical to the Notch sibling neuron phenotype, none of the 11 spdo alleles show the excess neuroblast formation characteristic of Notch mutations. Mutations in two other genes, Delta (10 alleles) and mastermind (1 allele) have been identified that yield similar equalization of sibling neuron fates. Because both Delta and mastermind are in the well-characterized Notch signaling pathway, null and hypomorphic alleles of several 'Notch pathway' genes have been tested: Delta, Notch, mam, neuralized and E(spl). Mutations in all these genes result in an excess of neuroblasts due to failure of lateral inhibition within the neuroectoderm. However, mutations in neuralized and E(spl) have no effect on the identity of the sibling neurons that were assayed, despite strong defects in the earlier process of neuroblast formation. In contrast, Delta, Notch and mam mutations all yield similar sibling neuron phenotypes, in addition to excessive neuroblast formation. These results can be illustrated using embryos homozygous for a hypomorphic mam allele in which neuroblast formation is essentially normal but sibling neuron fates are equalized. Loss of mam does not affect eve expression in GMCs, but leads to the duplication of RP2, Usib, aCC and dMP2 fates at the expense of the RP2sib, U, pCC and vMP2 fates, respectively. Thus, mutations in three genes (Delta, Notch and mam) have precisely the same sibling neuron phenotype as spdo mutations, suggesting that spdo, Delta, Notch and mam act together to specify asymmetric sibling neuron fate (Skeath, 1998).

Previous studies of big brain (bib) genetic interactions and expression agree that bib acts as a channel protein in proneural cluster cells that adopt the epidermal cell fate and serves a necessary function in the response of these cells to the lateral inhibition signal. These prior studies had not revealed any interaction between big brain and the other neurogenic genes. The neural hypertrophy in big brain mutant embryos is less severe than that in embryos mutant for other neurogenic genes. This paper shows that bib cannot rescue the phenotype in embryos mutant for other neurogenic genes. Ectopic bib expression does not rescue the cuticle-defect of embryos mutant for Dl, N, E(spl), mam or neu. In reciprocal experiments, ectopic Dl and neu do not rescue the neurogenic phenotype in bib mutant embryos. In contrast, ectopically activated N still has an antineurogenic effect in bib mutant embryos. These results indicate that bib, Dl and neu cannot functionally replace one another and that bib functions upstream of or parallel to activated N. Using mosaic analysis in the adult, it has been demonstrated that big brain activity is required autonomously in epidermal precursors to prevent neural development. Ectopically expressed big brain acts synergistically with ectopically expressed Delta and Notch, providing the first evidence that big brain may function by augmenting the activity of the Delta-Notch pathway (Doherty, 1997).

Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and formation of an additional wing margin. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. Ectopic expression of Delta provokes wing outgrowth and induction of a new margin, both on the dorsal and ventral side. Serrate acts downstream of apterous and induces expression of wing margin patterning genes. Serrate also has the potential to repress margin-specific genes such as wg and cut. This repression results in the failure to differentiate a proper wing margin, visible as a notch in the distal-most part of the wing margin. Thus ectopic Serrate provokes one of two responses, depending on the time and place of expression: outgrowth of wing tissue and induction of a new margin, or repression of margin specific genes that results in a nick in the wing margin. Actions of both Serrate and Delta are mediated by Notch, suggesting that the effects of the two Notch ligands depend on the cellular context, since the capability to activate Notch is spatially and temporally restricted, and expression of ligands at other times and in other places results in repression of Notch activity (Jönsson, 1996).

It has been suggested that lateral specification of cell fate by Notch signaling depends on feedback on Notch (N) and Delta (Dl) transcription to establish reciprocal distributions of the receptor and its ligand at the protein level. In Drosophila neurogenesis the predicted reciprocal protein distributions have not been observed. Either the current model of lateral specification or the description of N and/or Dl protein distributions must be incomplete. R8 photoreceptor specification in the developing eye has been reexamined to resolve this question with regard to this example of lateral specification. N and Dl protein levels were assessed in the cell as a whole and at the cell surface, where these proteins were mostly found at the intercellular adherens junctions. Protein levels do not correspond to Notch signaling in wild type. Dl protein first appears on cell surfaces just ahead of the furrow and becomes elevated in column 0 on the posterior cells of the so-called rosettes, including the future photoreceptors and the posterior core cell. The R8 cell surface exposes much greater amounts of Dl than its neighbors. However, Dl transcription and protein levels do correlate with altered N signaling in mutant genotypes. These findings suggest the difference relates to the speed of lateral specification in vivo. The time required for N signaling to inhibit atonal expression, an indication of the time required for the cell to respond to N signaling, is at most 90 min, but changes in the Dl protein distribution in mutant genotypes arise more slowly. N expression is little regulated by N signaling, but protein encoded by the N^ts1 allele is temperature-sensitive for appearance at the cell surface. Some aspects of the pattern of Dl protein appears to be due to endocytosis. It is concluded that feedback of N signaling on Dl transcription does occur but is too slow to account for the pattern of R8 specification. Studies of the ommatidia mosaic for a Notch duplication, or for the N^ts1 allele at semi-restrictive temperatures, have found that cells beginning with less N activity are not necessarily predisposed to be selected for R8 differentiation. These results are inconsistent with the notion that small fluctuations in N signaling levels between equivalent cells are assessed and as a consequence initiate the process of R8 selection. The data argue that other signals may be responsible for the pattern of R8 cell fate allocation by N. Other genes are known where mutations affect the R8 pattern and may constitute a second signal. These include that coding for the EGF receptor, where certain alleles can block R8 specification. Additional genes affecting the R8 pattern include the one coding for the secreted protein Scabrous and another coding for the homeodomain protein Rough. It is also possible that lingering effects of any prior N signaling in the same cells might render them unequal to a competition to become R8 (Baker, 1998).

Integrins are evolutionarily conserved transmembrane alpha,beta heterodimeric receptors involved in cell-to-matrix and cell-to-cell adhesions. In Drosophila, the position-specific (PS) integrins (see Myospheroid) mediate the formation and maintenance of junctions between muscle and epidermis and between the two epidermal wing surfaces. Besides integrins, other proteins are implicated in integrin-dependent adhesion. In Drosophila, somatic clones of mutations in PS integrin genes disrupt adhesion between wing surfaces to produce wing blisters. To identify other genes whose products function in adhesion between wing surfaces, a screen was conducted for autosomal mutations that produce blisters in somatic wing clones. 76 independent mutations were isolated in 25 complementation groups, 15 of which contained more than one allele. Chromosomal sites were determined by deficiency mapping, and genetic interactions with mutations in the beta PS integrin gene myospheroid were investigated. Mutations in four known genes (blistered [Drosophila's Serum response factor implicated in the specification of intervein cells], Delta, dumpy and mastermind) were isolated. Mutations were isolated in three new genes (piopio, rhea and steamer duck) that affect myo-epidermal junctions or muscle function in embryos. Mutations in three other genes (kakapo, kiwi and moa) may also affect cell adhesion or muscle function at hatching. These new mutants provide valuable material for the study of integrin-dependent cell-to-cell adhesion. It is thought that blisters arise in Delta and mastermind clones because of a failure to maintain the normal properties of ectodermal cells within the clonal boundaries (Prout, 1997).

hedgehog is expressed in the extreme apical end of fly ovarioles in terminal filament cells and associated somatic cells. hh activity stimulates the proliferation of pre-follicule somatic cells, and promotes the specification of polar follicle cells. hh signaling during egg chamber assembly appears to be closely related to, or part of pathways involving the neurogenic genes. Egg chamber production involves the specification of several somatic cell types, including polar cells and stalk cells. Polar cells, somatic cells usually present at both the anterior and posterior poles of the oocyte, appear ectopically throughout egg chambers exposed to elevated levels of HH during their formation. Reduced activity of Notch and Delta also causes the production of an increased number of polar cells at the ends of egg chambers as well as the loss of stalk cell fate (Ruohola, 1994). hh signaling specifies the proper anterior-posterior orientation of polar cells, while cell-cell interactions mediated by N and Dl ensure that only two cells maintain this fate cells prior to egg chamber formation in Drosophila (Forbes, 1996).

In wild type germarium, egg chambers are formed by the inter-leaving of follicle cells between adjacent germ-line clusters, and subsequently, a small population of specialized follicle cells, called stalk cells, form a narrow stem between adjacent hambers. In germaria mutant for Notch and Delta both these processes are defective: Follicle cells do not properly interleaf between adjacent clusters, and stalk cells are morphologically abnormal and fail to form a stalk. In mutant somatic cells there is an overabundance of polar follicle cells, which normally are present as a pair of specialized cells in the terminal follicle cell cluster. The Notch system may function in the germarium to sort main body follicle cells, terminal follicle cells and stalk cells, and then, in the vitellarium to sort terminal follicle cells and polar follicle cells (Ray, 1996).

The functions of artificially constructed secreted forms of the two known Drosophila Notch ligands, Delta and Serrate, were examined by expressing them under various promoters in the Drosophila developing eye and wing. The phenotypes associated with the expression of secreted Delta (DlS) or secreted Serrate (SerS) forms mimic loss-of-function mutations in the Notch pathway. Both genetic interactions between DlS or SerS transgenics and duplications or loss-of-function mutations of Delta or Serrate indicate that DlS and SerS behave as dominant negative mutations. Expression of DlS and SerS in the eye results in a rough eye phenotype. This phenotype is enhanced by loss-of-function Delta and gain-of function Suppressor of hairless. These observations were extended to the molecular level by demonstrating that the expression of Enhancer of split mdelta, a target of Notch signaling, is down-regulated by SerS. The antagonistic nature of the two mutant secreted ligand forms in the eye is consistent with their behavior in the wing, where they are capable of down-regulating wing margin specific genes in an opposite manner to the effects of the endogenous ligands. For example, wingless expression is down-regulated where a SerS expressing stripe crosses the dorsal/ventral boundary. The secreted ligands also interfere with wing vein specification. This analysis uncovers secreted molecular antagonists of Notch signaling and provides evidence of qualitative differences in the actions of the two ligands DlS and SerS (Sun, 1997).

The Notch pathway plays a key role in the formation of many tissues and cell types in Metazoans. Notch acts in two pathways to determine muscle precursor fates. The first is the 'standard' Notch pathway, in which Delta activates the Notch receptor, which then translocates into the nucleus in conjunction with Su(H) to reprogram transcription patterns and bring about changes in cell fates. The second pathway is poorly defined, but known to be independent of the ligands and downstream effectors of the standard pathway. The standard pathway is required in many different developmental contexts; it was of interest to determine if there is a general requirement for the novel pathway. The novel Notch pathway is required for the development of each of five examined cell types. Holonull Notch mutants (mutants null for maternal and zygotic Notch) have a more extreme phenotype than null mutants for Su(H), Delta, neuralized or mastermind. In Notch holonull embryos, clusters of 10 or 15 eve expressing RP2-like cells are found in place of a normal single RP2. The phenotype for the other neurogenic genes is far less severe. Notch and other neurogenic genes are involved in the determination of the mesectoderm and the visceral mesoderm. The Notch holonull phenotype is more severe in both cases than that of other holonull embryos. These results indicate that the novel pathway is a widespread and fundamental component of Notch function. Both Notch pathways operate in the differentiation of the same cell types. In such cases, the novel pathway acts first and appears to set up or limit the size of equivalence groups. The standard pathway then acts within the equivalence groups to limit individual cell fates (Rusconi, 1999).

The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind(mam) gene have implicated mam as a component of the Notch signaling pathway. However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated. To address these questions, truncated versions of the Mam protein have been constructed that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation. By several criteria, these effects appear to phenocopy loss of function for the Notch pathway. When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage. Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss. The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg. These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins. Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway. This system should prove useful for the investigation of the role of Mam within the Notch pathway (Helms, 1999).

In the embryonic ventral neuroectoderm of Drosophila the proneural genes achaete, scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm before and during delamination of the first two populations of neuroblasts show that cells in all regions of proneural gene activity change their form prior to delamination. Furthermore, the form changes in the neuroectodermal cells of embryos lacking the achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of embryos overexpressing l'sc, suggest that these genes are responsible for most of the morphological alterations observed (Stollewerk, 2000).

