Wingless and organ development Wingless is required for cell proliferation in the Malpighian tubule anlage. The pattern of Wingless mRNA and protein in Wingless developing tubules is consistent with a requirement for wingless for cell division. Analysis of the temporal requirement for wingless using a temperature-sensitive allele confirms that the normal expression of wingless is necessary during cell proliferation in Malpighian tubules. Overexpression of wingless results in supernumerary cells in the tubules (Skaer, 1992).
The proventriculus is a multiply folded muscular organ of the foregut formed from a simple epithelial tube. It functions to grind and masticate food.
Coordinated cell movements are critical for tissue and organ morphogenesis in animal
development. Drosophila genes hedgehog and wg, which encode signaling
molecules, and the gene myospheroid, which encodes a beta subunit of the integrins, are required
for epithelial morphogenesis during proventriculus development. In contrast, this morphogenetic
process is suppressed by the decapentaplegic gene (Pankratz, 1995).
A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition
within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of
equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the
other, the sibling cell, achaete expression is lost. In this paper
the mechanisms are charted by which achaete expression is differentially maintained in the tip cell lineage to stabilize cell fate. Initially,
wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells.
Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Then,
Notch signaling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete
expression is maintained only in the tip cell. In embryos mutant for Notch, tip cells segregate at the expense of sibling cells,
whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when
numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the
developing tubules of embryos lacking Wingless, after the critical period for tip cell allocation, reveals an additional
requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect
on the final tubule cell number (Wan, 2000).
In order to establish when the wg product is required for
tip cell allocation, wg activity was manipulated using the
temperature-sensitive allele, wgIL114. At the permissive temperature tip cells develop
normally, while at the restrictive temperature Wg
function is lost and tip cells fail to appear. Temperature
shift experiments establish that embryos must
develop at the permissive temperature between 4 and 5 h
for tip cells to appear normally. In accordance with
these findings, embryos shifted to the permissive temperature
for this period develop with tip cells, while a
shift to the restrictive temperature results in tubules with
no tip cells.
Given that the restoration of wild-type wg protein has
been shown to take 20-30 min and that the removal of
functional protein takes a similar time after shifting temperature, these
results establish the window of requirement as 4.5-5.5 h into
embryogenesis for functional
Wg to be expressed in the tubules for tip cell allocation.
Wg is normally expressed in the tubule primordia as they
evert from the hindgut.
From 4.5 h, expression is higher in the posterior region of the
developing tubules than the anterior. The tip
mother cell segregates from this posterior region, where wg
continues to be expressed. Wg is lost from
tubules during stage 12. Manipulation of wg expression using the temperature sensitive (ts) allele reveals a requirement for wg in cell division, separate from
its role in tip cell specification. If Wg is removed after 5 or
6 h after egg lay (AEL) but before cell division ceases in the tubules, the
final tubule cell number is reduced compared to wild type. This indicates that in the absence of Wg, the appearance of tip cells is
insufficient to promote the normal
pattern of division in the tubules (Wan, 2000).
The tip cell progenitor is selected from a group of competent
cells by lateral inhibition and is demarcated by the
continued expression of ac. Further extrinsic and intrinsic cues (Wg signaling and
the asymmetric distribution of Nb) operate to ensure the
continued expression of ac and so confirm tip cell potential.
The selection of cell fate from an equivalence group by
lateral inhibition alone relies on chance fluctuations in the
equilibrium of signaling between cells and therefore may
not be completely reliable. The activity of other genes, by
biasing lateral inhibition, serves to make the selection of
cells to specific fates more robust. Such mechanisms have
been shown to confirm cell fate in the PNS and
of the anchor cell in the nematode gonad.
The results presented here indicate that wg and nb are required for the
specification of the tip cell and sibling cell fate in the
Malpighian tubules. The activity of these two genes biases
the outcome of intercellular signaling at separate stages in
this process, resulting in the reliable allocation of tip and
sibling cell fates, suggesting that this distinction is important
to the development of the tubules. However, it is clear
that continued cell division in the tubules relies only on the
allocation of the tip cell progenitor and not on the differentiation
of fate between the tip cell's daughter cells, in which nb plays
an important role (Wan, 2000).
This result is surprising, since Nb is active where sister
cells of specific lineages are allocated to separate cell fates,
for example, in the PNS, in the CNS, and in myogenesis. Separation between sister cell
fates involves the maintenance of gene expression in one
sibling and its repression in the other, for example, of Kr,
eve, and S59 in sibling muscle founder cells.
This pattern is also seen in the tubules: ac, Kr, and Dl
continue to be expressed in the tip cell but are repressed in
the tip cell's sibling. In the neural and myogenic lineages the correct
allocation of sibling cell fates underpins normal tissue
differentiation. In the tubules, the
separate roles of the tip cells and their siblings are not yet known; they both
appear to be active in regulating cell proliferation but later
only the tip cell expresses genes characteristic of neuronal
cells. The later function of both cell
types has yet to be elucidated. By manipulating nb, Malpighian tubules that lack sibling cells can be generated, but these have two
tip cells or have two sibling cells but lack tip cells, thus
providing an important tool for this analysis (Wan, 2000).
