From stage 8 of oogenesis, and persistently throughout the rest of oogenesis, a unique 3.2 kb Fused transcript is produced in low amounts in nurse cells. In embryos, this transcript is evenly distributed in all embryonic cells until the extended germ band stage [Images], after which it decreases even further. Ubiquitous expression is detected later in imaginal wing and leg discs (Therond, 1993).

Effects of Mutation or Deletion

In fused mutants, the expression of engrailed and wingless is normal until germ band extension but disappears either partially (en) or totally (wg) at germ band retraction (stage 10). Maternal fused is sufficient to correct the segmentation pattern caused by fused mutation. fused-naked double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In the fused-naked double mutant embryo, en is expressed as in nkd mutant at germ band extension, but later this expression is restricted and becomes normal at germ band retraction. On the contrary, wg expression disappears as in fu simple mutant embryos (Limbourg-Bouchon, 1991).

Mutations in the ci locus alter the regulation of ci expression and can be used to examine ci function during development. A wing defect is present in animals mutant for fused. In ciCe/+ and fused mutants, the deletions between wing veins three and four correlate with increased ci protein levels in the anterior compartment (Slusarski, 1995).

fused (fu) is a maternal effect segment polarity gene of Drosophila melanogaster. In addition, fu females have tumorous ovaries. Two ethyl methanesulfonate mutageneses were carried out in order to isolate suppressors of the fu phenotype. A new gene, Suppressor of fused [Su(fu)], was identified. It is located in the 87C8 region of the third chromosome. Su(fu) displays a maternal effect and is also expressed later in development. Although Su(fu)LP is a complete loss-of-function mutation, it is homozygous viable and produces no phenotype by itself. Su(fu) fully suppresses the embryonic and adult phenotypes of fu mutants. Su(fu) mutations are semidominant and a Su(fu)+ duplication has the opposite effect, enhancing the fused phenotype. It is proposed therefore that the Su(fu)+ product is involved in the same developmental step as the Fu+ kinase. Thus, a new gene interacting with the segment polarity pathway was identified using an indirect approach (Preat, 1992).

fused is a segment-polarity gene encoding a putative serine-threonine kinase. In a wild-type context, all fu mutations display the same set of phenotypes. Nevertheless, mutations of the Suppressor of fused [Su(fu)] gene define three classes of alleles: fuO, fuI, and fuII. The Fused (Fu) protein functions in vivo as a kinase. The N-terminal kinase and the extreme C-terminal domains are necessary for Fu+ activity, while a central region appears to be dispensable. A striking correlation is observed between the molecular lesions of fu mutant alleles and the phenotype displayed as a result of the interaction of these alleles with Su(fu). Indeed, fuI alleles, which are suppressed by Su(fu) mutations, are defined by inframe alterations of the N-terminal catalytic domain, whereas the C-terminal domain is missing or altered in all fuII alleles. An unregulated FuII protein, which can be limited to the 80 N-terminal amino acids of the kinase domain, would be responsible for the neomorphic costal-2 phenotype displayed by the fuII-Su(fu) interaction. It is proposed that the Fu C-terminal domain can differentially regulate the Fu catalytic domain according to cell position in the parasegment (Therond, 1996a).

Ci proteolysis is inhibited in fu mutants and cos2 mutants. In wild-type wing imaginal discs, full-length Ci is found at high levels in a stripe along the AP compartment boundary and at low levels throughout the rest of the anterior compartment. High level Ptc is expressed in a thin stripe along the boundary. In discs mutant for fu, the high level Ci stripe is expanded, and the Ptc stripe is more diffuse with a modest protein level. In loss-of-function cos2 clones, Ci protein level is elevated and these clones cell-autonomously express high level Ptc. To examine whether Ci protein level in fu and cos2 mutants is up-regulated through inhibition of proteolysis, extracts from fu and cos2 hypomorphic discs were analyzed by SDS-PAGE and western blot. Proteolysis of Ci is not detectable in cos2 hypomorphic wing extracts and is significantly inhibited in extracts from both class I and class II fu mutant discs. Despite the inhibition of Ci proteolysis in fu mutants, such animals display evidence of compromised Ci activity, both in reduced ptc expression and fusion between LV3 and 4 in adult wings (Wang, 1999).

The genomic DNA sequence of a 2.4-kb region of the X-linked developmental gene fused was determined in 15 Drosophila virilis strains. One common replacement polymorphism is observed, where a negatively charged aspartic amino acid is replaced by the noncharged amino acid alanine. This replacement variant is located within the serine/threonine kinase domain of the fused gene and is present in ~50% of the sequences in the sample. Significant linkage disequilibrium is detected around this replacement site, although the fused gene is located in a region of the D. virilis X chromosome that seems to experience normal levels of recombination. In a 600-bp region around the replacement site, all eight alanine sequences are identical; of the six aspartic acid sequences, three are also identical. The occurrence of little or no variation within the aspartic acid and alanine haplotypes, coupled with the presence of several differences between them, is very unlikely under the usual equilibrium neutral model. These results suggest that the fused alanine haplotypes have recently increased in frequency in the D. virilis population (Vieira, 2000).

The primitive gonad of the Drosophila embryo is formed from two cell types, the somatic gonad precursor cells (SGPs) and the germ cells, which originate at distant sites. To reach the SGPs the germ cells must undergo a complex series of cell movements. While there is evidence that attractive and repulsive signals guide germ cell migration through the embryo, the molecular identity of these instructive molecules has remained elusive. Evidence is presented suggesting that hedgehog (hh) may serve as such an attractive guidance cue. Misexpression of hh in the soma induces germ cells to migrate to inappropriate locations. Conversely, cell-autonomous components of the hh pathway appear to be required in the germline for proper germ cell migration (Deshpande, 2001).

Known cell-autonomous components of the Hh signaling pathway also appear to be required in germ cells for normal migration behavior. Germline clones were used to test four different hh pathway genes -- ptc, pka, smo, and fu. For all four, abnormalities in germ cell migration were observed in the progeny. In the case of both the ptc and smo germline clones, eggs fertilized by wild-type sperm developed into completely normal adults. Moreover, there are no apparent defects in the formation of the somatic gonad or in the pattern of Clift expression. These findings would support the view that the migration defects seen in ptcmat-zyg+ and smomat-zyg+ embryos arise from cell-autonomous deficiencies in the response to Hh by the germ cells. However, it should be pointed out that there could be some undetected nonautonomous problem in somatic hh signaling in these embryos that induces abnormalities in germ cell behavior (Deshpande, 2001).