Analysis of wild-type and Delta mutant embryos also suggests that the ASC genes are important for the maintenance of the morphology of the neuroectodermal cells. Despite the fact that the total area of the intermediate region does not change significantly between early and mid-stage 8, cell size changes can be detected in this region shortly before delamination of the SI neuroblasts. While 20% of the intermediate cells remain larger than the average, the cells that had an average cell size in the VNE of early stage 8 embryos now split into groups of smaller cells. The fact that the number of cells that remain larger than the average corresponds to the number of cells that express the ASC genes in the intermediate region suggests that the proneural genes are required to keep these cells enlarged. This view is confirmed by analyses of the VNE of Delta mutant embryos. In Delta mutant embryos all cells of a proneural cluster continue to express the proneural genes and become neuroblasts. This altered gene expression causes all cells of a proneural cluster to remain enlarged until proneural gene expression is turned off (Stollewerk, 2000).

The Notch receptor triggers a wide range of cell fate choices in higher organisms. In Drosophila, segregation of neural from epidermal lineages results from competition among equivalent cells. These cells express achaete/scute genes, which confer neural potential. During lateral inhibition, a single neural precursor is selected, and neighboring cells are forced to adopt an epidermal fate. Lateral inhibition relies on proteolytic cleavage of Notch induced by the ligand Delta and translocation of the Notch intracellular domain (NICD) to the nuclei of inhibited cells. The activated NICD, interacting with Suppressor of Hairless [Su(H)], stimulates genes of the E(spl) complex, which in turn repress the proneural genes achaete/scute. New alleles of Notch are described that specifically display loss of microchaetae sensory precursors. This phenotype arises from a repression of neural fate, by a Notch signaling distinct from that involved in lateral inhibition. The loss of sensory organs associated with this phenotype results from a constitutive activation of a Deltex-dependent Notch-signaling event. These novel Notch alleles encode truncated receptors lacking the carboxy terminus of the NICD, which is the binding site for the repressor Dishevelled (Dsh). Dsh is known to be involved in crosstalk between Wingless and Notch pathways. These results reveal an antineural activity of Notch distinct from lateral inhibition mediated by Su(H). This activity, mediated by Deltex (Dx), represses neural fate and is antagonized by elements of the Wingless (Wg)-signaling cascade to allow alternative cell fate choices (Raiman, 2001).

In a screen for flies associated with the loss of microchaetae, a number of mutations in Notch were isolated that result in a dominant loss of thoracic microchaetae, which are called N^Mcd, where Mcd stands for microchaetae defective. These mutations are lethal, and, for this reason, their behavior was analyzed in mosaics in which clones of mutant cells are juxtaposed with wild-type territories. In these mosaics, mutant cells are recognized by the use of both bristle and epidermal markers. All mutants behave genetically in a similar manner, the strongest alleles, N^Mcd1 and N^Mcd5 (collectively N^Mcd1/5), were chosen for further analysis. In clones for N^Mcd1 and N^Mcd5, 99% of the microchaetae are absent, whereas macrochaetae are not affected (Raiman, 2001).

Genetic analysis indicates that the dominant effects of the N^Mcd alleles are due to antagonism of the wild-type function of Notch. The mutant phenotype of N^Mcd is enhanced when N⁺ is lowered and is partially suppressed when N⁺ is increased. Thus, these gain-of-function alleles of Notch do not induce an aberrant function of the receptor (neomorphism), but rather produce receptors that are more active on the normal function of Notch. N^Ax alleles exhibit a similar genetic behavior and a similar phenotype to the N^Mcd alleles. However, several differences distinguish N^Ax from N^Mcd. The N^Ax mutant exhibits a variable loss of both thoracic microchaetae and macrochaetae, leading to irregular patterns. In contrast, N^Mcd affects only microchaetae. Furthermore, the remaining microchaetae of the N^Mcd/+ flies are arranged in fewer rows, which are organized in a regular pattern. Finally, N^Ax/+ flies exhibit broader wings with shortened veins. In contrast, the wings of the N^Mcd/+ flies appear as those of wild-type flies. In this study of the N^Mcd alleles, focus was placed on the bristle pattern (Raiman, 2001).

A further demonstration of the specificity of the N^Mcd mutations for microchaetae is seen by analysis of N^Mcd1/5clones with impaired function of either hairy or extramacrochaetae (emc), two negative regulators of ac/sc. Flies lacking hairy or its cofactor groucho (gro) exhibit ectopic microchaetae in the scutellum region of the thorax. In clones mutant for N^Mcd1/5 and lacking gro (N^Mcd1/5 gro^-cells), ectopic microchaetae are absent. In contrast, the N^Ax mutants again behave differently, since, in Ax^59b gro^- cells, ectopic microchaetae form. The ectopic macrochaetae, which develop in emc¹clones, also arise in N^Mcd1/5 emc¹clones, even when their precursors differentiate simultaneously to those of the microchaetae (Raiman, 2001).

In the absence of any component of lateral inhibition, an excess of neural precursors occurs at the expense of epidermis. In Notch^-, Su(H)^-, and Dl^-clones (mosaic animals), the neurogenic phenotype is extreme; all mutant cells adopt the neural fate, and no cells are left to form epidermis. The lack of epidermal mutant cells leads to a wound partially skinned up by wild-type epidermal-surrounding cells. In gro^- and E(spl)^-, as well as in the hypomorphic Dl clones, the neurogenic phenotype is less severe, and such clones can differentiate tufts of densely packed sensory bristles accompanied by few epidermal cells. Furthermore, mutant cells for loss-of-function alleles of Notch have an enhanced capacity to produce an inhibitory signal that forces neighboring wild-type cells to adopt the epidermal fate. This signal is mediated by Delta. Thus, along the borders of N mutant clones, no bristles are formed by wild-type cells (Raiman, 2001).

Alleles of Notch encoding constitutively activated receptors show the opposite phenotype, with wild-type bristles forming at the border of mutant territories that adopt epidermal fate. The phenotype of the N^Mcd mutants resembles that of classic gain-of-function alleles of Notch (among which are the N^Ax alleles) and therefore might result in an activation of the lateral inhibition function. If this were the case, removal of the function of some or all of the mediators of lateral inhibition will abolish the effects of the N^Mcdalleles. To test this, double-mutant clones were made using the loss-of-function mutations Dl^RevF10, Dl^9P39, Df(3R)E(spl)b32.2, gro^E48, and Su(H)^IB115. In this case, double-mutant clones for N^Mcd1,5 and components that mediate lateral inhibition [Delta; E(spl)-C; gro; Su(H)] would be predicted to inactivate lateral signaling; they would be predicted to display the neurogenic phenotypes characterized by the lack of mutant epidermal cells. Surprisingly, in all cases, the double-mutant clones display the N^Mcd1/5 phenotype with mutant epidermis and no microchaetae differentiated. Therefore, N^Mcdcells do not require Dl, Su(H), gro, or the E(spl)-C in order to adopt the epidermal fate. In contrast, neurogenic double-mutant clones are observed using Ax^59bor Ax^SX1and at least with Dl, gro, and E(spl)-C. The N^Mcd Ser and N^Mcd Dl Ser clones display the N^Mcdphenotype, suggesting that the N^Mcdphenotype does not require Serrate, the other ligand of Notch (Raiman, 2001).

The macrochaetae can differentiate normally in clones mutant for N^Mcd. In the absence of lateral signaling (double-mutant clones for N^Mcd1,5 and one of the components of lateral inhibition [Dl; E(spl)-C; gro; Su(H)]), mutant clones would be predicted to display tufts of macrochaetae (the neurogenic phenotype). Macrochaetae differentiating as single bristles are observed rather than as a neurogenic tuft. These results confirm that the N^Mcdmutants affect a function of Notch distinct from lateral inhibition (Raiman, 2001).

Drosophila hephaestus (heph) is required to attenuate Notch activity after ligand-dependent activation during wing development. The original male sterile heph allele was identified in a genetic screen for loci required for spermatogenesis. New lethal alleles of heph have been isolated that affect wing margin and wing vein pattern formation in genetic mosaics. Drosophila heph gene encodes the apparent homolog of mammalian polypyrimidine tract binding protein (PTB). PTB was first identified in vertebrates as a protein that binds to intronic polypyrimidine tracts preceding many 3' pre-mRNA splice sites. Many different functions have been identified for vertebrate PTB, including the control of alternative exon selection, translational control or internal ribosome entry site (IRES) use, mRNA stability and mRNA localization. PTB may also act as a transcriptional activator. The study of heph is the first genetic analysis of polypyrimidine tract binding protein function in any organism and the first evidence that such proteins may be involved in Notch signaling (Dansereau, 2002 and references therein).

Somatic clones lacking heph express the Notch target genes wingless and cut, induce ectopic wing margin in adjacent wild-type tissue, inhibit wing-vein formation and have increased levels of Notch intracellular domain immunoreactivity. Clones mutant for both Delta and hephaestus have the characteristic loss-of-function thick vein phenotype of Delta. These results led to the hypothesis that hephaestus is required to attenuate Notch activity following its activation by Delta (Dansereau. 2002).

To determine the epistatic relationship between heph and Dl, the wing vein phenotypes of double mutant clones were compared with clones lacking only heph or Dl. Clones of cells mutant for both heph and Dl cause a thick vein phenotype that is indistinguishable from the effects of Dl mutant clones. These phenotypes indicate that heph is not required for specification of vein fate, i.e., heph is not directly required for rho expression or Egfr activity. Two interpretations are suggested. The parsimonious interpretation that heph acts to repress Delta contradicts the loss of Dl staining in heph mutant tissue, and the lack of requirement for Dl in specifying vein fate. Another interpretation is that Notch must be activated by Dl before heph is required. That is, heph may attenuate the Notch signaling pathway in cells where Notch has already been activated by Dl (Dansereau. 2002).

How does heph regulate Notch activity? This study has linked together for the first time the PTB/hnRNPI RNA-binding proteins and the Notch signaling pathway. Given the strong sequence similarity shared between heph and vertebrate PTBs, it is probable that heph regulates the processing, stability or translation of a Notch pathway mRNA. However, the heph mosaic wing phenotypes most closely resemble the effects of low level ectopic Notch activation and cannot be easily correlated with an effect on any particular known element in the Notch pathway. The phenotypes of clones mutant for Delta and heph are most informative in explaining where heph acts in the Notch pathway. The epistasis of Dl over heph in double mutant clones indicates that the Notch activation in heph clones depends on Dl. This ligand dependency excludes the possibilities that Notch target genes are generally de-repressed, or that the Notch receptor is constitutively activated, in heph mutant cells. Rather, it suggests that in the absence of heph, existing Notch activity is amplified and/or maintained. Therefore, the favoured explanation is that heph is required to attenuate Notch activity after ligand-dependent activation (Dansereau. 2002).

The phenotypic consequences of heph are most prominent in the wing margin cells and wing vein cells. Both of these cell types require decreases in the levels of Notch activity during development and the heph phenotype results from persistent Notch activity in these cells. The wing margin cells lose Notch activation and wg expression during the refinement of wg and Dl/Ser expression during the late second and early third instar. During larval development, the cells that will ultimately give rise to the vein express low levels of Notch and Notch target genes such as E(spl)mß, indicating that these cells have low levels of Notch activation prior to the repression of Notch transcription in pupal vein cells. Although it is not certain how these cells normally lose Notch activation, one possibility is that NICD stability is tightly regulated in order for cells to change Notch activation states and that heph⁺ may be required for cells to degrade NICD following ligand activation of the Notch receptor (Dansereau. 2002).

The most intriguing possibility is that heph may negatively regulate the translation of E(spl)-C mRNAs. The E(spl) complex bHLH genes are transcribed in response to Notch signaling and this is counteracted by inhibition of translation by the 3'-UTR's of E(spl)-C mRNAs. This inhibition is presumably mediated through the binding of factors to conserved sequences found in most E(spl)-C mRNAs as well as in genes of the Bearded family, another group of Notch mediators. In this model, loss of heph function would increase the stability of E(spl)-C mRNAs, resulting in amplification of the effects of transcriptional activation by Notch signaling. Increased expression of E(spl)-C members has been demonstrated to inhibit wing vein differentiation, although the ectopic expression of individual E(spl)-C members has not been demonstrated to induce ectopic wing margin. However, it is possible that the stabilization of multiple E(spl)-C mRNAs could result in more dramatic effects on the wing margin. Furthermore, E(spl)-C members have different transcription patterns and may have divergent roles downstream of Notch. If heph were to regulate a subset of the E(spl)-C mRNAs, it would explain the limited requirement of heph in various Notch-mediated signaling events (Dansereau. 2002).