Wingless and mesoderm development In the mesoderm of Drosophila embryos, a defined number of cells segregate as progenitors of individual
body wall muscles. Progenitors and their progeny founder cells display lineage-specific expression of
transcription factors but the mechanisms that regulate their unique identities are poorly understood. The homeobox genes ladybird early and ladybird late are shown to be expressed in only one muscle progenitor and its progeny: the segmental border muscle (SBM) founder cell and two precursors of adult muscles. lb activity is associated with all stages
of SBM formation, namely the promuscular cluster, progenitor cell, founder cell, fusing myoblasts and syncytial fiber. The segregation of the ladybird-positive progenitor requires coordinate action of neurogenic genes and an
interplay of inductive Hedgehog and Wingless signals from the overlying ectoderm. The SBM progenitor corresponds to the most
superficial cell from the promuscular cluster,
thus suggesting a role for the overlying
ectoderm during its segregation. Since epidermal Wg and Hedgehog (Hh) signaling has been shown to
influence muscle formation, the SBM-associated lb expression was examined in embryos
carrying hh and wg thermosensitive mutations. Wg and Hh signalings, mutually dependent at this time, are shown to be required for the promuscular lb activity and/or the segregation of SBM
progenitors. The initial influence of these signals is no longer observed later in
development. In addition to signals from the epidermis, the
activity of the mesodermal gene tinman, initially expressed in the whole trunk mesoderm, is involved in the early events of myogenesis. In tin - embryos, the formation of SBM promuscular clusters and segregation of lb-positive progenitor cells are strongly affected, leading to the absence of
the majority of SBM fibers. During promuscular cluster
formation, since tin expression becomes restricted to the dorsal mesoderm, its influence on ventrolaterally located SBMs is likely to be indirect and mediated via an
unknown factor. The lack of neurogenic gene function, known to be
involved in cell-cell interactions during lateral inhibition, generates the opposite phenotype. Mastermind - and Enhancer of split - embryos fail to restrict promuscular lb expression to only one cell; in consequence, they display a hyperplastic lb pattern in later stages (Jagla, 1998).
During Drosophila embryogenesis, mesodermal cells are
recruited to form a stereotyped pattern of about 30
different larval muscles per hemisegment. The formation
of this pattern is initiated by the specification of a special
class of myoblasts, called founder cells, that are uniquely
able to fuse with neighbouring myoblasts. The COE transcription factor Collier plays a role in the
formation of a single muscle (muscle DA3[A] in the abdominal segments; DA4[T] in the thoracic segments T2 and T3). Col
expression is first observed in two promuscular clusters (in
segments A1-A7), corresponding to two progenitors and
then their progeny founder cells, but its transcription is
maintained in only one of these four founder cells, the
founder of muscle DA3[A]. This lineage-specific restriction
depends on the asymmetric segregation of Numb during
the progenitor cell division and involves the repression of
col transcription by Notch signaling. In col mutant
embryos, the DA3[A] founder cells form but do not
maintain col transcription and are unable to fuse with
neighbouring myoblasts, leading to a loss-of-muscle
DA3[A] phenotype. In wild-type embryos, each of the
DA3[A]-recruited myoblasts turns on col transcription,
indicating that this conversion, accomplished by the DA3[A] founder cell, induces the naive myoblasts to express founder cell distinctive patterns of
gene expression, activating col itself. Muscles DA3[A] and DO5[A] (DA4[T] and DO5[T] respectively)
derive from a common progenitor cell, the DA3[A]/DO5[A] progenitor. However, ectopic expression
of Col is not sufficient to switch the DO5[A] to a
DA3[A] fate. Together these results lead to a proposal that
specification of the DA3[A] muscle lineage requires both
Col and at least one other transcription factor, supporting
the hypothesis of a combinatorial code of muscle-specific
gene regulation controlling the formation and
diversification of individual somatic muscles (Crozatier, 1999a).
The col-expressing promuscular clusters and progenitor cells
have a distinctive position, as defined relative to morphological
landmarks and ectodermal Engrailed (En) expression. The DA3[A]/DO5[A] progenitor cell lies underneath the anterior epidermal compartment, whereas the DT1[A]/DO4[A] progenitor cell lies on the anterior edge of the posterior
compartment, consistent with mapping of the primordium for
the somatic mesoderm. Since Wingless (Wg) and Hedgehog (Hh) signaling have been
shown to be required for mesoderm segmentation and
formation of a subset of muscle founder cells, col expression was analyzed in wg
and hh mutant embryos. At stage 10, both mutant embryos
show changes in mesodermal col expression: rather than being
restricted to specific clusters in the anterior compartment, it
appears almost continuous along the anteroposterior axis. Therefore, both wg and hh signalings appear to restrict col transcription to specific clusters. Lack of Wg or Hh
activity does not seem, however, to impede specification of the
DA3[A]/DO5[A] progenitor, which is singled out in the mutant as well as the wild-type embryos. It was noticed, however, that, while the
DA3[A]/DO5[A] progenitor appears to be specified normally,
more than one cell is singled out from the DT1[A] /DO4[A]
cluster in hh mutant embryos (Crozatier, 1999a).