As would be expected from the known properties of these four genes in other well characterized hh pathways, the phenotypes produced by ptc and pka germline clones are similar and quite distinct from those observed for smo and fu. Moreover, the migration defects observed in ptc/pka and smo/fu germline clones can be explained by the antagonistic role of these genes in the hh signaling pathway. In the absence of maternal ptc or pka, smo and its downstream effectors in the hh pathway are activated in the germ cells independent of the Hh ligand. As a consequence, many of the germ cells clump together as they begin passing through the midgut, and then remain in place instead of migrating toward the SGP cells. Additionally, the mitotic cycle in ptcmat- (and to a lesser extent pkamat-) germ cells is inappropriately activated. Up regulation of cell division has been observed in somatic tumors that lack ptc function and in ptc mutant C. elegans germ cells. In the case of smo and fu, the germ cells can't respond to the Hh ligand, and they are unable to detect or associate with the SGP cells, and instead migrate randomly through the mesoderm (Deshpande, 2001).

During Drosophila wing development, Hedgehog (Hh) signaling is required to pattern the imaginal disc epithelium along the anterior-posterior (AP) axis. The Notch (N) and Wingless (Wg) signaling pathways organise the dorsal-ventral (DV) axis, including patterning along the presumptive wing margin. A functional hierarchy of these signaling pathways is described that highlights the importance of the competing influences of Hh, N, and Wg in establishing gene expression domains. Investigation of the modulation of Hh target gene expression along the DV axis of the wing disc has revealed that collier/knot (col/kn), patched, and decapentaplegic are repressed at the DV boundary by N signaling. Attenuation of Hh signaling activity caused by loss of fused function results in a striking down-regulation of col, ptc, and engrailed (en) symmetrically about the DV boundary. This down-regulation depends on activity of the canonical Wg signaling pathway. It is proposed that modulation of the response of cells to Hh along the future proximodistal (PD) axis is necessary for generation of the correctly patterned three-dimensional adult wing. These findings suggest a paradigm of repression of the Hh response by N and/or Wnt signaling that may be applicable to signal integration in vertebrate appendages (Glise, 2002).

Analysis of Hh target gene expression close to the DV boundary in fu mutants has revealed that activation of col and dpp and up-regulation of ptc transcription is lost from the center of the wing pouch when Hh signaling is attenuated. Inactivation of fu results in a complete loss of en expression from the anterior compartment; however, the ectopic activation of en induced by submaximal Hh pathway activation (achieved by removing Pka-C1 activity) is similarly sensitive to inhibitory influences emanating from the DV boundary. In each case, the repression of Hh targets extends up to six rows of cells away from the DV boundary in the anterior compartment. This down-regulation is due to activation of the canonical Wg signaling pathway. At what level is this global inhibition of Hh targets by Wg signaling achieved? One possibility is that Hh target genes are regulated by the opposing effects of activator and repressor complexes acting directly via cis-acting sequences. Such a situation has previously been described for the stripe gene, which is directly activated by Hh signaling and repressed by Wg signaling in the Drosophila embryo. This mechanism would imply the existence of cis-acting sites for both the Ci and dTCF/pangolin proteins within the regulatory regions of each of the Hh target genes -- col, ptc, dpp, and en -- that have been analysed, a possibility that cannot currently be verified due to the absence of sequence information for all of these regions. An alternative scenario, however, is that Wg signaling modulates the activity of the Hh signaling pathway itself, perhaps at the level of the Su(fu)/Fu/Ci protein complex to affect localization and/or activity of Ci. Consistent with this possibility, recent studies have provided evidence for a direct protein-protein interaction between the vertebrate Su(fu) and ß-Catenin/Armadillo proteins. Such an interaction might mediate the attenuation of Hh signaling activity, for instance, by promoting the cytoplasmic retention of Ci. It should be noted, however, that no modulation of Ci distribution has been detected along the DV axis using the available anti-Ci antibodies in the fu mutant backgrounds, the pka-C1, or the pka-C1;wg clones (Glise, 2002).

The development of multicellular organisms requires the establishment of cell populations with different adhesion properties. In Drosophila, a cell-segregation mechanism underlies the maintenance of the anterior (A) and posterior (P) compartments of the wing imaginal disc. Although engrailed (en) activity contributes to the specification of the differential cell affinity between A and P cells, recent evidence suggests that cell sorting depends largely on the transduction of the Hh signal in A cells. The activator form of Cubitus interruptus (Ci), a transcription factor mediating Hh signaling, defines anterior specificity, indicating that Hh-dependent cell sorting requires Hh target gene expression. However, the identity of the gene(s) contributing to distinct A and P cell affinities is unknown. A genetic screen based on the FRT/FLP system has been to search for genes involved in the correct establishment of the anteroposterior compartment boundary. By using double FRT chromosomes in combination with a wing-specific FLP source, 250,000 mutagenized chromosomes were screened. Several complementation groups affecting wing patterning have been isolated, including new alleles of most known Hh-signaling components. Among these, a class of patched (ptc) alleles was identified exhibiting a novel phenotype. These results demonstrate the value of this setup in the identification of genes involved in distinct wing-patterning processes (Végh, 2003).

A total of 250,000 mutant chromosomes covering the X chromosome and both major autosomes were screened. Four complementation groups were identified that affected wing patterning similar to mutations in smo. The largest of these groups represents alleles in smo itself. Two groups exhibiting a subset of smo phenotypes represent new alleles of fused and collier/knot. Fused is a positive regulator of Hh signaling, and collier/knot is an Hh target gene required for the formation of the L3/L4 intervein region. Surprisingly, the remaining complementation group turned out to consist of novel ptc alleles with striking characteristics. Molecularly, they represent point mutations causing an amino acid substitution in either the first or the second large extracellular loop. In contrast to ptc null alleles, homozygous mutant clones failed to upregulate Hh target genes even in the presence of Hh. Together these findings suggest that the mutant proteins repress Smo constitutively, most likely because they fail to bind Hh. Animals mutant for trans-heterozygous combinations of these new ptc alleles with ptcS2 are fully viable. The ptcS2 product lacks the ability to repress Smo but is able to sequester, and hence bind to, Hh. The intragenic complementation that was observed suggests that both functions of Ptc, binding of Hh and repression of Smo, can be provided by individual proteins that possess only one of each. Recently, it was shown that a combination of two proteins, one consisting of the N- and the other the C-terminal half of Ptc, reconstitutes Ptc function. Although these experiments cannot be directly compared with the findings in this study, together they do suggest that Ptc function can be separated intramolecularly into independent modules of N- vs. C-terminal and extra- vs. intracellular domains. One possible scenario that could explain the intragenic complementation would be if Ptc proteins act in a multimeric complex (Végh, 2003).