During neurogenesis in Drosophila, groups of ectodermal cells are endowed with the capacity to become neuronal precursors. The Notch signaling pathway is required to limit the neuronal potential to a single cell within each group. Loss of genes of the Notch signaling pathway results in a neurogenic phenotype: hyperplasia of the nervous system accompanied by a parallel loss of epidermis. Echinoid (Ed), a cell membrane associated Immunoglobulin C2-type protein, has been shown to be a negative regulator of the EGFR pathway during eye and wing vein development. Using in situ hybridization and antibody staining of whole-mount embryos, Ed has been shown to have a dynamic expression pattern during embryogenesis. Embryonic lethal alleles of ed reveal a role of Ed in restricting neurogenic potential during embryonic neurogenesis, and result in a phenotype similar to that of loss-of-function mutations of Notch signaling pathway genes. In this process Ed interacts closely with the Notch signaling pathway. Loss of ed suppresses the loss of neuronal elements caused by ectopic activation of the Notch signaling pathway. Using a temperature-sensitive allele of ed it has been shown that Ed is required to suppress sensory bristles and for proper wing vein specification during adult development. In these processes also, ed acts in close concert with genes of the Notch signaling pathway. Thus the extra wing vein phenotype of ed is enhanced upon reduction of Delta (Dl) or Enhancer of split [E(spl)] proteins. Overexpression of the membrane-tethered extracellular region of Ed results in a dominant-negative phenotype. This phenotype is suppressed by overexpression of E(spl)m7 and enhanced by overexpression of Dl. This work establishes a role for Ed during embryonic nervous system development, as well as adult sensory bristle specification and shows that Ed interacts synergistically with the Notch signaling pathway (Ahmed, 2003).

Genetic interactions between brahma and Delta

The Drosophila trithorax group gene brahma (brm) encodes the ATPase subunit of a 2-MDa chromatin-remodeling complex. brm was identified in a screen for transcriptional activators of homeotic genes and subsequently shown to play a global role in transcription by RNA polymerase II. To gain insight into the targeting, function, and regulation of the BRM complex, a screen was carried out for mutations that genetically interact with a dominant-negative allele of brm (brm^K804R). First, dominant mutations were screened that are lethal in combination with a brm^K804R transgene under control of the brm promoter. In a distinct but related screen, dominant mutations were identified that modify eye defects resulting from expression of brm^K804R in the eye-antennal imaginal disc. Mutations in three classes of genes were identified in the screens: genes encoding subunits of the BRM complex (brm, moira, and osa), other proteins directly involved in transcription (zerknullt and RpII140), and signaling molecules (Delta and vein). Expression of brm^K804R in the adult sense organ precursor lineage causes phenotypes similar to those resulting from impaired Delta-Notch signaling. These results suggest that signaling pathways may regulate the transcription of target genes by regulating the activity of the BRM complex (Armstrong, 2005).

A total of 17,146 mutant chromosomes were screened and 39 mutations were recovered that genetically interact with a dominant-negative allele of brm (brm^K804R). Of the 25 mutations that were positively identified, nearly half (48%) are alleles of genes encoding subunits of the BRM complex (brm, mor, or osa), suggesting that the other genes identified in the screens are also critical for brm function. Similar screens could be used to study any Drosophila chromatin-remodeling factor that functions as the ATPase subunit of a protein complex (Armstrong, 2005).

The screens identified a single allele of RpII140, which encodes the second largest subunit of RNA pol II. Other alleles of RpII140 also dominantly enhanced eye defects resulting from expression of brm^K804R. This finding complements the observation that the BRM complex is required for global transcription by RNA pol II and suggests that the BRM complex may interact more closely than previously thought with the general transcriptional machinery. These findings are consistent with the observation that yeast TFIID and RNA pol II are required for the recruitment of SWI/SNF to the RNR3 promoter. No physical interaction between RNA pol II and the BRM complex was detected by co-immunoprecipitation, however, and SWI/SNF recruitment does not depend upon RNA pol II at all yeast promoters. Why the basal transcription machinery targets chromatin-remodeling complexes to some, but not all, promoters remains to be determined (Armstrong, 2005).

Two distinct BRM complexes (called BAP and PBAP) have been identified in Drosophila (Mohrmann, 2005). Both complexes contain the BRM ATPase (related to the yeast SWI2/SNF2 and RSC ATPases), the SANT-domain protein Moira (MOR), the HMG-domain protein BAP111, the actin-related protein BAP55, actin, BAP60, and SNR1. The BAP complex contains Osa, while the PBAP complex lacks Osa and instead contains Polybromo (Baf180, CG11375) and the ARID-domain, zinc-finger protein BAP170. BAP may represent the Drosophila counterpart of the yeast SWI/SNF and human BAF complexes, while PBAP appears more highly related to the yeast RSC and human PBAF complexes (Mohrmann, 2005). Both BAP and PBAP are abundant and are widely associated with transcriptionally active chromatin in larval salivary glands. Both complexes use the BRM ATPase; the expression of BRM^K804R should therefore interfere with the functions of both the BAP and PBAP complexes (Armstrong, 2005).

The presence or absence of the Osa subunit distinguishes the BAP complex from PBAP. Two osa alleles were isolated from the male-specific lethality screens, suggesting that this screen has the potential to identify factors important for BAP function. The osa alleles fail to modify the eye defects caused by expression of dominant-negative brm (as does a deficiency spanning osa), suggesting that the eye-based screen may select for genes important for PBAP function. In agreement with these observations, it has been found that while osa interacts with brm in the wing, it acts in opposition to brm in the eye. The elucidation of the relative roles of BAP and PBAP in vivo will require the isolation of mutations in genes encoding unique subunits of this complex, including polybromo and BAP170 (Armstrong, 2005).

Numerous recent studies have revealed close functional relationships between chromatin-remodeling complexes and histone-modifying enzymes. For example, the MOF histone acetyltransferase functionally antagonizes the Drosophila ISWI chromatin-remodeling factor; bromodomains within the yeast RSC chromatin-remodeling complex recognize acetylated histone H3 and methylation of lysines 4 and 9 of H3 and lysine 20 of H4 by Ash1 may recruit the BRM complex. Histone modification, including methylation of lysine 4 of H3, is also required for expression of Notch target genes (Armstrong, 2005).

However, to date no E(brm) mutations have been identified in genes encoding histone-modifying enzymes. Also no genes were recovered encoding structural components of chromatin or subunits of other chromatin-remodeling complexes. Why weren't mutations in these classes of genes recovered in these screens? Recover of mutations in histone genes was not expected in these screens since they are present in many copies in flies. The eye-based screen was limited to the third chromosomes, and genes on the X chromosome would have escaped detection in both of screens. Furthermore, it is not believed that either one of the genetic screens was taken to saturation. It is also possible that chromatin-remodeling and modifying enzymes that interact with brm are redundant or are not expressed in limiting quantities (Armstrong, 2005).

Dl represented the largest E(brm) complementation group; over a third of the mutations (36%) were alleles of Dl. These findings suggest that the functions of the BRM complex and the Notch signaling pathway are intimately related. Notch signaling is one of the most extensively studied signaling pathways. It is essential for the development of most tissues and is likely present in all metazoans, although this study focuses on the pathway in Drosophila. A transmembrane ligand (either Delta or Serrate) on the signaling cell binds the Notch receptor on the signal-receiving cell, resulting in two proteolytic cleavages of the Notch transmembrane protein. This proteolysis causes the release of the Notch ICD, which translocates to the nucleus to regulate gene expression. Once in the nucleus, the ICD forms a complex with the Suppressor of Hairless [Su(H)] transcription factor (a CSL protein) to activate Notch target genes. In the absence of signaling (and therefore the absence of ICD), Su(H) complexes with corepressors that deacetylate histones to repress transcription of target genes. The role of Notch signaling is particularly well understood in regard to cell fate determinations within the adult SOP lineage. Loss of Dl-Notch signaling can result in an increase of neurons or glia at the expense of other cell types (Armstrong, 2005).

Previous work suggested that the BRM complex is critical for the development of the peripheral nervous system; somatic clones of brm mutant tissue throughout the fly showed duplicated, stunted, or fused mechanosensory bristles. Expression of the dominant-negative allele of brm results in similar bristle defects, as well as alterations in the number and identities of campaniform sensilla, sensory organs used for flight. The identification of numerous alleles of Dl in these screens as well as the observation of increased penetrance of a variety of phenotypes in individuals heterozygous for alleles of both brm and Dl is consistent with these observations and points to a close functional connection between the Notch signaling pathway and the BRM complex (Armstrong, 2005).

To explore further the connection between the BRM complex and Dl-Notch signaling, the role of the BRM complex was investigated in cell fate specification within the adult SOP lineage, where every stage of development is regulated by Dl-Notch signaling. Reduced Dl-Notch signaling within the imaginal disc proneural cluster that gives rise to the SOP leads to formation of ectopic SOPs that form perfectly normal sense organs, leading to bristle/socket duplications, a phenotype similar to the bristle defects seen in brm mutant clones. In contrast, reduced Dl-Notch specifically within the SOP lineage results in loss of external cell types and production of ectopic internal cell types such as glia or neurons. This is precisely the phenotype observed following expression of brm^K804R within the SOP lineage (Armstrong, 2005).

What is the role of the BRM complex in the Notch signaling pathway? Since the BRM complex plays a global role in transcription by RNA pol II, it is possible that the genetic interactions and phenotypes that were observed are the result of decreased Dl expression. This is thought unlikely due to the selectivity of the screens. Indeed, no genetic interactions were observed between Dl and RpII140 mutations. It is also possible that the BRM complex and the Dl-Notch pathway are independently regulating the same target genes. If both pathways are limiting, a reduction in Dl-Notch signaling may enhance a brm phenotype. A more intriguing possibility is that Dl-Notch signaling may regulate the activity or targeting of the BRM complex. As a ubiquitous complex that is critical for the transcription of most genes by RNA pol II genes, the BRM complex is a logical target for the signaling pathways. Once the ICD of Notch is in the nucleus, it may form complexes not only with Su(H), but also with the BRM complex, thus regulating its activity or its association with Notch target genes. Strong support for this model is provided by recent biochemical studies of the human BRM (hBRM) protein. hBRM physically interacts with the ICD of Notch and both hBRM and ICD are found to be associated with the promoters of Notch target genes (Kadam, 2003). On the basis of these findings, further analyses of the interactions between Dl-Notch signaling and the BRM chromatin-remodeling complex are clearly warranted (Armstrong, 2005).

The data suggest that the BRM complex may play an important role in another signal transduction pathway. An allele of vn, which encodes a secreted protein related to the mammalian neuregulin family of ligands for the EGF receptor, was recovered as an enhancer of eye defects resulting from the expression of brm^K804R. Many signal pathways intersect and complex interactions between EGF receptor signaling and the Notch pathway have been reported in Drosophila. EGF receptor signaling can work in concert with or antagonistically to Notch signaling. The current findings suggest that the BRM complex interacts with one or both of these pathways during eye development, but the precise nature of these interactions remains to be determined (Armstrong, 2005).

In conclusion, unbiased genetic screens have led to an unexpected connection between the BRM chromatin-remodeling complex and Dl-Notch signaling. Both the BRM complex and the Dl-Notch signaling pathway are conserved in mammals; these results therefore suggest that similar interactions may be critical for mammalian development. In mice, loss of Notch activity leads to tumor formation; similarly the genes encoding subunits of the mammalian BRM complexes also act as tumor suppressors. Further work is required to determine the precise nature and extent of interactions between the BRM chromatin-remodeling complex and signaling pathways (Armstrong, 2005).