Muscle founder cells arise from the asymmetric division of muscle progenitor cells, each of which develops from a group of cells in the somatic mesoderm that express lethal of scute. All the cells in a cluster can potentially form muscle progenitors, but owing to lateral inhibition, only one or two develop as such. Muscle progenitors, and the subsequent founder cells, then express transcription factors such as Krüppel, S59 and Even-skipped, all of which confer identity on the muscle. Definition of some muscle progenitors, including three groups that express S59, depends on Wingless signaling. Lateral inhibition requires Delta signaling through Notch and the transcription factor Suppressor of Hairless. Since the Wingless and lateral-inhibition signals are sequential, one might expect that muscle progenitors would fail to develop in the absence of Wingless signaling, regardless of the presence or absence of lateral-inhibition signaling. The development of the S59-expressing muscle progenitor cells has been examined in mutant backgrounds in which both Wingless signaling and lateral inhibition are disrupted. Progenitor cells fail to develop when both these processes are disrupted. This analysis also reveals a repressive function of Notch, required before or concurrent with Wingless signaling that is unrelated to its role in lateral inhibition (Brennan, 2000).
During wild-type development, expression of S59 is first seen during stage 10 in a single muscle progenitor cell either side of the midline in every segment. By stage 11, this pattern has evolved in abdominal segments such that S59 expression is seen both in the nervous system and in two groups of muscle progenitor cells. During stage 12, a third muscle progenitor cell starts to express S59. These muscle progenitor cells give rise to three muscle founder cells that maintain the expression of S59. Fusion of these founder cells with myoblasts results in the S59-expressing muscles seen in late stages of embryogenesis (Brennan, 2000).
Disruption of lateral-inhibition signaling, in either Notch (N) germline-clone, suppressor of Hairless germline-clone or Delta zygotic mutant embryos, increases the number of cells expressing S59 compared with wild type at stage 11. Because of general degeneration of these embryos during germ-band retraction, however, it is difficult to examine the expression of S59 after stage 11, but the mesoderm clusters that can be identified are expanded (Brennan, 2000).
Unlike the disruption of lateral-inhibition signaling, attenuation of Wingless signaling, by removing either wingless (wg) or dishevelled function, blocks the expression of S59 in the mesoderm. In contrast, increasing Wingless signaling, either by overexpressing the Wingless protein in the mesoderm using the GAL4/UAS system (twist-GAL4>UASwg embryos), or by removing shaggy function (sggm11 germline-clone embryos), leads to enlarged groups of S59-expressing muscle progenitor cells during stage 11. However, during germ-band retraction, the groups are reduced in size. In the twist-GAL4>UASwg embryos the reduction in cluster size leads to a largely normal set of three muscles, whereas in the sggm11 embryos the reduction is more extreme and leads to the loss of S59-expressing muscles (Brennan, 2000).
Since Wingless signaling is required for the initiation of S59 expression in the mesoderm and lateral-inhibition signaling is required for the subsequent restriction of S59 expression to one or two cells within each cluster, it is expected that in the absence of Wingless signaling S59 will not be expressed, even if lateral-inhibition signaling is also blocked. This appears to be the case in wgS107.5;DlFX3 zygotic and wgS107.5,Su(H)SF8 germline-clone embryos. In contrast, mesodermal S59 expression is observed in Df(1)N81k1,dshv26 and Df(1)N81k1;wgCX4 germline-clone embryos, in which Wingless signaling is blocked and Notch function is removed. Finally, as with the single-mutant embryos, the double-mutant embryos degenerate during germ-band retraction, making it difficult to examine S59 expression after stage 11 (Brennan, 2000).
These results first confirm that Wingless signaling is required for the initiation of S59 expression and that a Delta-initiated lateral-inhibition signal is required for the restriction of S59 expression to one or two cells of each initial cluster. They also confirm the prediction that, in the absence of a Wingless signal, S59 is not expressed, regardless of whether lateral-inhibition signaling is occurring. Also, even though hyperactivating Wingless signaling leads to initially enlarged groups of S59-expressing muscle progenitor cells, a reasonably normal muscle pattern is obtained (Brennan, 2000).
The observed S59 expression in Df(1)N81k1, dshv26 and Df(1)N81k1; wgCX4 embryos can be explained if it is assumed that Notch has a repressive function that precedes Wingless signaling. In this situation, removal of Notch function will lead to the derepression of S59 expression before Wingless signaling. Consequently, it does not matter whether or not Wingless signaling occurs. This repressive function cannot be related to Delta signaling, however, because the removal of Delta or Su(H) function in embryos where Wingless signaling is not occurring does not result in S59 expression. The repressive function of Notch uncovered in these experiments must therefore be distinct from its repressive role during lateral inhibition (Brennan, 2000).