Fused, Hedgehog signaling and oogenesis

The fused gene encodes a serine/threonine kinase involved in Hedgehog signal transduction during Drosophila embryo and larval imaginal disc development. Additionally, fused mutant females exhibit reduced fecundity that is associated with defects in three aspects of egg chamber formation: encapsulation of germline cysts by prefollicular cells in the germarium, interfollicular stalk morphogenesis and oocyte posterior positioning. Using clonal analysis it has been shown that fused is required cell autonomously in prefollicular and pre-stalk cells to control their participation in these aspects of egg chamber formation. In contrast to what has been found for Hedgehog and other known components of Hedgehog signal transduction, fused does not play a role in the regulation of somatic stem cell proliferation. However, genetic interaction studies, as well as the analysis of the effects of a partial reduction in Hedgehog signaling in the ovary, indicate that fused acts in the classical genetic pathway for Hedgehog signal transduction, which is necessary for somatic cell differentiation during egg chamber formation. Therefore, a model is proposed in which Hedgehog signals at least twice in germarial somatic cells: initially through a fused-independent pathway to control somatic stem cell proliferation, and next, through a classical fused-dependent pathway to regulate prefollicular cell differentiation (Besse, 2002).

Egg chamber formation in the ovary requires a somatic cell developmental program that involves (1) somatic stem cell self-renewing divisions; (2) prefollicular cell morphogenesis for germline cyst encapsulation, anchoring/positioning of the oocyte posteriorly, and interfollicular stalk formation, and (3) prefollicular cell differentiation into three cell types -- stalk, polar and follicular epithelial cells. However, it is not clear as yet how prefollicular cell morphogenesis and differentiation are integrated. This study of fu mutant ovaries, which produce ovarioles containing multicyst and apposed egg chambers, has revealed that, in contrast to what has been shown for other components of Hh signal transduction, fu function is not required for the first step of this program, the proliferation of somatic stem cells. In addition, unlike other genes that, when mutated, lead to defective egg chamber formation (e.g. mutations that affect components of the Notch/Delta signaling pathway), fu is not required for the third step in this program -- stalk and polar cell specification and formation of the follicular epithelium. Rather, this analysis revealed several specific defects in prefollicular cell behavior during egg chamber formation, all involving cell-cell recognition and adhesion, cell shape changes, and cell motility (Besse, 2002).

Germline cyst encapsulation requires extension of cellular processes by prefollicular cells in regions 2a/2b of the germarium, such that they can recognize and adhere to mature 16-cell germline cysts, and subsequently migrate centripetally between individual cysts. Interfollicular stalk formation requires that pre-stalk cells in regions 2b/3 lose heterotypic adhesion to germline cells and gain homotypic adherence and the capacity to intercalate. The effector molecules implicated in these processes have not been characterized to a great extent, though several surface membrane and cytoskeletal proteins that have been shown to exhibit dynamic expression patterns in prefollicular cells and their descendants are likely to be involved. For example, several proteins (actin, Fas III, Hts, alpha-Spectrin, Filamin and others) are localized specifically to the cellular processes that prefollicular cells extend over germline cysts, and most of these are subsequently concentrated apically in pre-stalk cells just before their intercalation. Once the interfollicular stalk is formed, the expression of some of these proteins is downregulated in stalk cells (Fas III), while other proteins are expressed laterally in these cells (actin, Hts, alpha-Spectrin and PS1-ß integrin). Finally, proper expression of the Shotgun (DE-Cadherin), Armadillo/ß-Catenin and alpha-Catenin cell-cell adhesion complex at the membrane of both the posterior follicle cells and the oocyte probably mediates contact between these two cell types and posterior positioning of the oocyte (Besse, 2002).

In fu mutant ovarioles, encapsulation of multiple cysts in a single egg chamber is associated with absence of prefollicular cell extensions around germline cysts and impaired centripetal migration of these cells. In addition, stalk formation in fu mutants is, in less affected individuals (young females with normal egg chambers), slow/delayed and, in more severely affected individuals (older females with multicyst egg chambers), very irregular (leading to abnormal stalk morphology). By following the expression of Shotgun, which marks the apical membrane of pre-stalk cells, it has been shown that, in fu mutants, pre-stalk cells that have migrated centripetally between germline cysts are blocked before the intercalation process. Induction of fu mutant clones in prefollicular cells leads to the same types of encapsulation and stalk morphogenesis defects, indicating cell autonomous function in these cells for these processes. This study highlighted the impaired ability of fu mutant prefollicular cells to migrate between germline cysts and to participate to interfollicular stalk formation. This mosaic analysis also showed that fu mutant and wild-type cell populations have a tendency to remain segregated, implying that surface differences between these cells prevent their intermixing. Finally, fu function in prefollicular cells is implicated in another process involving specific cell-cell interactions, posterior positioning of the oocyte in the egg chamber. Taken together, these results suggest a function for fu in prefollicular cells for appropriate expression of one or several surface membrane or cytoskeletal proteins necessary for several aspects of prefollicular cell morphogenesis during egg chamber formation. Although the expression of a number of cytoskeletal and membrane proteins was examined in prefollicular cells in fu mutants (for example, Shotgun, Fas III and others), so far it has not been possible to relate the anomalies observed to a loss in expression or in polarized localization of any of these proteins. The actual cell adhesion effectors that may be regulated by differential Hh signal transduction in wing development, as is the case for germ cell migration and egg chamber formation, remain to be determined (Besse, 2002).