Delta function in tracheal differentiation

The Drosophila tracheal system consists of a stereotyped network of epithelial tubes formed by several tracheal cell types. By the end of embryogenesis, when the general branching pattern is established, some specialized tracheal cells then mediate branch fusion, while others extend fine terminal branches. Evidence is presented that the Notch signaling pathway acts directly in the tracheal cells to distinguish individual fates within groups of equivalent cells. Notch helps to single out those tracheal cells that mediate branch fusion by blocking their neighbours from adopting the same fate. This function of Notch would require the restricted activation of the pathway in specific cells. In addition, and probably later, Notch also acts in the selection of those tracheal cells that extend the terminal branches. Both the localized expression and the mutant phenotypes of Delta, coding for a known ligand for Notch, suggest that Delta may activate Notch to specify cell fates at the tips of the developing tracheal branches (Llimargas, 1999).

Zygotic N mutant embryos show abnormalities in most of the ectodermal derivatives, including the tracheal system, where some of the tracheal cells are converted into neuroblasts; the embryos exhibit only rudimentary branches, most probably due to the loss of tracheal cells. In addition, abnormalities in fusion and terminal branching are also detected when luminal or cellular markers are used. These phenotypes could be due to early requirements for the N pathway before tracheal fate is allocated. To detect late N requirements, N^ts mutant embryos (N^ts over a null N allele) were shifted to the restrictive temperature at stage 11. The terminal branching and fusion phenotypes produced resemble those observed in zygotic null N mutants, yet the defects in the primary branching pattern are milder. The phenotypes observed both in N^ts combinations and in null N mutants suggest that, in addition to its early role in tracheal specification, N acts later in both fusion and terminal branching programs (Llimargas, 1999).

How is the activity of N regulated in the tracheal system? Dl has been shown to act as a ligand for N. Zygotic null N mutants have a weaker tracheal phenotype than that of null Dl mutants, possibly because the maternal N product rescues the zygotically mutant embryos. The tracheal branches in Dl mutants are so truncated that it is not possible to determine effects on fusion or terminal branching. The overexpression of Dl driven by the breathless-Gal4 line affects fusion and terminal branching in ways similar to both the gain and loss of N function. Thus, ectopic fusions and unfused branches are observed with variable frequency, as well as missing or extra terminal branches. These phenotypes correlate with both the lack and the excess of cells expressing fusion and terminal markers. The expression pattern of Dl in the tracheal cells was studied in detail using both in situ hybridization and antibody staining to further clarify Dl function. The Dl protein accumulates in vesicles at higher levels in the tip cells of the primary branches from stage 12 to 13. The localized accumulation of the protein is coextensive with a localized expression of the gene in the tip cells. At later stages, large amounts of transcripts and protein are found in the DT. The early accumulation of Dl depends on N activity. Dl accumulates at low levels at the tips of the branches in embryos with constitutively active N, while in null N mutants, there is a broader accumulation of Dl protein. The coincidence of the early Dl expression with the activity of the N pathway and the phenotypes of Dl overexpression, suggests that Dl activates N to diversify the tip cells of the primary branches (Llimargas, 1999).

A model is presented for the establishment of tracheal fates at the tips of the branches. bnl, coding for the FGF ligand, is expressed in clusters of cells outside the tracheal system and, in addition to guiding branch migration, it is necessary to pattern the tips of the primary branches. Bnl activates the Breathless receptor in a gradient, leading to the expression of the pantip, terminal and fusion markers. Two pieces of evidence indicate that the tip cells are initially equivalent: (1) they all express the pantip markers in response to bnl and, (2) they can all behave in the same way in different mutant backgrounds. The expression of the pantip markers becomes restricted to those cells at the leading ends of the branches, since the Breathless (Btl) activity is higher there due to the proximity of the Bnl source. Among these tip cells, some become fusion cells while others can differentiate as terminal cells, even though they all receive similar amounts of Bnl. Therefore, the bnl/btl pathway is not sufficient to account for the diversification of the tip cells, and it is proposed that the N signaling acts to achieve this. The pnt gene (Pantip-1) has been shown to repress the fusion markers, yet even though the tip cells all express pnt, only one of them acquires the fusion fate. A possible resolution of this paradox might be that there is a balance between the bnl/btl pathway, which promotes the fusion fate, and the N signal and pnt, which represses it. The N pathway shifts this balance: its presumed inactivity in one cell would allow that cell to overcome the repression by pnt, while its activity in the remaining cells would allow them to overcome the activation by bnl/btl. It is possible that biasing differences in the tip cells are also required to shift the balance. Once the fusion cell is specified, the expression of some pantip markers decays in that cell, suggesting that the fusion factors repress the pantip genes. A piece of evidence supports this: in N mutants, where all the cells express the fusion markers, Pantip-1 and Pantip-2 expressions are extinguished. Simultaneously, the fusion cell seems to signal via Dl to its neighbors to repress the fusion genes, allowing them to express the antifusion or the terminal genes. The data suggest that the fusion genes repress the antifusion ones, because in the wild type the antifusion and the fusion genes are expressed in complementary patterns and, in null N mutants (where all the cells express the fusion markers), the antifusion genes are repressed. Similarly, the specification of the terminal fate also depends on a balance between inductive signals, mediated by bnl/btl and the pantip genes, and repressive signals, mediated by N, spry and hdc. Cells closest to the Bnl source that also express high levels of pantip markers and that do not acquire the fusion fate, can become terminal cells. The remaining cells receive negative signals from the already specified terminal cell (mediated by Dl and spry) and from the also specified fusion cell (mediated by the novel protein Headcase, the Fusion 6 marker) to repress the terminal fate. Thus, the acquisition of fates among a group of cells located at the tips of each primary branch depends on the integration of positive and negative effects from different signaling pathways (Llimargas, 1999 and references).

Delta function in oogenesis

During Drosophila oogenesis the body axes are determined by signaling between the oocyte and the somatic follicle cells that surround the egg chamber. A key event in the establishment of oocyte anterior-posterior polarity is the differential patterning of the follicle cell epithelium along the anterior-posterior axis. Both the Notch and epithelial growth factor (EGF) receptor pathways are required for this patterning. To understand how these pathways act in the process, an examination was made using markers for anterior and posterior follicle cells accompanying constitutive activation of the EGF receptor, loss of Notch function, and ectopic expression of Delta. A constitutively active EGF receptor can induce posterior fate in anterior but not in lateral follicle cells, showing that the EGF receptor pathway can act only on predetermined terminal cells. Furthermore, Notch function is required at both termini for appropriate expression of anterior and posterior markers, while loss of both the EGF receptor and Notch pathways mimic the Notch loss-of-function phenotype. Ectopic expression of the Notch ligand, Delta, disturbs EGF receptor dependent posterior follicle cell differentiation and anterior-posterior polarity of the oocyte. These data are consistent with a model in which the Notch pathway is required for early follicle cell differentiation at both termini, but is then repressed at the posterior for proper determination of the posterior follicle cells by the EGF receptor pathway (Larkin, 1999).

To further investigate the interplay between Notch and Delta in follicle cell differentiation the effect of overexpression of Delta in the germarium was studied. The Drosophila ovary consists of 15-20 ovarioles: strings of egg chambers aligned in developmental order. At the anterior end of each ovariole lies the germarium, where the germ line stem cells divide to form 16 cell cysts. These cysts are enveloped by a somatic follicle cell layer and released from the germarium as a subset of follicle cells intercalates to form an interfollicular stalk. Expression of constitutively active Notch generates long stalks in the germarium by virtue of holding the stalk cells and polar cells in a precursor stage. Loss of Notch or Delta activity results in the opposite phenotype: lack of stalks. Overexpression of Delta in the germarium leads to the formation of long stalklike structures. These long stalks do not contain differentiated stalk or polar cells. Instead the markers Fasciclin III (FasIII) and Big Brain (Bib) are expressed as in the stalk cell precursors. These data suggest that overexpression of Delta produces long stalks due to a prolonged precursor stage for stalk and polar cells, a phenotype observed previously due to expression of constitutively active Notch; thus the phenotype produced by overexpression of Delta mimics that of the constitutively active Notch receptor in this developmental process (Larkin, 1999).

The data presented here show that the function of the EGF receptor pathway in posterior follicle cells requires functional Notch, but that the Notch pathway can act in these cells without an active EGF receptor pathway. A target for the EGF receptor pathway, pointed P1 is not activated in temperature sensitive Notch egg chambers, but the Notch-dependent termini are established in grk mutants. In addition, in Notch and grk double mutant experiments, the Notch loss-of-function phenotype is observed. However, if a ligand for Notch, Delta is overproduced at stage 6, posterior follicle cell development is compromised. The simplest model to explain these data is one in which the Notch pathway acts in both termini for differentiation of the terminal follicle cells and is subsequently repressed at the posterior for the EGF receptor-dependent posterior follicle cell differentiation. Therefore, proper function of the EGF receptor pathway in the posterior follicle cells requires the cessation of Delta expression in these follicle cells, suggesting that the Notch pathway can modulate cellular responses to the EGF receptor pathway (Larkin, 1999).

The body axes of Drosophila are established during oogenesis through reciprocal interactions between the germ line cells and the somatic follicle cells that surround them. The Notch pathway is required at two stages in this process: first, for the migration of the follicle cells around the germ line cyst and, later, for the polarization of the anterior-posterior (A-P) axis of the oocyte. Its function in these events, however, has remained controversial. Using clonal analysis, it has been shown that Notch signaling controls cell proliferation and differentiation in the whole follicular epithelium. Notch mutant follicle cells remain in a precursor state and fail to switch from the mitotic cell cycle to the endocycle. Furthermore, removal of Delta from the germ line produces an identical phenotype, showing that Delta signals from the germ cells to control the timing of follicle cell differentiation. This explains the axis formation defects in Notch mutants, that arise because undifferentiated posterior follicle cells cannot signal to polarize the oocyte. Delta also signals from the germ line to Notch in the soma earlier in oogenesis to control the differentiation of the polar and stalk follicle cells. The germ line therefore regulates the development of the follicle cells through two complementary signaling pathways: Gurken signals twice to control spatial patterning, whereas Delta signals twice to exert temporal control (Lopez-Schier, 2001).

The observation that loss of Notch activity does not lead to the formation of extra polar cells raises the question of whether Notch is actually required in the polar follicle cells themselves. A screen was carried out for Notch mutant clones in these cells using the polar cell-specific marker A101. In control experiments, wild-type clones were recovered that include the polar follicle cells in 18% of the egg chambers, whereas no case were found in which a Notch mutant cell expressed A101 out of 200 egg chambers screened. In addition, clones were recovered in the stalk cells. Thus, the Notch pathway seems to be required for the differentiation of the polar and stalk follicle cells, as well as the epithelial cells (Lopez-Schier, 2001).

Several lines of evidence indicate that the encapsulation of the germ line cysts depends on the polar and stalk cells. Consistent with this, adjacent egg chambers are often fused in ovaries containing Notch mutant clones, and the polar cells and stalk cells are always absent in these cases. In contrast, large Notch mutant clones in the epithelial follicle cell layer have no effect on cyst encapsulation. In rare cases, adjacent egg chambers are only partially fused, with a single or double layer of follicle cells between them. In these examples, the boundary between the partially fused cysts is covered by mutant epithelial follicle cells, but there are no A101 positive cells where the polar follicle cells would be expected to lie. In contrast, adjacent cysts are separated normally when the polar cells are wild type but the epithelial cells are mutant. These results are consistent with a model in which Notch pathway mutants disrupt the encapsulation of the germ line cysts because Notch signaling is required for the differentiation of the polar and stalks cells that mediate this process (Lopez-Schier, 2001).

The differentiation of the epithelial follicle cells is first apparent at stage 7 and occurs after the cells have exited the mitotic cell cycle and entered the endocycle to become polyploid. Because Notch mutations arrest these cells at the precursor stage, whether they also prevent this switch in the cell cycle was examined by analyzing the division patterns of mutant clones. At later stages of oogenesis, mutant clones always contain many more cells than the wild-type twin spot clones that were induced at the same time. The mutant clones still occupy the same area as the twin spot clones, however, because the Notch mutant cells are much smaller than their wild-type siblings. Thus, the loss of Notch causes the cells to go through extra cell divisions, without a corresponding increase in growth rate, to generate a larger number of smaller cells (Lopez-Schier, 2001).