The second observation suggests that in response to increased Wingless signaling there is a linked increase in lateral-inhibition signaling. This would mean that increased Wingless signaling will only lead to a significant increase in the number of muscle progenitors if lateral inhibition cannot occur. The observed difference in the final muscle pattern between twist–GAL4>UASwg and sggm11 embryos is probably due to the difference in how Wingless signaling is activated in the different embryos. In the twist–GAL4>UASwg embryos, Wingless signaling is activated only transiently and is restricted to the mesoderm. In contrast, Wingless signaling is activated globally and throughout embryogenesis in sggm11 germlineclone embryos. This difference, along with the proposed linkage between Wingless signaling and lateral inhibition would mean that lateral inhibition is much greater in the sggm11 embryos. This situation would explain the greater reduction in the size of the groups of S59-expressing muscle progenitor cells observed in the sggm11 embryos and the loss of muscles if the restriction is too great (Brennan, 2000).
The link between Wingless signaling and lateral inhibition could occur in a number of ways. For example, Wingless signaling may directly alter a component of the Delta signaling pathway that would then increase the ability of this pathway to transduce the Delta signal. Alternatively, Wingless signaling could affect Delta signaling by altering the transcription of one of the components of the pathway. Either of these mechanisms would allow the organism to generate a lateral-inhibition signal appropriate to the input signal: a strong Wingless signal would lead to a strong lateral-inhibition signal and prevent unnecessary and unwanted development, whereas a weak Wingless signal would lead to a weak lateral-inhibition signal that allows development to proceed even though the input signal is weak. This would allow normal development to occur even if there are fluctuations in the input signal (Brennan, 2000).
It is thought that the muscle progenitor cells develop from a large pool of developmentally equivalent cells that is refined through two steps to produce one muscle progenitor cell. A very large group of cells is initially defined that have the potential to become muscle progenitor cells but are prevented from doing so by the novel function of Notch identified here. Wingless signaling then alleviates this repressive function of Notch within a few cells of the cluster to establish an equivalence group. This triggers the process of lateral inhibition, which subsequently selects a single cell to become a muscle progenitor. In this situation, overexpressing Wingless or constitutively activating Wingless signaling will alleviate the initial repressive function of Notch in all the cells is observed, revealing the larger extent of the initial cluster. The linked increase in lateral-inhibition signaling, however, ensures that the normal number of muscle progenitor cells develop (Brennan, 2000).
This model contrasts with others in which Wingless signaling is instructive and defines the position at which muscle progenitor cells will develop, but can explain why overexpressing Wingless leads to the development of S59-expressing muscles in their normal position. In this model the Wingless signal is permissive and not instructive: it does not define where S59 will be expressed but merely reveals places defined by earlier mechanisms. Finally, these data suggest that the loss of S59 expression in the absence of a Wingless signal is due to the early repression mediated by Notch (Brennan, 2000).
Mutations in wingless leads
to the complete loss of a subset of muscle founder cells characterised by the expression of NK1/S59.
Wingless acts directly on the mesoderm to ensure the formation of
NK1-expressing founder cells. Wg can signal across germ
layers: in the wild-type embryo, Wg from the ectoderm constitutes an inductive signal
for the initiation of the development of a subset of somatic muscles (Baylies, 1995).
The somatic muscles, the heart, the fat body, the somatic part of the gonad and most of the
visceral muscles are derived from a series of segmentally repeated primordia in the
Drosophila mesoderm. This work describes the early development of the fat body and its
relationship to the gonadal mesoderm, as well as the genetic control of the development of
these tissues. The first sign of fat body development is the expression of serpent in segmentally repeated clusters within the trunk mesoderm in parasegments 4-9. Segmentation and dorsoventral patterning genes define three regions in each parasegment
in which fat body precursors can develop. The primary and secondary dorsolateral fat body primordia are formed ventral to the visceral muscle primoridium in each parasegment. The ventral secondary cluster forms more ventrally in the posterior portion of each parasegment. Fat body progenitors in these regions are
specified by different genetic pathways. Two dorsolateral regions require engrailed and hedgehog (within the even-skipped domain) for
their development while the ventral secondary cluster is controlled by wingless. Ubiquitous mesodermal en expression leads to an expansion of the primary clusters into the sloppy-paired domain, resulting in a continuous band of serpent-expressing cells in parasegments 4-9. The observed effect of en on fat body development is seen not only on mesodermal overexpression but also when en is overexpressed in the ectoderm. Loss of wingless leads to an expansion of the dorsolateral fat body primordium. decapentaplegic and one or more
unknown genes determine the dorsoventral extent of these regions. High levels of Dpp repress serpent, resulting in the formation of visceral musculature, an alternative cell fate (Reichmann, 1998).
