fused
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).
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).
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).
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fused:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
date revised: 20 July 2013
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