In the ovary, Hh signals from the terminal filament and cap cells and is required for somatic stem cell (SSC) proliferation and subsequently for egg-chamber budding. SSC self-renewing properties are not maintained in the absence of Hh signaling, whereas excessive Hh signaling produces supernumerary stem cells. In addition to the membrane receptors Ptc and Smo, Ci has been implicated in this process as a component of Hh signal transduction. However, in a hh loss-of-function context, SSC proliferation is restored by induction of low levels of somatic Hh signaling in SSC (achieved by removing protein kinase A function, an inhibitor of Ci activity, in these cells). It has therefore been suggested that, as in wing imaginal disc development, where fu activity is required for transducing high but not low levels of Hh signaling, fu activity may not be endogenously required for regulation of SSC division. Results obtained upon induction of fu mutant clones, as well as the quantitation of the mitotic activity of somatic cells in fu mutant germaria, confirm that fu is not necessary for SSC proliferation, suggesting that Hh signals to SSC through a Fu-independent pathway (Besse, 2002).

fu endogenous function is required in prefollicular cells for acquisition of specific morphogenetic properties. In addition, several lines of evidence are provided for a role for fu in a classical Hh signal transduction pathway within prefollicular cells for their participation in egg chamber formation. (1) fu and hh mutant ovarian phenotypes overlap, since both result in aberrant somatic cell behavior and formation of multicyst and apposed egg chambers. (2) fu is necessary, downstream of hh, for the expression of an ovarian somatic ptc-lacZ enhancer-trap. (2) fu ovarian phenotypes can be partially suppressed by removing either one or two copies, respectively, of two negative regulators of Hh signaling [Cos2 and Su(fu)], or by overexpressing the transcription factor Ci. (4) The morphogenetic defects described for fu mutant prefollicular cells can be phenocopied by somatic overexpression of either Cos2 or the inhibitory Cicell proteins. A model is therefore proposed in which Hh signals at least twice in germarial somatic cells: once through a fu-independent pathway to control SSC proliferation and again through a classical fu-dependent pathway to regulate early aspects of prefollicular cell differentiation. Therefore, fu loss-of-function mutations, which affect only prefollicular cell morphogenesis, allow the analysis of the specific role of Hh signal transduction in this process (Besse, 2002).

Interestingly, previous studies focusing mostly on the effects of excessive Hh signal transduction in the ovary also indicated that two different stages of somatic ovarian cell development in the germarium are targeted by this signaling molecule: early on (region 2a/2b), SSC proliferation and oocyte posterior positioning are affected; and later (region 2b/3) there is an apparent delay in the prefollicular cell development program, which, when combined with early effects on SSC proliferation, leads to the formation of giant stalks comprising poorly differentiated prefollicular cells between early egg chambers, delayed polar cell specification (stage 4 instead of 2) and an excess of these cells, and continued follicular epithelial cell division after stage 6. In fu mutants there is no effect on SSC or follicular cell proliferation, but some of the defects affecting prefollicular cells are similar, including non-posterior oocyte positioning, delayed prefollicular cell differentiation leading to delayed egg chamber budding and delayed polar cell specification. In addition, both somatic fu and ptc mutant clones show striking segregation from wild-type cells, fu mutant clones preferentially localized to the follicular epithelium, whereas ptc mutant clones localized to the stalks. These results indicate that cellular differences in Hh signal transduction levels, whether reduced (fu) or increased (ptc) compared with wild-type levels, affect the cell-cell recognition and adhesive properties of prefollicular cells. Taken together, these studies show that there is an overlap between the ovarian phenotypes associated with a reduction and an increase in Hh signaling, indicating that crucial levels of Hh signaling are required for prefollicular cell morphogenesis (Besse, 2002).

Nonetheless, fu mutations do not completely arrest egg chamber budding, rather causing a delay in several aspects of the prefollicular cell developmental program, including stalk and polar cell specification. Even fu mutant clones induced in prefollicular cells using the strong hypomorphic allele, fumH63, did not provoke more severe anomalies than the other alleles used in this study. These results suggest that prefollicular cell development does not depend solely on fu-dependent Hh signaling and that there is possibly some redundancy in the regulation of this process. Indeed, other studies have shown the importance of germline-emitted signals, in particular the secreted molecules Egghead, Brainiac and Gurken/TGFalpha, for the encapsulation of germline cysts by prefollicular cells. In addition, specification of polar and stalk cells via germline-to-soma signaling involving Delta/Notch, is also necessary for proper egg chamber formation. It is possible then that the correct timing of events in the mid-germarial region for proper encapsulation and egg chamber budding is achieved by two signaling sources, the terminal filament (Hh signaling) and mature 16-cell germline cysts (Egghead, Brainiac, Gurken/TGF-alpha, and Delta signaling). The integration of all of these signals by prefollicular cells would be necessary for these cells to go through their developmental program in the appropriate time frame, thus allowing synchronous germline cyst maturation and encapsulation by prefollicular cells (Besse, 2002).

Polar cells are pairs of specific follicular cells present at each pole of Drosophila egg chambers. They are required at different stages of oogenesis for egg chamber formation and establishment of both the anteroposterior and planar polarities of the follicular epithelium. Definition of polar cell pairs is a progressive process since early stage egg chambers contain a cluster of several polar cell marker-expressing cells at each pole, while as of stage 5, they contain invariantly two pairs of such cells. Using cell lineage analysis, it has been demonstrated that these pre-polar cell clusters have a polyclonal origin and derive specifically from the polar cell lineage, rather than from that giving rise to follicular cells. In addition, selection of two polar cells from groups of pre-polar cells occurs via an apoptosis-dependent mechanism and is required for correct patterning of the anterior follicular epithelium of vitellogenic egg chambers. Prevention of pre-polar cell death and subsequent generation of supernumerary polar cells may lead to production of an excess of signaling molecules, such as Unpaired, and alteration of endogenous morphogen gradients which could explain why both squamous cells and border cells exhibit aberrant behavior when pre-polar cell death is blocked (Besse, 2003).