In each of
parasegments 10-12 one of these primary dorsolateral regions generates somatic gonadal precursors instead of fat body. The balance between fat body and somatic gonadal fate in these serially
homologous cell clusters is controlled by at least five genes. A model is suggested in which
tinman, engrailed and wingless are necessary to permit somatic gonadal develoment, while
serpent counteracts the effects of these genes and promotes fat body development. In wg mutant embryos, all dorsolateral mesodermal cells, including those in parasegments 10-12, acquire fat body fate. This phenotype can be interpreted as the combined effects of two separate functions of wg: (1) wg is necessary to repress fat body development in the dorsolateral mesoderm underlying the wg domain in all parasegments; (2) wg is required in the primary cluster to permit somatic gonadal precursor instead of fat body development in parasegments 10-12. Loss of engrailed results in the absence of demonstrable somatic gonadal precursors, similar to the situation in tinman mutants. Ubiquitous mesodermal en expression leads to the formation of additional somatic gonadal precursor cells in parasegments 10-12. The
homeotic gene abdominalA limits the region of serpent activity by interfering in a mutually
repressive feed back loop between gonadal and fat body development. It is unlikely that abdA represses srp directly, since srp can be expressed in cells in which abdA is active. abdA might prevent srp from inhibition of a somatic gonadal precursor competence factor (Riechmann, 1998).
Wingless and terminalia This study reports the expression pattern of Dll in the genital disc, the requirement of Dll activity for the development of the terminalia and the activation of Dll by the combined action of the morphogenetic signals Wingless (Wg) and Decapentaplegic (Dpp). In Drosophila, the terminalia comprise the entire set of
internal and external genitalia (with the exception of the
gonads), and includes the hindgut and the anal structures. They arise from a single imaginal disc of ventral
origin which is of complex organization and shows bilateral symmetry. The genital disc
shows extreme sexual dimorphism. Early in development,
the anlage of the genital disc of both sexes consists of three
primordia: the female genital primordium (FGP); the male
genital primordium (MGP), and the anal primordium (AP).
In both sexes, only two of the three primordia develop: the
corresponding genital primordium and the anal primordium.
These in turn develop, according to the genetic sex, into
female or male analia. The undeveloped genital primordium
is the repressed primordium (either RFP or RMP,
for the respective female and male genital primordia) (Gorfinkiel, 1999).
During the development of the two components of the anal primordium -- the hindgut and the analia -- only the latter is dependent on Dll and hedgehog (hh) function. The hindgut is defined by the expression of the homeobox gene even-skipped. The lack of Dll function in the anal primordia transforms the anal tissue into hindgut by the extension of the eve domain. Meanwhile targeted ectopic Dll
represses eve expression and hindgut formation. The Dll requirement for the development of both anal plates in males and only for the dorsal anal plate in females, provides further evidence for the previously held idea that the analia arise from two primordia. In addition, evaluation was made of the requirement for the optomotor-blind (omb) gene which, as in the leg and antenna, is located downstream of Dpp. These results suggest that the terminalia show similar behavior as the leg disc or the antennal part of the eye-antennal disc, consistent with both the proposed ventral origin of the genital disc and the evolutive consideration of the terminalia as an ancestral appendage (Gorfinkiel, 1999).
The requirement for the Hh signal in Dll activation might
be mediated by Wg and Dpp signals. This occurs in other
ventral discs. Dll expression arises at the juxtaposition of
Wg and Dpp expressing cells as revealed by double staining
for Dll and Dpp, and Dll and Wg. In both genital and anal
primordia, Dll expressing cells overlap those that
express wg and dpp. It has been previously
reported that the ectopic expression of both Wg and Dpp
produces several phenotypic alterations in both female and
male terminalia. Similar types of
transformations are also induced by the lack of function of
either patched (ptc) or Protein kinase A (Pka). In these
mutants, the Hh pathway is constitutively active giving
rise to the derepression of Wg and Dpp. The lack
of Pka function in the genital disc induces ectopic Dll. This Dll induction requires both Wg and Dpp
signals in the same cells since Dll is not activated in Pka2;dpp2 and in Pka2;wg2
double mutant clones, as occurs in other discs of ventral origin (Gorfinkiel, 1999).
In the male repressed primordium (RMP) of the female
genital disc, wg is expressed but not dpp. Consequently, Dll
is not expressed because Dll is only activated in cells that
express both dpp and wg. Ectopic Dpp expression in the wg
expression domain driven by the MS248-GAL4 line
induces Dll 'de novo' in the RMP, which
shows an increase in size. However, these changes do not
allow the development of adult structures from this primordium since there is no activation of the male specifc cyto-differentiation genes because the genetic sex has not changed. Dll
is not activated in the repressed female primordium (RFP)
of the male genital disc despite the fact that, in this primordium, both wg and dpp are normally expressed. This activation does not occur even if the levels of Dpp are increased.
These results suggest that specific genes expressed in the
RFP can exert a negative control of Dll expression (Gorfinkiel, 1999).
In order to find other genes involved in the development
of the terminal structures, the expression
pattern and the functional requirement for optomotor-blind
(omb) were examined. This gene encodes a protein with a DNA-binding
domain (T domain) and behaves
as a downstream gene of the Hh pathway in other imaginal
discs. In the genital disc,
Omb is detected in the dpp expression domains, abutting
the wg expressing cells. This behaviour
of omb expression is similar to that found in the leg and
antennal discs. In the genital
disc, omb is also regulated by the Hh signaling pathway
since Pka2
clones also ectopically express omb.