To determine when these extra divisions occur, an antibody against the phosphorylated form of the Histone-H3, which labels cells in late G2 and mitosis but not cells in the endocycle, was used. Before stage 6, there is no obvious difference between the frequency of mitoses in wild-type and mutant cells. Wild-type cells never stain for phospho-Histone H3 after stage 6, however, whereas mutant cells continue to divide up until stages 10B or 11. Furthermore, mutant cells have a lower DNA content and smaller nuclei than do wild-type cells. These results indicate that Notch mutant cells fail to switch from the mitotic cell cycle to the endocycle, and carry on dividing instead of becoming polyploid (Lopez-Schier, 2001).

Clonal analysis of Delta mutation indicates that Delta signals from the germ line to activate Notch in the somatic follicle cells twice during oogenesis: once in the germarium to induce the differentiation of the polar/stalk cell lineage and then later to induce the differentiation of the epithelial follicle cells. To see if this correlates with expression of the two proteins, wild-type ovaries were stained for Notch and Delta. Notch protein is expressed from the germarium up to stage 7 and localizes to the apical membrane of the follicle cells, in close contact with the germ line. This apical localization disappears after this stage, leaving behind a faint punctate cytoplasmic staining. Delta protein expression shows a different but complementary pattern of expression. Delta is expressed by the germ line and the soma, but it is particularly abundant in the nurse cells and oocyte, where it seems to accumulate at the plasma membrane, and in large particles in the cytoplasm. Delta protein is present at low levels during early oogenesis but increases in abundance from stage 5 onward to reach its highest levels at stage 7, right at the time when Notch protein disappears from the apical membrane of follicle cells. This suggests that there is a burst of Delta signaling during stages 5 through 7, which removes most Notch protein from the membrane, either because the receptor is cleaved on binding to Delta or because Notch is down-regulated in response to the activation of the pathway. In either case, the loss of Notch protein from the follicle cell membranes should depend on Delta signaling, and the distribution of Notch protein was therefore examined in Delta germ line clones, using antibodies directed against the extracellular and intracellular portions of the receptor. Both antibodies reveal that Notch is not down-regulated in Delta mutant egg chambers. This is particularly clear in chimaeric egg chambers that contain both wild-type and mutant germ cells, where Notch remains associated with the apical membrane of all follicle cells in contact with the mutant germ cells but disappears from those overlying the wild-type germ line cells. This suggests that the Delta signals to the epithelial follicle cells at around stages 5 to 7, which coincides with when these cells cease dividing and start to differentiate (Lopez-Schier, 2001).

Drosophila oogenesis provides an excellent system in which to analyze the Notch signaling pathway for several reasons. (1) Delta signals to activate Notch in a large number of follicle cells at the same time, because ~1000 epithelial follicle cells receive the second signal during stages 5 through 7. (2) The epithelial cells constitute one of the rare examples where the down-regulation of Notch in response to Delta can be observed directly, and this allows one to see when and where signaling takes place. (3) The cells that send the signal are clearly distinct from the cells that receive it, because Notch is not required in the germ line, nor Delta in the epithelial cells. The germ cells form a separate lineage from the rest of the organism very early in embryogenesis, and it is therefore straightforward to determine whether other genes in the pathway act in the signaling or responding cells. Two other neurogenic genes, egghead and brainiac, have previously been shown to produce Notch-like phenotypes during oogenesis. Because both genes are required in the germ line, this leads to the clear prediction that they are involved in the production of functional Delta. Brainiac shows sequence similarity to Fringe, and related mammalian proteins have been characterized as glucosyltransferases. This raises the interesting possibility that Brainiac is a glucosyltransferase that adds sugar residues to Delta, in much the same way that Fringe modifies Notch (Lopez-Schier, 2001).

In many developmental processes, polyploid cells are generated by a variation of the normal cell cycle called the endocycle in which cells increase their genomic content without dividing. How the transition from the normal mitotic cycle to endocycle is regulated is poorly understood. The transition from mitotic cycle to endocycle in the Drosophila follicle cell epithelium is regulated by the Notch pathway. Loss of Notch function in follicle cells or its ligand Delta function, in the underlying germline, disrupts the normal transition of the follicle cells from mitotic cycle to endocycle: mitotic cycling continues, leading to overproliferation of these cells. The regulation is at the transcriptional level, since Su(H), a downstream transcription factor in the pathway, is also required cell autonomously in follicle cells for proper transitioning to the endocycle. One target of Notch and Su(H) is likely to be the G2/M cell cycle regulator String, a phosphatase that activates Cdc2 by dephosphorylation. String is normally repressed in the follicle cells just before the endocycle transition, but is expressed when Notch is inactivated. Analysis of the activity of String enhancer elements in follicle cells reveals the presence of an element that promotes expression of String until just before the onset of polyploidy in wild-type follicle cells but well beyond this stage in Notch mutant follicle cells. This suggests that it may be the target of the endocycle promoting activity of the Notch pathway. A second element that is insensitive to Notch regulation promotes String expression earlier in follicle cell development, which explains why Notch, while active at both stages, represses String only at the mitotic cycle-endocycle transition (Deng, 2001).

To investigate the possibility that the Notch pathway is involved in the regulation of the mitotic cycle-to-endocycle transition, the expression of the Notch ligand, Delta, was examined in oogenesis. Weak Delta expression is observed in follicle cells and germline from the germarium to stage 5 in oogenesis. At around stage 6, a dramatic upregulation of Delta expression is observed in the germline cells reaching the highest level at stage 7. This upregulation of Delta in the germline coincides with the transition from mitotic cell cycle to endocycle in the follicle cells. To determine if Delta plays a role in regulating the mitotic-to-endocycle-transition, a mosaic analysis was undertaken. Mitotic recombination was employed to create clones of cells lacking Delta function, then cell cycle stage was analyzed by examining Phospho-Histone 3 (PH3) and CycB. The mutant cell clones were marked by lack of green fluorescence protein (GFP) expression, while the wild-type cells were labeled by GFP. Since wild-type follicle cells lack PH3 and CycB after stage 6, owing to the transition into the endocycle, the expression of these markers was analyzed in follicle cells adjacent to Delta germline clones after stage 6. This analysis revealed defects in cell cycle regulation in follicle cells surrounding the Delta germline clones. In almost all cases, follicle cells surrounding the Delta germline clones expressed PH3 and CycB beyond stage 7, suggesting that these cells remain in the mitotic cell cycle program. Although dividing, these follicle cells appeared to maintain their apical-basal polarity, as shown by normal apical localization of Armadillo. In Delta follicle cell clones, no PH3 and CycB staining was detected in follicle cells after stage 6, suggesting that the germline but not the follicle cell contribution of Delta is essential for the follicle cell mitotic cycle transition into the endocycle program (Deng, 2001).

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 ovary of Drosophila is composed of about 16-20 ovarioles, each of which contains a series of egg chambers that proceed through the 14 stages of oogenesis as they move from the anterior germarium toward the oviduct at the posterior. The germline stem cells reside at the anterior tip of the germarium and divide asymmetrically to produce a new stem cell and a cystoblast, which then undergoes four consecutive mitoses with incomplete cytokinesis to give rise to a cyst of 16 interconnected germ cells. One of these cells is selected to become the oocyte and moves to the posterior of the cyst in region 3 of the germarium. This asymmetric arrangement of the germ cells generates the first anterior-posterior (A-P) polarity in development and leads to the polarization of the A-P axis of the embryo through two signaling events between the oocyte and the somatic follicle cells. At stage 6, Gurken signals from the oocyte to induce the adjacent follicle cells to adopt a posterior rather than an anterior fate. The posterior cells then send an unknown signal back to the oocyte at stage 7 to induce the formation of a polarized microtubule cytoskeleton, which directs the transport of bicoid mRNA to the anterior of the oocyte and of oskar mRNA to the posterior. The localization of these transcripts defines the A-P axis of the embryo, since bicoid mRNA encodes the anterior morphogen that patterns the head and thorax of the embryo, and oskar mRNA defines the site of formation of the pole plasm, which contains the abdominal and germline determinants (Torres, 2003 and references therein).

Mutants that disrupt the movement of the oocyte to the posterior of the cyst give rise to bipolar egg chambers with symmetric oocytes that localize bicoid mRNA to both poles and oskar mRNA to the center, indicating that all subsequent anterior-posterior asymmetries depend on the positioning of the oocyte. This morphogenetic movement occurs as the cyst moves from region 2b to 3 of the germarium. The cyst flattens to form a lens-shaped disc in region 2b, and somatic follicle cells migrate to separate the cyst from the preceding older egg chamber. As the cyst enters region 3, it rounds up with the oocyte at the posterior and eventually protruding into the surrounding follicle cell layer. This process requires the preferential adhesion of the oocyte to the follicle cells that surround the posterior of the cyst. Both the oocyte and these follicle cells independently upregulate the homophilic adhesion molecule Shotgun, and removal of Shotgun from either cell disrupts oocyte positioning. This has led to a model in which the upregulation of Shotgun in the oocyte allows it to outcompete the nurse cells for adhesion to the posterior follicle cells, thereby anchoring it at the posterior, as the cyst changes shape. Thus, the first cue for anterior-posterior polarity is the increased adhesiveness of the posterior follicle cells, although it is not known why these cells behave differently from the other follicle cells (Torres, 2003 and references therein).

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).

The analysis of follicle cell patterning raises an intriguing paradox. On the one hand, the anterior-posterior polarity of the follicle cell layer depends on the positioning of the oocyte, since this determines the direction of the Gurken signaling that makes the posterior cells different from the anterior ones. On the other hand, the positioning of the oocyte depends on the fact that oocyte adheres more strongly to the posterior follicle cells than to the other follicle cells, indicating that these posterior cells must already be different before the oocyte is positioned. Although the follicle cells that adhere to the oocyte have not been unambiguously identified, it has recently been proposed that they correspond to the posterior polar cells. In order to investigate the source of this early asymmetry, the function of the polar cells has been analyzed by disrupting Delta signaling from single germline cysts. The results resolve this paradox by showing that the anterior and posterior polar cells are not equivalent in the germarium. However, the positioning of the oocyte does not depend on the posterior polar cells but on the anterior polar cells of the adjacent older cyst and the stalk that they induce. This leads to the proposal of a novel model for anterior-posterior axis formation in Drosophila, in which each cyst transmits polarity to the adjacent younger cyst (Torres, 2003).

It has been thought that the polar and terminal follicle cells at each end of the egg chamber are equivalent until around stage 5 to 6 of oogenesis, when Gurken signals from the oocyte to induce the adjacent cells to adopt a posterior fate. The current results reveal, however, that there are a number of differences between the anterior and posterior pairs of polar cells when they differentiate during stages 1 to 2: (1) these cells arise asynchronously, since the anterior polar cells appear about 12 hr before the posterior ones; (2) about four cells initially express polar cell markers at the anterior, whereas only two, or very occasionally three, cells express these markers at the posterior; (3) only the anterior polar cells are competent to induce the formation of the stalk, since normal stalks form at the posterior of a Delta mutant cysts, which lack posterior polar cells, and (4) posterior polar cells are not sufficient for stalk formation, since they are unable to induce a stalk when the older cyst lacks anterior polar cells (Torres, 2003).

It is unclear why posterior polar cells differentiate later than the anterior ones, since both depend on the same Delta signal from the germline. One possibility is that this difference occurs because the Delta signal itself is asymmetric. The oocyte has already reached the posterior by the time that this induction occurs, and it is therefore possible that Delta signals more strongly or earlier from the nurse cells at the anterior of the germline cyst than from the posterior oocyte. Alternatively, the asynchrony could reflect an intrinsic difference in the competence of the polar cell precursors to respond to Delta. Clonal analysis indicates that the precursors of the anterior polar cells of one cyst, the stalk cells, and the posterior polar cells of the adjacent younger cyst are closely related, indicating that they all arise from the group of polar/stalk precursors that migrate between the cysts in region 2b of the germarium. Thus, the posterior polar cell precursors migrate at the same time as those of the anterior polar cells of the adjacent older cyst, whereas the precursors of the anterior polar cells of the same cyst only arrive about 12 hr later, when the next cyst reaches region 2b. This means that posterior polar cell precursors are significantly older than their anterior counterparts, and this may reduce their competence to respond to the Delta signal, leading to a delay in their differentiation. One reason why this might occur is because these cells have already been exposed to Unpaired signaling from the anterior polar cells of the older cyst and have therefore started to differentiate as stalk, which may make it more difficult to induce them to switch into the polar cell pathway (Torres, 2003).