Thus, each pair of mature polar cells derives from a pool of precursor pre-polar cells within which supernumerary cells are eliminated via an apoptosis-dependent mechanism. This mechanism probably requires both caspase activity and the 'death' gene reaper, since death is inhibited by ectopic expression of the bacculoviral p35 protein and is associated with specific induction of reaper expression. However, whereas the self-death machinery appears to be evolutionary conserved, a wide range of distinct signaling mechanisms can be used to elicit apoptosis. Cellular interactions within or without the pre-polar cell cluster may also be crucial for regulation of the selective pre-polar cell loss. In the present study, no correlation could be made between pre-polar cell position and cell removal, at least for apoptosis events occurring after egg chamber budding. It would be interesting nonetheless to examine Notch signaling as a survival factor in this system. Indeed, induction of Notch loss-of-function clones in prefollicular cells is associated with absence of polar cells. Conversely, egg chambers with terminal clones expressing an activated form of Notch contain up to 6 polar cell marker-positive cells. Such phenotypes, interpreted as reflecting a role for Notch signaling in polar cell specification, could also correspond to a Notch-dependent control of apoptosis within the pre-polar cell lineage (Besse, 2003).

The fused gene encodes a serine/threonine kinase identified as a positive effector of the Hedgehog signal transduction pathway. In the ovary, Hedgehog signal transduction controls somatic stem cell (SSC) proliferation. Indeed, SSC self-renewing properties are not maintained in the absence of Hh signaling, whereas excessive Hh signaling produces supernumerary stem cells and leads to the accumulation of poorly differentiated somatic cells between egg chambers. Analysis of fu mutations had indicated that fu function is not involved in this process. Rather, fu-dependent Hedgehog signal transduction is necessary for somatic prefollicular cell differentiation and morphogenesis. In particular, fu function seems to be required for correct timing of the polar cell differentiation program. Indeed, fu mutant females exhibit a global shift in the dynamics of A101 staining, as visualized after anti-ß-galactosidase staining of fuJB3/fuJB3; A101/+ females. (1) The appearance of A101 staining is delayed, since 28% of stage 2 fu egg chambers do not exhibit any marked anterior cells compared to 19% in heterozygous sisters. (2) Restriction of A101 staining to 2 polar cells is also delayed, since 60% of stage 3 and 19% of stage 4 fu egg chambers contained 3 or more stained anterior cells compared to 33% and 4%, respectively, for fu+ egg chambers. Strikingly, 100% of stage 5 fu mutant egg chambers exhibit only 2 A101+ cells, indicating that restriction in the number of polar cells does eventually occur as in wild-type ovarioles. Altogether, these results suggest that fu mutations lead to a delay in the polar cell differentiation program (Besse, 2003).

Close examination of fu ovarioles has revealed that a higher proportion of groups containing 4 A101+ cells, 5 A101+ cells (8/156), and even 6 A101+ cells (1/156) can be found in stage 2 as well as stage 3 fu mutant egg chambers compared to the wild-type situation. It was reasoned that the presence of such groups of cells could result either from abnormally slow apoptosis-dependent elimination of pre-polar cells, or from an overproduction of pre-polar cells or their precursors. The first hypothesis could not be tested directly because the relatively low number of TUNEL-positive cells found in both wild-type and fused females (possibly due to rapid elimination of apoptotic cells) made it impossible to compare quantitatively the dynamics of polar cell apoptotic cell death between these two contexts. Therefore the second hypothesis was tested; defects were sought in polar and pre-polar cell proliferation, or in the number of polar cell precursor cells. First, polar cell proliferative properties do not seem to be altered in the vitellarium of fu ovarioles since (1) no increase in the size of A101+ terminal clusters is observed with increasing age of fu egg chambers from stage 2 to 5, and (2) no prolongation beyond stage 6 of somatic cell mitotic activity is observed in fu ovarioles. Second, using a dominantly marked clone approach, it has been shown that early clusters of 4-6 A101+ cells found in fu females never contain more than 2 GFP+ cells, and therefore that they do not result from extra divisions of precursor cells within the germarium. Third, it was reasoned that preventing apoptosis in the polar cell lineage in a fu mutant context should give an indication about the number of polar cell precursors present in these flies. If the number of such precursors is greater in fu females than in wild-type females, then blocking apoptosis should result in a greater number of 'rescued' cells in polar cell clusters than in a wild-type context (that is more than 6 cells). Therefore, the flp-out/Gal4 system was used to generate large somatic clones of p35 overexpressing cells in a fu mutant context. Although an increase in the average size of the terminal Fas III+ cell cluster was observed after p35 overexpression, only groups containing 2 to 6 cells were recovered. This indicates that fused females contain the same number of polar cell precursors as wild-type females (Besse, 2003).

Therefore, the supernumerary polar cells in both wild-type and fused mutant contexts is interpreted to represent pre-polar cells, and it is proposed that slower apoptosis-mediated reduction in the number of these cells in a fused context allows easier visualization of these cells. Thus, fused mutations, by delaying the somatic cell differentiation program, confirm the existence of pre-polar cell clusters and allow detection of up to 6 pre-polar cells. However, restriction in the final number of polar cells is achieved by stage 5 and is probably also mediated by apoptosis since TUNEL-positive A101+ cells are found in fused females (Besse, 2003).

fused regulates germline cyst mitosis and differentiation during Drosophila oogenesis

The fused gene encodes a serine-threonine kinase that functions as a positive regulator of Hedgehog signal transduction in Drosophila embryogenesis, wing morphogenesis, and somatic cell development during oogenesis. This study characterizes the germline ovarian tumors present in adult ovaries of fused mutant females, a phenotype not observed upon deregulation of any other component of Hedgehog signaling. In the strongest fused mutant contexts, tumorous ovarian follicles accumulate early spectrosome-containing germ cells corresponding to germline stem cells and/or early cystoblasts as evidenced by activated Dpp signal transduction and transcriptional repression of bag-of-marbles, encoding the cystoblast determination factor. These early germ cells are maintained far from their usual position in a specialized niche of somatic cells in the apical part of the germarium, which appears normal in size in fused mutant ovarioles. Therefore, these results indicate a novel function for fused in downregulation of Dpp signaling which is necessary for de-repression of bag-of-marbles and consequent cystoblast determination. The abnormal accumulation of these early germ cells seems to be due primarily to defects in differentiation, since germline stem cell proliferation in the germarium is not affected. A later block in germline development, at the 16-cell cyst stage before significant nurse cell and oocyte differentiation, is also observed in tumorous follicles when fused function is only partially lowered. Finally, fused mutant ovaries exhibit some germline cysts having undergone a supernumerary fifth mitotic division. Through clonal analysis, evidence is provided that fused regulates these cystocyte divisions cell autonomously, while the tumorous phenotype probably reflects both a somatic and germline requirement of fused for cyst and follicle development (Narbonne-Reveau, 2006).