The phenotypes produced due to omb lack of
function using the allele omb282 were examined; homozygous females for this allele could not be obtained
but some male pharates were analyz ed. In males, the dorsal
bristles of the claspers and the hypandrium bristles are
absent. Also, the hypandrium is
devoid of hairs and the hypandrium fragma is reduced. Surprisingly, the anal plates are mostly
somewhat enlarged in the ventral region and reduced in the
dorsal areas. The structures affected in omb2
are
duplicated when omb is overexpressed in the dpp domain
using the dpp-GAL4/UAS-omb combination. In males, the
dorsal bristles of the clasper and the hypandrium
bristles are duplicated. These phenotypes are
similar to the ones obtained as a result of ectopic Dpp (Gorfinkiel, 1999).
The hindgut of the Drosophila embryo is subdivided into three major domains, the small intestine, large intestine, and rectum, each of
which is characterized by specific gene expression. The expression of wingless, hedgehog, decapentaplegic, and engrailed corresponds to the generation or growth of particular domains of the hindgut. wg, expressed in the prospective anal pads, is necessary for activation of hh in the adjacent prospective rectum. hh is expressed in the prospective rectum, which forms anterior to the anal pads, and is necessary for the expression of dpp at the posterior end of the adjacent large intestine. wg and hh are also necessary for the development of their own expression domains, anal pads, and rectum, respectively. dpp, in turn, causes the growth of the large intestine, promoting DNA replication. en defines the dorsal domain of the large intestine, repressing dpp in this domain. A one-cell-wide domain, which delineates the anterior and posterior borders of the large intestine and its internal border between the dorsal and ventral domains, is
induced by the activity of en. A model is proposed for the gene regulatory pathways leading to the subdivision of the hindgut into domains (Takashima, 2001).
The term 'tissue compartments' can be used to indicate the domains of the gut. In this report, the term 'domain' is used in order to avoid confusion with
the term 'developmental compartment', which has been defined
by clonal analysis of the wing disc. To clarify the use of
anatomical descriptions, the organization of the hindgut
domains, as revealed by specific gene expression patterns is described. The most anterior domain of the hindgut, which is just posterior to the midgut, is the small
intestine. The small intestine is followed by the large intestine, then the rectum. The large intestine is further subdivided into a ventral and a dorsal domain. A one-cell-wide domain, which was designated as h4, forms at the anterior and posterior borders of the large intestine, as well as at the border between the dorsal
and ventral domains of the large intestine. The
cells in these regions are designated collectively 'border cells'. Until the
end of stage 12, the hindgut tube is situated on the midline of
the body, and is left-right symmetric. During early stage 13,
the hindgut rotates to the left, resulting in the original dorsal
and ventral domains coming to face the left and right side of
the body, respectively. The orifice of the rectum (the anal
slit) is surrounded by the anal pads, the development of
which is tightly linked to that of the hindgut (Takashima, 2001).
wg, hh, and dpp are expressed in the hindgut of the Drosophila embryo. The expression patterns of these genes have been re-examined in detail to define their exact spatial relationship. wg is expressed throughout the proctodeum at stage 9, then soon becomes restricted to two separate
regions: (1) the primordium of the anal pads, which
surrounds the posterior opening of the hindgut, and (2) a
narrow ring anterior to the small intestine. The expression in these two
domains persists throughout embryogenesis (Takashima, 2001).
hh is expressed throughout the hindgut primordium at
stage 10. Subsequently, as in the case of wg, the expression
is divided into two separate regions at stage 11: the region just posterior to the anterior wg domain, which corresponds to the small intestine, and the posterior-most region of the hindgut, which corresponds to the prospective rectum and is situated just anterior to the anal pads (Takashima, 2001).
The border cells differentiate at the anterior and posterior
border of the large intestine and at the border between the
dorsal and ventral domains of the large intestine. The border
cells are first detected at stage 12 by lacZ expression of
some enhancer-trap strains, and after stage 14, the cells are distinguished by marked expression of Crb and dead ringer. By double staining for En and beta-galactosidase protein of border cell-specific enhancer trap
lines, the border cells are found to abut the En-positive
domain and to express no En protein, suggesting that dpp-positive cells abutting the en-positive domain differentiate into border cells. It is
noteworthy that the spatial organization of en, hh, wg, and
dpp domains is quite different from that of the segmented
epidermis or the imaginal discs, suggesting that a different
patterning mechanism is working in the hindgut (Takashima, 2001).