The difference in the timing of the differentiation of anterior and posterior polar cells probably accounts for all the other asymmetries between these two sets of otherwise identical cells. For example, the posterior polar cells may be unable to induce the stalk because, by the time they are specified, the polar/stalk cell precursors have lost their competence to respond to the inductive signal. Activation of the JAK/STAT pathway is both necessary and sufficient to induce polar/stalk precursors to adopt the stalk cell fate, and the ligand for this pathway, Unpaired, is expressed in both sets of polar cells. The posterior polar cells express Unpaired only at stage 2, however, whereas the stalk is induced about a day earlier, when the cyst enters region 3 of the germarium. Although the expression of Unpaired by the posterior polar cells plays no role in stalk formation, it is not redundant because Unpaired also induces the epithelial follicle cells around the polar cells to adopt a terminal fate, and this is essential later in oogenesis to render these cells competent to respond to Gurken by becoming posterior. The delay in the differentiation of the posterior polar cells can also explain why only two, or very occasionally three, cells are initially specified at this end of the egg chamber, compared to the four or more cells that arise at the anterior, since most of the stalk/polar precursors have already adopted the stalk fate by this stage, and there will therefore be many fewer uncommitted precursors that are still competent to become polar cells. Finally, this delay can account for the later differentiation of the posterior polar cells, which is reflected in the fact that they round up and detach from the stalk during stages 4 to 5, whereas the anterior polar cells do this during stages 2 to 3. Thus, all of the differences between the anterior and posterior polar cells are temporal, and the two pairs of cells are identical in terms of their differentiation and gene expression from stage 2 until stage 6, when Gurken signals to break this symmetry (Torres, 2003).

The positioning of the oocyte at the posterior of the germline cyst generates the first anterior-posterior polarity in Drosophila development and ultimately leads to the formation of the A-P axis of the embryo. The results indicate that the asymmetric induction of the polar cells plays a key role in generating this polarity and leads to the proposal of a model in which each cyst induces the positioning of the oocyte in the following younger cyst. (1) By the time a germline cyst reaches region 3 of the germarium, it has already positioned its oocyte to the posterior and is separated from the adjacent younger cyst in region 2b by a pool of uncommitted polar/stalk precursors. At this point, Delta signals from the germline cyst to activate Notch in the adjacent anterior polar/stalk precursors, thereby inducing them to develop as polar cells. (2) These anterior polar cells turn on Unpaired and induce the more anterior polar/stalk precursors to differentiate as stalk cells. (3) The stalk cells intercalate with each other and converge toward the middle of the ovariole to generate a two cell-wide stalk. This morphogenetic movement causes the younger anterior cyst to round up, as it is pulled into region 3. In parallel, the stalk somehow induces the upregulation of Shotgun in the follicle cells that contact the oocyte of the younger cyst, and they therefore adhere preferentially to this cell. This positions the oocyte by causing it to protrude into the follicle cell layer, which anchors it at the posterior as the cyst changes shape. (4) By the time that the younger cyst has positioned its oocyte, it has entered region 3 of the germarium and activated Delta signaling. This then induces polar cell fate in the polar/stalk precursors that have migrated to cover its anterior, and the cycle begins again. It is only at this stage that polar cells differentiate at the posterior of the older cyst, which has now exited the germarium and is at stage 2 (Torres, 2003).

The oocyte is therefore positioned by a relay mechanism that involves a series of posterior to anterior inductions. The older cyst induces the anterior polar cells, the anterior polar cells induce the stalk, and the stalk induces the positioning of the oocyte of the younger anterior cyst. Each ovariole functions as a production line for new egg chambers, since the germline stem cells lie at one end and divide to provide a constant source of new cysts. This relay can therefore be repeated over and over again to position the oocyte at the posterior of each new cyst that passes through the germarium. In this way, anterior-posterior polarity is transmitted from one cyst to the next, to define the anterior-posterior axis of each egg chamber. This type of relay represents a simple mechanism for propagating a repeated asymmetric pattern (Torres, 2003).

One open question that remains is the identity of the cells that preferentially adhere to the oocyte to anchor it at the posterior. Although it has been previously proposed that the posterior polar cells fulfill this function, several pieces of evidence demonstrate that they are not required for oocyte positioning. (1) The oocyte is always correctly localized at the posterior of Delta mutant cysts, which lack posterior polar cells. Large Notch mutant follicle cell clones at the posterior of the egg chamber also block posterior polar cell specification but have no effect on oocyte positioning. (2) The oocyte always becomes mislocalized in the wild-type cysts anterior to a Delta mutant cyst, even though the posterior polar cells are present, indicating that these cells are not sufficient for oocyte positioning. In fact, the oocyte shows no preference for contact with the posterior polar cells, when it moves to the side of the cyst. (3) The oocyte is positioned as it enters region 3 of the germarium, while the posterior polar cells are only specified a day later at stage 2. (4) Electron micrographs reveal that the oocyte contacts about six follicle cells as it protrudes from the posterior of the cyst on entering region 3, whereas there are only two polar cells (Torres, 2003).

Since the posterior polar cells do not position the oocyte, some other cells must fulfill this function. One possibility is that the stalk induces the epithelial follicle cells at the posterior of the cyst to upregulate Shotgun and adhere to the oocyte. Alternatively, the stalk cells could adhere to the oocyte directly. This would be consistent with the fact that all stalk cells upregulate Shotgun as the stalk forms and remove the requirement for a third induction from the stalk to the epithelia cells. Furthermore, such a direct contact would help to explain why the formation of the stalk causes the anterior cyst to round up. The cells that eventually become the posterior polar cells may also participate in this adhesion, and then switch to the polar fate at stage 2, because they remain in contact with the oocyte and are therefore exposed to the next round of Delta signaling (Torres, 2003).

The relay between cysts represents the most upstream event in Drosophila axis formation that has been discovered so far and can account for the origin of anterior-posterior polarity in the vast majority of egg chambers. It cannot explain, however, how the first cyst in each ovariole is polarized. This leads to the prediction that other cells must fulfill the function of the posterior stalk in positioning the oocyte of this first cyst, and good candidates would be the basal stalk cells of the pupal ovariole. It is worth noting that the polarization of each cyst does not require that the previous cyst has correctly positioned its oocyte, since wild-type cysts anterior to a Delta mutant cyst induce oocyte positioning in the next cyst, even though their own oocyte is misplaced. Thus, the polarization of the first egg chamber in each ovariole is not necessary for the polarity of all subsequent egg chambers (Torres, 2003).

arrest mutants have pleiotropic phenotypes, ranging from an early arrest of oogenesis to irregular embryonic segmentation defects. One function of arrest is in translational repression of oskar mRNA; this biochemical activity is presumed to be involved in other functions of arrest. To identify genes that could provide insight into how arrest contributes to translational repression or that may be targets for arrest-dependent translational control, deficiency mutants were screened for dominant modification of the arrest phenotype. Only four of the many deficiencies tested, which cover ~30% of the genome, modified the starting phenotype. One enhancer, identified fortuitously, is the Star gene. Star interaction with arrest results in excess Gurken protein, supporting the model that gurken is a target of repression. Two modifiers were mapped to individual genes. One is Lk6, which encodes a protein kinase predicted to regulate the rate-limiting initiation factor eIF4E. The second is Delta. The interaction between arrest and Delta mimics the phenotype of homozygous Delta mutants, suggesting that arrest could positively control Delta activity. Indeed, arrest mutants have significantly reduced levels of Delta protein at the interface of germline and follicle cells (Yan, 2004).

A screen of third chromosome deficiencies was screened for dominant modifiers of aret mutants. About three-quarters of the third chromosome was screened, corresponding to ~30% of the genome. Only four deficiencies dominantly modified the aret mutant phenotype, suggesting that the total number of genes in the genome with this property is small. For two of the four deficiencies the gene responsible for the interaction was identified, and a third interacting gene was fortuitously discovered while preparing for the screen. It was anticipated that two different types of modifiers might be detected by the screen: those in genes that act in the same process as Bru and those in genes that are themselves regulated by Bru or act in a process in which a limiting component is regulated by Bru. Characterization of the interacting genes suggests that examples of each type of modifier were discovered (Yan, 2004).

The combination of aret^PD/aret^QB with Dl^9P/+ produces a variety of ovarian defects, complicating interpretation of the phenotype. Nevertheless, one striking feature is the similarity of many of the defects to those seen when Dl activity is largely or completely eliminated, suggesting that the aret mutations are enhancing the Dl phenotype. Dl is a component of the Notch/Dl signaling pathway, which acts in many signaling events in a wide range of cell types. In the ovary Dl is required in the germline cells for control of differentiation and proliferation of the somatic follicle cells and for setting up anteroposterior polarity. The earliest and, at least initially, most dramatic consequence of loss of Dl activity is the fusion of cysts—the phenotype most apparent in the aret^PD/aret^QB; Dl^9P/+ ovaries (Yan, 2004).

Large germline clones of strong Dl mutant alleles cause a complete fusion of egg chambers into a single egg chamber with multiple cysts, reminiscent of the complete fusions described here. Smaller clones retain a more regular ovariole organization. Individual egg chambers with Dl germline clones often fuse with the adjacent anterior wild-type egg chamber. Fusion can be incomplete, resulting in a double layer of follicle cells that separate the egg chambers, much as observed for the A/P partial fusions reported in this study. However, the similarities are not perfect. For example, Dl mutant clones upregulate FasIII in the follicular epithelium, but aret^PD/aret^QB; Dl^9P/+ egg chambers do not. Other features of the Dl mutant phenotype, such as the defects in anteroposterior polarity, are difficult to detect in the aret^PD/aret^QB; Dl^9P/+ ovaries, because of their arrest of oogenesis. The lack of perfect correspondence between the Dl germline clones and the aret^PD/aret^QB; Dl^9P/+ ovaries is not surprising for several reasons: (1) there is substantial phenotypic variation even among the Dl germline clones, if both large and small clones are considered; (2) the clones are homozygous for Dl^–, while in the aret mutant background one wild-type copy of Dl remains; (3) the Dl-like defects in aret^PD/aret^QB; Dl^9P/+ ovaries are superimposed on the aret mutant phenotype (Yan, 2004).

The simplest interpretation of these results is that the aret mutations are reducing the activity of the N/Dl signaling pathway, which in combination with mutation of one copy of Dl leads to phenotypes similar to those resulting from loss of Dl. This model is fully supported by the finding that in aret mutants the amount of Dl protein concentrated at the border between germline cells and follicle cells is reduced. What remains unclear is how this reduction occurs. Assuming that Bru is acting as a translational repressor, in the aret mutant the target protein should be present at elevated levels. By this model the target should be a gene that normally has a negative effect on Dl expression or delivery to the membrane. Alternatively, Bru could also have a role in translational activation, in which case Dl could be a direct target. This seems quite unlikely, as the Dl 3'-UTR lacks any recognizable BREs, the sequences to which Bru is known to bind. Nevertheless, a role for Bru in translational activation is possible, and the target could normally have a positive effect on provision of Dl activity (Yan, 2004).

Drosophila follicle cells are patterned by multiple levels of Notch signaling and antagonism between the Notch and JAK/STAT pathways

The specification of polar, main-body and stalk follicle cells in the germarium of the Drosophila ovary plays a key role in the formation of the egg chamber and polarisation of its anterior-posterior axis. High levels of Notch pathway activation, resulting from a germline Delta ligand signal, induce polar cells. This study shows that low Notch activation levels, originating from Delta expressed in the polar follicle cells, are required for stalk formation. The metalloprotease Kuzbanian-like, which cleaves and inactivates Delta, reduces the level of Delta signaling between follicle cells, thereby limiting the size of the stalk. Notch activation is required in a continuous fashion to maintain the polar and stalk cell fates. Mutual antagonism between the Notch and JAK/STAT signaling pathways provides a crucial facet of follicle cell patterning. Notch signaling in polar and main-body follicle cells inhibits JAK/STAT signaling by preventing STAT nuclear translocation, thereby restricting the influence of this pathway to stalk cells. Conversely, signaling by JAK/STAT reduces Notch signaling in the stalk. Thus, variations in the levels of Notch pathway activation, coupled with a continuous balance between the Notch and JAK/STAT pathways, specify the identity of the different follicle cell types and help establish the polarity of the egg chamber (Assa-Kunik, 2007).