The fused ovarian tumor phenotype, like that of other ovarian tumor mutants in Drosophila, was discovered several decades ago, but the precise characterization of the associated germline defects has not been carried out until now. The characteristics of fused ovarian tumors, based on past and present studies can be summarized as follows:

  • (1) fused mutations affect female, but not male, germline development;
  • (2) fused ovarian tumors likely reflect both a somatic and germline requirement for fused function;
  • (3) fused mutations affect female germline development at three points: (i) cystoblast determination, (ii) nurse cell/oocyte differentiation and (iii) four rounds of cystocyte mitosis;
  • (4) fused function in germline cyst development is largely Hh-independent, while the present work shows that Dpp signaling is deregulated by fused mutations (Narbonne-Reveau, 2006).

This analysis of ovarian tumors from females bearing strong and weaker fused mutant alleles reveals blocks at two points in germline development, cystoblast determination and nurse cell/oocyte differentiation, respectively. In females carrying strong hypomorphic fu alleles (class I or II) over a small deficiency fuZ4 (class 0), the majority of the ovarioles are abnormal and tumorous germaria and follicles are filled with spectrosome-containing germ cells expressing the dad-lacZ Dpp target reporter transgene and not expressing the bamp-Bam:GFP construct, characteristics shared with germline stem cells and early undetermined cystoblasts. Indeed, maintenance of germline stem cells has been shown to require repression of bam transcription via Dpp/BMP signal transduction and consequent phosphorylated-Mad binding to a silencer element in the bam promoter. In wild-type, bam repression occurs only at the apical end of the germarium because of the existence of a somatic cell niche (terminal filament and cap cells), in which dpp/bmp is specifically expressed, while cystoblast differentiation requires bam de-repression as the germ cells move away from the Dpp-expressing niche. In fu tumorous ovarioles, dad-lacZ expression and bam promoter repression is maintained at a considerable distance from the apical end of the germarium, even though the size of the somatic cell niche is not altered as assayed by specific expression of a hh-lacZ reporter construct. In addition, the excess in Dpp-activated early germ cells in fu mutant ovarioles cannot be attributed to elevated mitotic activity of the germline stem cells in the niche. It is not clear, however, whether the Dpp-activated early germ cells in fu mutants are latent or continue dividing with complete cytokinesis to give two spectrosome-containing daughter cells. Nonetheless, the results suggest a novel Hh-independent function for fu in Dpp signaling: fu function may be necessary to restrict Dpp signal production in somatic cells or to downregulate Dpp signal transduction in early germ cells so that bam transcription may be de-repressed allowing consequent cystoblast differentiation to occur. In fu mutants, an elevated level of Dpp signal or of Dpp signal transduction would lead to extended repression of bam and maintenance of the GSC/early cystoblast state beyond the niche. Unlike fu, however, mutations in bam affect both female and male germ cell development in Drosophila. Since germline stem cell maintenance and bam repression in the testis depends primarily on Gbb/BMP signaling, rather than on Dpp signaling as in the ovary, it is possible that fu mutations specifically perturb Dpp signaling and, therefore, only affect oogenesis (Narbonne-Reveau, 2006).

Analysis of weaker fu hypomorphic contexts revealed a different type of tumorous follicle exhibiting a later block in germline development. Females homozygous for different hypomorphic fu alleles (class I and II) present a low to moderately high proportion of tumorous follicles. In these females, tumorous follicles contain dozens of disorganized, immature germline cysts, including numerous 16-cell cysts (and probably 32-cell cysts) blocked before significant nurse cell and oocyte maturation. Although these germline cysts are present in follicles that have separated from the germarium, they exhibit characteristics of cysts normally found only in the germarium, like the presence of branched fusomes and indicators of mitotic activity (bamp-Bam:GFP construct and Phosphohistone H3). Even though oocyte determination does occur in some of the germline cysts in these fu tumorous follicles, as evidenced by oocyte-specific expression of Orb and the synaptonemal complex component C3G, further growth and maturation of nurse cells and oocyte is impeded. It is not possible to affirm, however, whether the block in nurse cell and oocyte development in these follicles reflects a direct role for fu in this specific step of the differentiation program or an indirect effect of the perturbation of interdependent processes. Indeed, one possibility is that inclusion of numerous cysts in one follicle leads to a block in further cyst development due to reduced efficiency of crucial intercellular communication between somatic and germ cells. In favour of this hypothesis, when only a few germline cysts are present in a follicle (2-4 cysts) due to defects in encapsulation by fu mutant prefollicular cells, significant nurse cell and oocyte development is observed (Narbonne-Reveau, 2006).

Finally, analysis of fu mutant ovaries revealed another defect concerning germline cyst development, in particular, the presence of cysts having undergone a supernumerary mitotic division, that is five rounds of mitosis instead of four, producing 31 nurse cells and one oocyte with five ring canals. Significantly, this defect is detected upon induction of fu mutant germline clones, but is not encountered upon induction of fu mutant prefollicular cell clones, thereby indicating that cell autonomous fu function contributes to cell cycle arrest after the fourth round of cystocyte divisions. This mutant phenotype is of particular interest since few factors controlling the number of germline cyst divisions have been identified so far. However, the existence of a ‘counting factor’, whose quantity would be limiting after four rounds of mitosis has long been postulated. The cytoplasmic Bam protein has some characteristics of such a factor since it accumulates specifically in dividing cystocytes. In addition, a genetic interaction has been shown between bam and encore, the latter encoding a gene function specifically required for restriction of germline cyst division. Using a bamp-bam:GFP transgene it was shown that expression of this reporter persists abnormally in 16-cell cysts in fu tumorous follicles. In addition, other markers that are normally expressed only in germline cysts in the germarium, including the fusome, also linger on in germline cysts in fu tumorous follicles indicating severely delayed development of these cysts. These are therefore likely important elements contributing to the disturbance of a counting mechanism and, consequently, occurrence of a fifth mitotic division in some germline cysts in fu mutant ovarioles. How fu function participates, directly or indirectly, to this counting mechanism is not clear, but the results provide a link between fu and cell cycle control (Narbonne-Reveau, 2006).

fused is expressed in both somatic and germline cells of the ovary. Although induction of either fu mutant germline clones or fu mutant prefollicular cell clones, produces cell autonomous defects in cyst development (fifth mitotic division) and cyst encapsulation by prefollicular cells, respectively, this type of analysis did not lead to the production of ovarian tumors. It is possible that the tumorous phenotype in fu mutants reflects both a somatic and germline requirement for fu function. In view of these results, one other attractive hypothesis is that fu function may be necessary in anterior somatic cells of the germarium (difficult to study using clonal analysis) for regulating soma to germline signaling, such as that mediated by Dpp, for germline cyst development (Narbonne-Reveau, 2006).