Strong defects occur in the hindgut of wg mutant embryos, and it has been
argued that such defects are likely due to an early effect on
cell proliferation. The effects of the hypomorphic mutation wg17en40 on development of hindgut
patterning was examined in detail. In this mutant, proctodeal invagination
is almost normal until stages 9-10, but much of the proctodeum, except the anterior-most region including the small intestine, begins to degenerate after the onset of germband shortening, resulting in a very tiny epithelial sac. The anal pads, which express wg, also degenerate in this mutant. The expression of hh in the
prospective rectum is abolished in this wg mutant, while
the expression of hh in the small intestine is not affected. The hindgut of a null allele of wg (wgPY40) shows
essentially the same defects though overall morphology of
the embryo is affected more severely. This result suggests
that hh expression in the prospective rectum is activated by
wg signaling. To prove this, the effect of ectopic expression
of wg was examined by using the GAL4-UAS system. The UAS-wgts strain, in which functional wg can be induced at the permissive temperature of 17°C, was
mated with the byn-GAL4 strain, in which GAL4 is
expressed throughout the whole hindgut and anal pads. It
was found that hh expression is expanded throughout
hindgut upon misexpression of wg. Except for a
short anterior portion corresponding to the small intestine,
the lumen of the hindgut is characteristically enlarged,
with no morphological boundary between the large intestine
and rectum. Similar results were obtained when an active
form of Armadillo is misexpressed throughout the hindgut. These results strongly
suggest that the ectopic wg activity induces hh expression at
the prospective large intestine, and the latter develops as a
part of the rectum. Conversely, in the hh mutant, there are no
drastic changes in anal pad development or wg expression (Takashima, 2001).
In wg mutants, dpp expression in the large intestine is
completely abolished. The ectopic expression of wg slightly upregulates the initial dpp expression in the large intestine at stage 11, but has repressed dpp throughout the hindgut by late stage 12. The relationship between wg activity in the anal pads and dpp expression in the large intestine seems to be indirect and complicated (Takashima, 2001).
en expression in the dorsal domain of the large intestine, in contrast to that of dpp and hh, is not affected in wg embryos. These results suggest that the defects of the hindgut in wg mutants are partly mediated by failure of hh expression in the future rectum. It should be noted that the defects of the large intestine in the wg mutant are more drastic than those in either hh or dpp mutants. There may exist some pathway of wg action that is not mediated by hh and dpp (Takashima, 2001).
It should be noted that wg and hh mutations result in a
short hindgut, and these mutations are associated with the
reduction of dpp expression in the large intestine. It is very
likely that suppression of the growth of the large intestine
correlates with the decrease in dpp expression. The effect of dpp
At the end of germband retraction, the dorsal epidermis of the Drosophila embryo exhibits a discontinuity that is covered by the amnioserosa. The process of dorsal closure (DC) involves a coordinated set of cell-shape changes within the epidermis and the amnioserosa that result in epidermal continuity. Polarization of the dorsal-most epidermal (DME) cells in the plane of the epithelium is an important aspect of DC. The DME cells of embryos mutant for wingless or dishevelled exhibit polarization defects and fail to close properly. The role of the Wingless signalling pathway in the polarization of the DME cells and DC was investigated. The ß-catenin-dependent Wingless signalling pathway is required for polarization of the DME cells. Although the DME cells are polarized in the plane of the epithelium and present polarized localization of proteins associated with the process of planar cell polarity (PCP) in the wing, e.g., Flamingo, PCP Wingless signalling is not involved in DC (Morel, 2004).
The initiation of DC in Drosophila embryos correlates with the
elongation and polarization of the DME cells in the DV axis of the embryo. In parallel with this polarization, a cable of F-actin assembles on the
dorsal-most surface of these cells and promotes the formation of filopodia and lamellae during the final phases of the process. A functional link between the polarization and the assembly of the cable of actin is supported by the observations that in mutants in which the DME cells do not elongate, there is no actin cable and no dynamic protrusions. As
a consequence, these embryos display defects and delays in the closure
process. Embryos mutant for wg and dsh are good examples of
this class (Morel, 2004).
The polarization of the DME cells occurs in the plane of the epithelium and can be seen as a manifestation of the phenomenon of planar cell polarity (PCP). Since a specific branch of Wg signalling has been implicated in PCP and there is evidence for an interaction between Dishevelled and JNK signalling during dorsal closure, whether there is a role for this mode of Wg
signalling in the process of DC was tested. The results clearly show that the 'canonical'
Wg signalling pathway that leads to activation of Armadillo and of the
transcription of target genes is necessary and sufficient to restore the
polarity of the DME cells and to promote a normal process of dorsal closure in
a wg mutant embryo. Surprisingly, it was found that the PCP pathway does
not appear to play a major role in DC or the polarization of the DME cells, since
activation of the 'canonical' pathway in the absence of dsh activity
rescues the polarity and function of the DME cells.
Dsh contains three highly conserved domains, the DIX, PDZ and DEP domains. The DEP domain mediates interaction of Dsh with the cell
cortex and is required for PCP but not 'canonical' Wg signalling,
while the DIX domain is required for the 'canonical' Wg signalling but seems dispensable for PCP. To investigate an involvement of the PCP pathway in the activities of the DME cells during DC, rescue experiments
of wg– embryos were carried out using truncated forms of Dsh deleted for either the DEP (DshDeltaDEP) or the DIX (DshDeltaDIX) domain. Although overexpression of DshDeltaDEP leads to the partial rescue of naked cuticle and of En expression, neither
naked cuticle nor rescue of En expression are observed in
wg>da>DshDeltaDIX (using da-GAL4 to drive DshDeltaDIX in wg mutants) embryos. This
thus confirms that DshDeltaDEP is able to signal within the 'canonical' Wg
pathway but not DshDeltaDIX (Morel, 2004).