Stalk formation between adjacent egg chambers is induced by directional signaling from the anterior polar cells of the older (posterior) egg chamber. Signaling via the JAK/STAT pathway provides an essential component of this process, but various indications have suggested a role for the Notch pathway as well. To verify the requirement for Notch signaling in the induction of stalk cells, follicle cell clones were generated that are mutant for Dl, the primary Notch ligand during oogenesis. Despite the proper specification of polar cells, egg chambers containing Dl follicle cell clones often failed to form a stalk on their anterior side, and as a result fused to the neighboring egg chamber. Such clones always encompassed follicle cells at the anterior portion of the egg chamber, indicating that Dl produced by anterior follicle cells is necessary to form an anterior stalk. However, the stalk positioned on the posterior side of these egg chambers was normal, even when the Dl clone surrounded the entire germline cyst. This is in keeping with the observation that posterior follicle cells do not contribute to stalk formation (Assa-Kunik, 2007).

In order to determine which cells of the anterior follicle cell population provide the signal for stalk formation, small anterior Dl-mutant follicle cell clones were analyzed. In all cases where Dl-mutant clones led to loss of the stalk, the anterior polar cells were included in the mutant clone, suggesting that these cells are the source of Dl signaling. A few instances were observed in which an anterior stalk formed even though both polar cells were mutant for Dl. Since the polar cell population defined by expression of Fng is initially larger, and is reduced to two cells by programmed cell death, this most probably resulted from the presence of wild-type Dl-expressing polar cells that provided the signal prior to their apoptosis. No phenotype was observed when the stalk cells themselves were mutant for Dl, indicating that Dl production by the stalk cells is not required for stalk specification (Assa-Kunik, 2007).

These results indicate that Notch signaling is required for at least two processes of follicle cell patterning during early oogenesis: specification of polar cells induced by Dl from the germ line and induction of stalk by Dl provided by anterior polar cells. How are these two signals distinguished, and what is the temporal relationship between them (Assa-Kunik, 2007)?

The universal Notch transcriptional reporter Gbe+Su(H)_m8-lacZ was to follow the activation profile of Notch signaling throughout oogenesis. During stages 2-3 of oogenesis, variations were observed in the strength of Notch pathway activation within different anterior follicle cell types. Activation of Notch was observed in the polar cells, but no activation could be detected at this resolution in the stalk cells. These observations indicate that the level of Notch activation in the stalk cells is significantly lower than in the polar cells. Utilization of a second Notch reporter (m7-lacZ) identified essentially the same pattern. However, as this reporter appears to be more sensitive than Gbe+Su(H)_m8-lacZ, low levels of Notch activation in the stalk cells at early stages could also be observed (Assa-Kunik, 2007).

Expression of Fng specifically in the future polar cells, provides a possible basis for the enhanced magnitude of Notch signaling in these cells. Polar cells are also part of the follicle cell population adjacent to the germline nurse cell complex, in which overall levels of Dl protein appear relatively high. However, the fraction of Dl localized to the nurse-cell membranes is difficult to quantify, preventing attribution with confidence the differences in signaling levels during early oogenesis to this parameter (Assa-Kunik, 2007).

To define the temporal sequence of polar and stalk cell induction, the expression of specific markers was followed for each cell type. Polar and stalk cell markers are first detected in stage 1 egg chambers (region 3 of the germarium). Markers of both cell types could be detected simultaneously in some egg chambers, where they were aligned as broad adjacent bands, with the polar cell marker always positioned towards the posterior. All other egg chambers at this stage displayed expression of the polar cell marker alone. These observations imply that polar cells are induced first, and, in agreement with the genetic evidence, are properly positioned to signal and induce stalk cell formation at the anterior end of the egg chamber (Assa-Kunik, 2007).

Taken together, these data suggest that distinctions in both the strength of signaling via the Notch pathway and the temporal sequence of pathway activation contribute to distinct cell-fate outcomes within the population of anterior follicle cells during early Drosophila oogenesis (Assa-Kunik, 2007).

It has been shown that the metalloprotease Kuzbanian-like (Kul) cleaves Dl in a cell-autonomous manner, leading to its downregulation. Modulation of Kul levels therefore provides a sensitive tool for manipulating Dl signaling activity in vivo. Attempts were made to determine whether Kul functions within follicle cells during early oogenesis. The expression pattern of Kul during oogenesis was monitored by fluorescent RNA in situ hybridization. Whereas Kul RNA was not detected in the germ line, prominent expression was observed in follicle cells, up to stage 3 (Assa-Kunik, 2007).

Kul levels can be effectively reduced by expression of a specific UAS-dsRNA construct. Since expression of Kul dsRNA by various GAL4 drivers resulted in lethality, expression of this construct was restricted to adult stages through the use of a temperature-sensitive GAL80 inhibitor system. This approach was used throughout the study to enable expression of various UAS-based transgenes during oogenesis. The GAL80^ts system was used in conjunction with the neur-GAL4 driver (A101-GAL4) to specifically express Kul dsRNA in polar cells, and assess the effect of Kul on Notch signaling in early follicle cells. Notch transcriptional reporter activity was examined in these egg chambers, and the position and intensity of staining compared with wild-type egg chambers that were processed under identical conditions. Following expression of dskul in polar cells, Notch reporter levels were significantly elevated, both in the germarium and in stage 1-3 egg chambers. These observations indicate that Kul acts as an attenuator of Dl signaling in early-stage follicle cells. Interference with Kul function in this fashion thus provides a means to address the significance of follicle cell Dl levels for proper stalk cell induction. Indeed, expression of dskul in the polar cells led to a significant increase in stalk-cell number, from an average of 7.0 to 10.3 cells per stalk (Assa-Kunik, 2007).

These results indicate that the size of the stalk is highly sensitive to the amount of Dl signaling between follicle cells. This is in agreement with previous experiments, in which the size of the stalk was dramatically increased following a mild hyperactivation of Notch. Consistent with these data, ovaries from heterozygous Dl females have a reduced number of stalk cells, underscoring the sensitivity of the system to levels of Dl signaling (Assa-Kunik, 2007).

To determine whether stalk cells remain sensitive to Notch pathway signaling following their differentiation, dskul was expressed in the stalk cells themselves, using the 24B-GAL4 stalk cell-specific driver, and an increase was observed in the number of stalk cells to an average of 9.0. Kul thus attenuates Dl levels even after the stalk is formed, implying that stalk-cell number is regulated by Dl signaling from both polar cells and the stalk cells themselves. In a converse experiment, Notch signaling was reduced or eliminated from the stalk cells. Expression of dsNotch, or of a dominant-negative Notch construct, by the 24B-GAL4 stalk cell-specific driver led to the disappearance of the stalk marker Big brain (Bib). Thus, persistent, low level activation of Notch is required to maintain stalk cell fate. The low levels of Dl employed for this purpose are presented initially at the polar cell-stalk cell boundary, but as the stalk becomes elongated they might be displayed by neighboring stalk cells (Assa-Kunik, 2007).

Dl is required for establishment and maintenance of the stalk cell fate. The sensitivity of stalk size to the levels of Dl provided by the stalk cells themselves suggests that Dl also affects stalk cell proliferation or survival. To examine this possibility, the anti-apoptotic protein p35 was expressed in both polar and stalk cells using the 109-53-GAL4 driver. A greater abundance was observed of cells not properly arranged into a one-cell-wide stalk. This suggests that excess stalk cells are normally eliminated by apoptosis, and would support a model in which Dl is required for stalk cell survival, as well as stalk differentiation (Assa-Kunik, 2007).

The above observations suggest that different levels of Notch signaling determine the final fate of cells from within the polar/stalk precursor population - a strong germline signal induces the polar cell fate, whereas a weaker follicle cell signal induces the stalk. As an additional test of this model, the effects were examined of strongly elevating the Notch follicle cell signal, by overexpression of Dl specifically in polar cells. Overexpression of Dl using polar cell-specific GAL4 drivers had dramatic effects on anterior follicle cell fate and tissue morphology. Significantly, this alteration in Notch signaling resulted in an excess of polar cells. Supernumerary polar cells formed primarily at the expense of stalk cells, as evidenced by their expression of both polar and stalk cell markers, and as fusions between adjacent egg chambers. Some of the excess polar cells expressed the main-body follicle cell marker Eya, suggesting that the elevated Dl signal was capable of recruiting polar cells from this neighboring population as well. Furthermore, overexpression of Dl within the stalk cells themselves, using the 24B-GAL4 driver, induced the expression of a polar cell marker within the stalk (Assa-Kunik, 2007).

The JAK/STAT ligand Upd is expressed in polar cells, and like Dl is required for induction of the stalk. The binding of Upd to its receptor, Domeless, activates the JAK kinase Hopscotch, which then phosphorylates STAT (Stat92E) to induce its translocation into the nucleus, where it regulates transcription. The observed shift from stalk to polar cell fate upon overexpression of Dl implies that Notch activation has the capacity to antagonize JAK/STAT signaling. To explore this issue further, the Notch m7-lacZ and the STAT92E-GFP transcriptional reporters were used to simultaneously monitor Notch and JAK/STAT signaling in the ovary. Two distinct distributions of transcriptional activation wee observed. During early stages of oogenesis, Upd signaling from the polar cells is capable of inducing strong STAT activation in stalk cells, but fails to elicit activation in either the polar cells themselves, or in the neighboring main-body follicle cells. At later stages, however, follicle cell populations, including main-body and border cells, exhibited concomitant Notch and STAT activation. This analysis highlights a continuous requirement for both the Notch and JAK/STAT signaling pathways during follicle cell differentiation, throughout oogenesis. As predicted, Notch signaling can antagonize STAT activation in follicle cells, but this capacity is spatially and temporally restricted (Assa-Kunik, 2007).

The antagonistic effect of Notch signaling in early egg chambers was further pursued by following nuclear localization of STAT as an assay for JAK/STAT pathway activity. Nuclear STAT staining was pronounced throughout the stalk separating the germarium from the polar cells of the adjacent, posterior egg chamber in wild-type ovaries. Consistent with the STAT92E-GFP reporter pattern, the anterior polar cells did not exhibit nuclear localization of STAT, indicating that although they produce the Upd ligand, they themselves are refractory to this signal. STAT also remained cytoplasmic in the main-body follicle cells adjacent to the polar cells (Assa-Kunik, 2007).

When Upd was overexpressed using a polar cell-specific driver, the anterior range of nuclear STAT localization was significantly increased. Consistent with this enhanced activation of JAK/STAT signaling, longer stalk-like structures were observed. In spite of the higher levels of Upd, nuclear STAT was still only seen in cells anterior to the source, including the future stalk and posterior polar cells of the adjacent younger egg chamber. By contrast, JAK/STAT signaling in the anterior polar cells themselves, and in the neighboring main-body follicle cells, was not activated (Assa-Kunik, 2007).

In light of the suggestion of an antagonistic relationship between Notch and JAK/STAT signaling, one possible explanation for failure of the polar and main-body follicle cells to respond to Upd is the higher level of Notch activation in these cells. To test this hypothesis, Notch-mutant clones were generated in the main-body follicle cells, and the nuclear localization of STAT was monitored. Elimination of Notch in these cells led to nuclear accumulation of STAT in mutant cells situated within four cell-diameters of the polar cells. No nuclear localization was detected in Notch-mutant cells situated further away, presumably owing to restricted diffusion of Upd from the polar cells (Assa-Kunik, 2007).

These results indicate that moderate to high levels of Notch activation inhibit JAK/STAT signaling, and that this inhibition acts before the nuclear translocation of activated STAT. Furthermore, the results demonstrate that correct specification of the polar, main-body and stalk follicle cells depends on crosstalk between distinct levels of Notch activity and the JAK/STAT pathway. High Notch activation induces polar cell fate, including expression of Upd, and antagonizes JAK/STAT signaling. Intermediate levels of Notch activation in the main-body follicle cells antagonize JAK/STAT signaling, without inducing expression of Upd. Finally, low levels of Notch activation synergize with Upd signaling to induce stalk cell fate and to regulate the size of the stalk (Assa-Kunik, 2007).