Alves, G., et al. (1998). Modulation of Hedgehog target gene expression by the Fused serine--threonine kinase in wing imaginal discs. Mech. Dev. 78(1-2): 17-3. PubMed Citation: 9858670

Besse, F., Busson, D. and Pret, A.-M. (2002). Fused-dependent Hedgehog signal transduction is required for somatic cell differentiation during Drosophila egg chamber formation. Development 129: 4111-4124. 12163413

Besse, F. and Pret, A.-M. (2003). Apoptosis-mediated cell death within the ovarian polar cell lineage of Drosophila melanogaster. Development 130: 1017-1027. 12538526

Blanchet-Tournier, M. F. et al. (1995). The segment-polarity gene fused is highly conserved in Drosophila. Gene 161: 157-162. PubMed Citation: 7665071

Claret, S., Sanial, M. and Plessis, A. (2007). Evidence for a novel feedback loop in the Hedgehog pathway involving Smoothened and Fused. Curr. Biol. 17(15): 1326-33. Medline abstract: 17658259

Delattre, M., et al. (1999). The Suppressor of fused gene, involved in Hedgehog signal transduction in Drosophila, is conserved in mammals Dev. Genes Evol. 209: 294-300. 11252182

Deshpande, G., et al. (2001). Hedgehog signaling in germ cell migration. Cell 106: 759-769. 11572781

Dussillol-Godar, F., et al. (2005). Modulation of the Suppressor of fused protein regulates the Hedgehog signaling pathway in Drosophila embryo and imaginal discs. Dev. Biol. 129: 53-66. 16413525

Glise, B., Jones, D. L. and Ingham, P. W. (2002). Notch and Wingless modulate the response of cells to Hedgehog signaling in the Drosophila wing. Dev. Bio. 248: 93-106 . 12142023

Friggi-Grelin, F., Lavenant-Staccini, L. and Therond, P. (2009). Control of antagonistic components of the Hedgehog signaling pathway by microRNAs in Drosophila. Genetics 179: 429-439. PubMed Citation: 18493062

Hooper, J. E. (1994). Distinct pathways for autocrine and paracrine Wingless signalling in Drosophila embryos. Nature 372: 461-464. PubMed Citation: 7984239

Hooper, J. E. (2003). Smoothened translates Hedgehog levels into distinct responses. Development 130: 3951-3963. 12874118

Horabin, J. I., et al. (2003). A positive role for Patched in Hedgehog signaling revealed by the intracellular trafficking of Sex-lethal, the Drosophila sex determination master switch. Development 130: 6101-6109. 14597576

Humke E. W., et al. (2010). The output of Hedgehog signaling is controlled by the dynamic association between Suppressor of Fused and the Gli proteins. Genes Dev. 24: 670-682. PubMed Citation: 20360384

Ingham, P. W. (1993). Localized hedgehog activity controls spatial limits of wingless transcription in the Drosophila embryo. Nature 366: 560-2. PubMed Citation: 8255293

Jia, J., Tong, C. and Jiang, J. (2003). Smoothened transduces Hedgehog signal by physically interacting with Costal2/Fused complex through its C-terminal tail. Genes Dev. 17: 2709-2720. 14597665

Kogerman, P., et al. (1999). Mammalian Suppressor-of-Fused modulates nuclear-cytoplasmic shuttling of GLI-1. Nat. Cell Biol. 1: 312-319. PubMed Citation: 10559945

Kuzhandaivel, A., Schultz, S. W., Alkhori, L. and Alenius, M. (2014). Cilia-mediated Hedgehog signaling in Drosophila. Cell Rep 7(3): 672-80. PubMed ID: 24768000

Lefers, M. A., Wang, Q. T. and Holmgren, R. A. (2001). Genetic dissection of the Drosophila Cubitus interruptus signaling complex. Dev. Bio. 236: 411-420. 11476581

Limbourg-Bouchon, B., Busson, D. and Lamour-Isnard, C. (1991). Interactions between fused, a segment-polarity gene in Drosophila, and other segmentation genes. Development 112: 417-29. PubMed Citation: 1794312

Lum, L., et al. (2003). Hedgehog signal transduction via Smoothened association with a cytoplasmic complex scaffolded by the atypical kinesin, Costal-2. Molec. Cell 12: 1261-1274. 14636583

Maloverjan A., et al. (2010). Identification of a novel serine/threonine kinase ULK3 as a positive regulator of Hedgehog pathway. Exp. Cell Res. 316: 627-637. PubMed Citation: 19878745

Methot, N. and Basler, K. (1999). Hedgehog controls limb development by regulating the activities of distinct transcriptional activator and repressor forms of Cubitus interruptus. Cell 96: 819-831. PubMed Citation: 10102270

Methot, N. and Basler, K. (2000). Suppressor of Fused opposes Hedgehog signal transduction by impeding nuclear accumulation of the activator form of Cubitus interruptus. Development 127(18): 4001-4010. 10952898

Monnier, V., et al. (1998). Suppressor of fused links fused and Cubitus interruptus on the hedgehog signalling pathway. Curr. Biol. 8(10): 583-6

Motzny, C. K. and Holmgren, R. (1995). The Drosophila cubitus interruptus protein and its role in the wingless and hedgehog signal transduction pathways. Mech Dev 52: 137-150

Murone, M., et al. (2000). Gli regulation by the opposing activities of Fused and Suppressor of Fused. Nat. Cell Biol. 2: 310-312. 10806483

Narbonne-Reveau, K., Besse, F., Lamour-Isnard, C., Busson, D. and Pret, A. M. (2006). fused regulates germline cyst mitosis and differentiation during Drosophila oogenesis. Mech. Dev. 23(3): 197-209. 16516445

Ogden, S. K., et al. (2006). Smoothened regulates activator and repressor functions of Hedgehog signaling via two distinct mechanisms. J. Biol. Chem. 281(11): 7237-43. 16423832

Ohlmeyer, J. T. and Kalderon, D. (1998). Hedgehog stimulates maturation of Cubitus interruptus into a labile transcriptional activator. Nature 397(6713): 749-53

Pearse, R. V., et al. (1999). Vertebrate homologs of Drosophila Suppressor of fused interact with the gli family of transcriptional regulators. Dev. Biol. 212(2): 323-36

Pham, A., et al. (1995). The Suppressor of fused gene encodes a novel PEST protein involved in Drosophila segment polarity establishment. Genetics 140: 587-598

Preat, T., et al. (1990). A putative serine/threonine protein kinase encoded by the segment-polarity fused gene of Drosophila. Nature 347(6288): 87-9

Preat, T. (1992). Characterization of Suppressor of fused, a complete suppressor of the fused segment polarity gene of Drosophila melanogaster. Genetics 132(3): 725-36

Preat, T., et al. (1993). Segmental polarity in Drosophila melanogaster: genetic dissection of fused in a Suppressor of fused background reveals interaction with costal-2. Genetics 135: 1047-62

Raisin, S., et al. (2010). Dynamic phosphorylation of the kinesin Costal-2 in vivo reveals requirement of fused kinase activity for all levels of hedgehog signalling. Dev. Biol. 344: 119-128. PubMed Citation: 20435030

Ramírez-Weber, F.-A., et al. (2000). Hedgehog signal transduction in the posterior compartment of the Drosophila wing imaginal disc. Molec. Cell 6: 479-485

Robbins, D. J., et al. (1997). Hedgehog elicits signal transduction by means of a large complex containing the kinesin-related protein costal2. Cell 90(2): 225-234

Ruel, L., et al. (2007). Phosphorylation of the atypical kinesin Costal2 by the kinase Fused induces the partial disassembly of the Smoothened-Fused-Costal2-Cubitus interruptus complex in Hedgehog signalling. Development 134(20): 3677-89. Medline abstract: 17881487

Sánchez-Herrero, E., et al. (1996). The fu gene discriminates between pathways to control dpp expression in Drosophila imaginal discs. Mech. Dev. 55: 159-170

Shi, Q., Li, S., Jia, J. and Jiang, J. (2011). The Hedgehog-induced Smoothened conformational switch assembles a signaling complex that activates Fused by promoting its dimerization and phosphorylation. Development 138(19): 4219-31. PubMed Citation: 21852395

Slusarski, D. C., Motzny, C. K. and Holmgren R. (1995). Mutations that alter the timing and pattern of cubitus interruptus gene expression in Drosophila melanogaster. Genetics 139: 229-240

Stegman, M. A. et al. (2000). Identification of a tetrameric hedgehog signaling complex. J. Biol. Chem. 275: 21809-12.

Stone, D. M., et al. (1999). Characterization of the human Suppressor of fused, a negative regulator of the zinc-finger transcription factor Gli. J. Cell Sci. 112: 4437-4448

Tang, L., et al. (2008). tsunami, the Dictyostelium homolog of the Fused kinase, is required for polarization and chemotaxis. Genes Dev. 22: 2278-2290. PubMed Citation: 18708585

Thérond, P. P., et al. (1993). Molecular organization and expression pattern of the segment polarity gene fused of Drosophila melanogaster. Mech Dev 44: 65-80. PubMed Citation: 8155575

Thérond, P., et al. (1996a). Functional domains of fused, a serine-threonine kinase required for signaling in Drosophila. Genetics 142(4): 1181-98. PubMed Citation: 8846897

Thérond, P. P., et al. (1996b). Phosphorylation of the fused protein kinase in response to signaling from hedgehog. Proc. Natl. Acad. Sci. 93: 4224-28. PubMed Citation: 8633045

Thérond, P. P., et al. (1999). Differential requirements of the Fused kinase for Hedgehog signalling in the Drosophila embryo. Development 126: 4039-4051. PubMed Citation: 10457013

Tukachinsky, H., Lopez L. V. and Salic, A. (2010). A mechanism for vertebrate Hedgehog signaling: recruitment to cilia and dissociation of SuFu-Gli protein complexes. J. Cell Biol. 191: 415-428. PubMed Citation: 20956384

Végh, M. and Basler, K. (2003). A genetic screen for hedgehog targets involved in the maintenance of the Drosophila anteroposterior compartment boundary. Genetics 163: 1427-1438. 12702686

Vieira, J. and Charlesworth, B. (2000). Evidence for selection at the fused locus of Drosophila virilis. Genetics 155: 1701-1709. PubMed Citation: 10924468

Wang, Q. T. and Holmgren, R. A. (1999). The subcellular localization and activity of Drosophila Cubitus interruptus are regulated at multiple levels. Development 126: 5097-5106. PubMed Citation: 10529426

Xia, L., Jia, S., Huang, S., Wang, H., Zhu, Y., Mu, Y., Kan, L., Zheng, W., Wu, D., Li, X., Sun, Q., Meng, A. and Chen, D. (2010). The Fused/Smurf complex controls the fate of Drosophila germline stem cells by generating a gradient BMP response. Cell 143: 978-990. PubMed ID: 21145463

Xia, L., et al. (2012). The niche-dependent feedback loop generates a BMP activity gradient to determine the germline stem cell fate. Curr. Biol. 22(6): 515-21. PubMed Citation: 22365848

Zhou, Q., Apionishev, S. and Kalderon, D. (2006). The contributions of protein kinase A and smoothened phosphorylation to hedgehog signal transduction in Drosophila melanogaster. Genetics 173(4): 2049-62. 16783001

Zhou, Q. and Kalderon, D. (2011). Hedgehog activates fused through phosphorylation to elicit a full spectrum of pathway responses. Dev. Cell 20(6): 802-14. PubMed Citation: 21664578

fused: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology

date revised: 20 July 2013

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.