Maintaining the moderate level of Notch signaling that is induced by Dl expressed in the follicle cells, is essential for producing a stalk with the correct cell number, and this is achieved at least in part by the activity of Kul in the signal-sending cells. The possibility that Notch signaling is also attenuated in the signal-receiving cells by the activity of JAK/STAT was examined by monitoring oogenesis in hopscotch (hop) hypomorphs, in which JAK/STAT signaling is compromised. Stalks formed at early stages of oogenesis in hop^mv1/GA32 females, and the oocyte moved to the posterior of the egg chamber as in wild type. However, stalk cells failed to intercalate, and the stalk consisted of two rows of cells linked by adherens junctions. At later stages, the stalk collapsed and, as was observed for strong hop alleles, the stalk cells reverted to the polar cell fate. These cells now clustered at the anterior corners of the older cyst, whilst remaining in contact with the oocyte of the younger egg chamber (Assa-Kunik, 2007).

The conversion of stalk cells to polar cells when the level of JAK/STAT signaling was compromised suggests that Notch signaling in the stalk cells is normally attenuated by the JAK/STAT pathway. When this inhibition is relieved in hop hypomorphs, the increase in the level of Notch signaling leads to their conversion to polar cells. Since the entire polar/stalk precursor cell population expresses Fng, even activation by the lower levels of Dl produced by these cells may be sufficient to give rise to polar cells, in the absence of repression by JAK/STAT (Assa-Kunik, 2007).

Insulin signals control the competence of the Drosophila female germline stem cell niche to respond to Notch ligands

Adult stem cells reside in specialized microenvironments, or niches, that are essential for their function in vivo. Stem cells are physically attached to the niche, which provides secreted factors that promote their self-renewal and proliferation. Despite intense research on the role of the niche in regulating stem cell function, much less is known about how the niche itself is controlled. Previous work has shown that insulin signals directly stimulate germline stem cell (GSC) division and indirectly promote GSC maintenance via the niche in Drosophila. Insulin-like peptides are required for maintenance of cap cells (a major component of the niche that are directly attached to GSCs through E-cadherin) via modulation of Notch signaling, and they also control attachment of GSCs to cap cells and E-cadherin levels at the cap cell-GSC junction. This study has further dissected the molecular and cellular mechanisms underlying these processes. Insulin and Notch ligands were shown to directly stimulate cap cells to maintain their numbers and indirectly promote GSC maintenance. It is also reported that insulin signaling, via phosphoinositide 3-kinase and FOXO, intrinsically controls the competence of cap cells to respond to Notch ligands and thereby be maintained. Contrary to a previous report, it was also found that Notch ligands originated in GSCs are not required either for Notch activation in the GSC niche, or for cap cell or GSC maintenance. Instead, the niche itself produces ligands that activate Notch signaling within cap cells, promoting stability of the GSC niche. Finally, insulin signals control cap cell-GSC attachment independently of their role in Notch signaling. These results are potentially relevant to many systems in which Notch signaling modulates stem cells and demonstrate that complex interactions between local and systemic signals are required for proper stem cell niche function (Hsu, 2011).

The Notch pathway plays a central role in many stem cell systems, and how systemic signals impact Notch signaling in stem cell niches is a question of wide relevance to stem cell biology. Notch controls cap cell number in the Drosophila female GSC niche, and recent studies showed that insulin-like peptides control Notch signaling in the niche (Hsu, 2009), although the underlying cellular mechanisms remained unclear. This study dissected the specific cellular requirements for Notch pathway components and the insulin receptor and reveals that insulin signaling controls cell–cell communication via Notch signaling within the niche (Hsu, 2011).

To summarize, from this study in combination with previous work, a fairly complex model emerges of how insulin-like peptides -- systemic signals influenced by diet -- impact the function of GSCs and their niche through multiple mechanisms. In adult females under favorable nutritional conditions, insulin-like peptides signal directly to GSCs via PI3K to inhibit FOXO and thereby increase their division rates by promoting progression through G2. In parallel to this direct effect on GSC proliferation, insulin-like peptides also act directly on cap cells (a major cellular component of the GSC niche) to control two separate processes. Stimulation of the insulin pathway, also via PI3K inhibition of FOXO, within cap cells intrinsically increase their responsiveness to the Notch ligand Delta (likely at a step upstream of nuclear translocation of the intracellular domain of Notch), which is likely produced by neighboring cap cells. (A similar process likely occurs during niche formation in larval/pupal stages, although in this case, Delta produced in basal terminal filament cells clearly contributes to the specification of cap cells.) Notch signaling within cap cells leads to their maintenance and, indirectly, to GSC maintenance. Independently of its effect on Notch signaling, insulin/PI3K/FOXO pathway activation in cap cells intrinsically promotes stronger cap cell-GSC adhesion (presumably via E-cadherin; Hsu, 2009), which also promotes GSC maintenance. Further, aging also appears to influence insulin signaling levels in Drosophila females (Hsu, 2009), suggesting that physiological changes caused by diverse factors can impinge on this GSC regulatory network. Together, these studies underscore the importance of investigating how whole organismal physiology impacts stem cell function via effects on stem cells and on their niche, potentially via changes in local signaling (Hsu, 2011).

Notch signaling requires direct cell-cell contact because Notch ligands are membrane-bound proteins that induce Notch activation in neighboring cells. In addition to transactivating Notch in adjacent cells, the Notch ligand Delta also inhibits Notch in cis, thus creating a potent switch between high Delta expression/low Notch activity and high Notch activity/low Delta expression (Sprinzak, 2010). Differential Notch activation often underlies binary cell fate decisions. For example, during Drosophila sensory organ development, cells with high levels of Delta and low Notch activity become neurons, while those with elevated Notch activity and low Delta become epidermal cells (Hsu, 2011).

In the Drosophila GSC niche, Notch activity is detected in all cap cells, and Dl-lacZ is expressed in all terminal filament cells. A subset of cap cells also expresses Dl-lacZ, suggesting that some cap cells may express Delta and have high Notch activity simultaneously. The basal terminal filament cell, in which Dl is required for cap cell formation, does not contact all cap cells directly, and it was also found that Dl and Ser are not required within GSCs for cap cell formation or maintenance. It is therefore proposed that cap cells may signal to each other via Delta to activate Notch signaling, and that, in cap cells, Delta might not consistently act in cis to inhibit Notch activation (Hsu, 2011).

The observation that a subset of cap cells can express Dl-lacZ and Notch activity simultaneously is consistent with recent findings. Human eosinophils express both Notch and its ligands, and autocrine Notch signaling controls their migration and survival (Radke, 2009). Similarly, Notch is co-expressed with its ligands in rat hepatocytes following partial hepatectomy and also in normal human breast cells, although it is unclear if autocrine signaling occurs. It is therefore conceivable that Delta expressed in cap cells may stimulate Notch signaling via both paracrine and autocrine manners (Hsu, 2011).

Alternatively, Notch ligands might be secreted from terminal filament cells to stimulate Notch signaling in all cap cells and thereby promote their maintenance. In fact, a soluble form of Delta capable of stimulating Notch has been identified in Drosophila S2 cell cultures, and the ADAM disintegrin metalloprotease Kusbanian is required for the production of soluble Delta in culture. Further, Dl and kuzbanian genetically interact, raising the possibility that soluble forms of ligands might modulate Notch signaling in vivo (Hsu, 2011).

neur encodes an E3 ubiquitin ligase that mediates the endocytosis of Notch ligands in signal-sending cells, thereby enhancing their signaling strength. Contrary to a previous report, this study found no evidence that Notch ligands produced from GSCs are required for self-renewal. In contrast, neur is intrinsically required for GSC maintenance. Similarly, in the Drosophila testis, neur, but not Dl and Ser, is required for GSC maintenance, further indicating that Neuralized maintains GSCs via a Notch-independent pathway (Hsu, 2011).

neur mutant cysts exhibit large and highly branched fusomes, another Notch-independent phenotype. In principle, this aberrant fusome morphology might result from a defect in fusome growth and/or partitioning, or be secondary to an excessive number of cyst division rounds. Nevertheless, the close association of some of these abnormal fusomes with the cap cell interface suggests that fusome defects might lead to GSC loss. Ubiquitination regulates many processes, including protein degradation and vesicular trafficking. It is therefore possible that Neuralized ubiquitinates specific substrates that regulate fusome-related vesicular trafficking during cyst division. Future studies should test whether E3 ligase activity is indeed required for the role of neur in early germline cysts, identify key ubiquitination targets, and elucidate the molecular mechanisms they regulate (Hsu, 2011).

Under low insulin signaling, the FOXO transcriptional factor is required for extended longevity, reduced rates of proliferation, and stress resistance, among other processes. FOXOs are conserved from yeast to humans, and they control many target genes, different subsets of which modulate distinct processes. Drosophila FOXO negatively controls GSC division when insulin signaling is low (Hsu, 2008). It was also shown that insulin signaling modulates niche-stem cell interactions and Notch signaling in the niche (to control cap cell number), and that insulin signaling declines as females become older, leading to stem cell loss (Hsu, 2009). This study has shown that FOXO is required to negatively regulate Notch signaling within cap cells under low insulin activity and that FOXO also modulates the physical interaction between cap cells and GSCs. The multiplicity of FOXO roles in stem cell regulation is further underscored by studies in other stem cell systems. For example, FOXOs regulate several processes, including cell cycle progression, oxidative stress, and apoptosis, in the hematopoietic stem cell compartment, thereby influencing stem cell number and activity. It will be important to investigate how the specificity of FOXO is controlled and also whether or not FOXO regulates other stem cell niches, perhaps acting as a mediator of changes in niche size and/or activity during aging or cancer development (Hsu, 2011).

This study suggests a potentially novel mechanism by which the Notch and insulin pathways interact. In the Drosophila female GSC niche, insulin signaling does not control ligand transcription, and it is not required for ligand function (i.e., Dl is required in basal terminal filament cells during cap cell formation, but InR is not). Instead, both InR and N are cell autonomously required for cap cell maintenance, and insulin receptor function (via repression of FOXO) is required for proper Notch signaling. Expression of the intracellular domain of Notch rescues the low cap cell and GSC numbers of InR mutants (Hsu, 2009), and ovarian Notch expression does not appear altered in InR mutants. Therefore, it is speculated that FOXO inhibits the ability of cap cells to respond to Notch ligands by regulating a target that negatively regulates the series of proteolytic events responsible for the release of the intracellular domain of Notch. It cannot, however, be rulef out that Notch and FOXO normally interact at the level of target gene regulation but that overexpression of the intracellular domain of Notch overrides the normal inhibition by FOXO (Hsu, 2011).

These findings contrast with other types of interactions between FOXO and Notch that have been reported. During muscle differentiation in myoblast cultures, FOXO promotes (instead of antagonizing) Notch activity via a physical interaction that leads to activation of Notch target genes. Positive interactions between Notch and PI3K signaling have also been reported. Specifically, activation of the PI3K pathway potentiates Notch-dependent responses in CHO cells, T-cells, and hippocampal neurons. The suggested mechanism, however, involves the inactivation of GSK3 by Akt phosphorylation upstream of FOXO, which is distinct from the involvement of FOXO in the insulin-Notch signaling interaction within the GSC niche. These examples illustrate the diversity of modes of interaction between Notch and insulin signaling. It is conceivable that the positive interaction that is describe between insulin and Notch signaling pathways in the GSC niche may occur in other stem cell niches (Hsu, 2011).

Deregulated Notch signaling is associated with many types of cancers and, in some cases, it is thought that altered Notch signaling promotes cancer development by overstimulating the self-renewal of normal stem cells (Wang, 2009). Hyperactivation of insulin/IGF pathway is also linked to increased cancer risk and poor cancer prognosis. The Notch and insulin/IGF pathways have been reported to interact in cancerous cells via yet another mechanism. Specifically, upregulation of the Notch ligand Jagged 1 leads to PI3K activation in human papillomavirus-induced cancer lines. It is speculated that additional types of interactions between Notch and insulin/IGF signaling, such as the positive regulation of Notch activity by the insulin/PI3K/FOXO pathway that occurs in the Drosophila GSC niche, may also contribute to cancer progression (Hsu, 2011).

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