Then the ability of either protein to rescue DC in
wg– embryos was tested. wg>da>DshDeltaDEP
embryos are longer than wg mutants and their dorsal cuticle is
improved; no hole is observed and only occasional warts can be seen. The DME cells are oriented in the DV direction and most of them show a slight elongation in the DV direction when the zippering process has started. Simultaneously, Fmi is observed at the membrane and accumulates at the level of the ANCs. Although no clear elongation of DME or ventral epidermal cells is observed, DC process is improved; two zippers, at the anterior and posterior ends of the embryo, are
initiated, whereas only the posterior one is observed in
wg– embryos. By contrast,
wg>da>DshDeltaDIX embryos have a shorter cuticle than
wg mutants and show a more severe puckering and hole on the dorsal
side. Furthermore, neither the shape nor the polarization of DME cells is improved in these embryos (Morel, 2004).
Thus, although DshDeltaDEP can rescue partially the DC defects of
wg mutants, ubiquitous overexpression of DshDeltaDIX does not
rescue any of the observed features confirming the requirement for the Wg
'canonical' pathway during DC. Thus, the conclusion that the PCP pathway does not appear to play a role in DC or in the polarization of the DME is
supported by the observation that although a moiety of Dishevelled that
promotes Armadillo signalling is capable of rescuing the defects of
wg mutants, a moiety that promotes JNK signalling and PCP does not.
Altogether, these results indicate that the polarization and activity of the
DME cells during dorsal closure requires Armadillo/ß-catenin-dependent Wg
signalling. Furthermore, this requirement is restricted to the epidermis because activation of Wg signalling in the amnioserosa has no effect on the
epidermis (Morel, 2004).
The polarization of the DME cells and subsequent dynamics of actin at the
LE can be construed since the development of the leading edge of a motile cell
and to a certain extent is akin to an epidermal/mesenchymal transition (EMT), as one of the features of this process is the reorganization of the actin cytoskeleton and the acquisition of motility by the cells. In this regard, it is interesting to note that ß-catenin-dependent Wnt signalling has been implicated in EMT both in normal and cancerous cells and that therefore there are precedents for the involvement of the
ß-catenin-mediated transcriptional regulation in the development of actin dynamics. However, the targets of the Wnt pathway mediating this process are not known (Morel, 2004).
It has been suggested that Dpp is a central effector of dorsal closure.
Embryos mutant for dpp signalling exhibit defects in dorsal closure. dpp is expressed in the DME cells and has been proposed to act as a long range signal for the elongation of the more ventral cells. Wingless is shown to be required for the correct
maintenance of dpp expression in the DME cells, although in these
experiments the input is less significant than has been reported before.
Altogether, these observations suggest that some of the activity of Wingless during DC is mediated by Dpp. Indeed, when the Dpp pathway was ubiquitously activated by the means of an activated form of its receptor Tkv, some rescue of the polarity of the DME cells was observed. However, although in this case the DME cells orient themselves in the DV direction and Fmi localises as it does in wild type, neither the DME nor the ventral epidermal cells elongate, and the DC process is not substantially improved. This contrasts with the full rescue of both the polarization of DME cells and the DC process following ubiquitous activation of the ß-catenin-dependent Wg pathway. Thus, if Dpp contributes to DC, it is not as the only target of Wg signalling (Morel, 2004).
Expression of Wingless from the amnioserosa in wg mutants induces high and continuous levels of dpp in the DME cells together with some rescue of the polarity of the DME cells but without any effect on the elongation of these or the more ventral cells. This rescue is very similar to the one observed with ubiquitous expression of the activated Tkv. These results indicate that Dpp does not act as a long-range signal for the elongation of the more ventral epidermal cells; rescue of Dpp expression in the DME cells or activation of Dpp signalling throughout the epidermis in wg mutants does not lead to the elongation of the more ventral cells. A similar conclusion had been suggested from the observation that epidermal cells initially elongate in the absence of Dpp signalling but resume their
polygonal shape soon after. However, an alternative explanation for these observations is that the elongation of the ventral epidermal cells requires inputs from both Dpp and Wingless signalling (Morel, 2004).
Altogether, these observations indicate that Dpp is not the only effector of Wingless during DC and indicates that Wingless signalling via Armadillo controls genes that act either in parallel or together with those regulated by JNK and Dpp (Morel, 2004).
Wingless is required in the epidermal cells but does not
act as a polarizing signal, since ubiquitous activation of the pathway rescues the defects of wg mutants. An important observation of these experiments is that the DME cells of wg mutant embryos display a polarity and an elongation at the very final stages of DC, suggesting that the polarization signal is received correctly by the DME cells but that in the absence of Wingless signalling there is a delay either in its interpretation
or in its materialization. This, together with the lack of importance of a
fixed source of Wingless for the polarization of the DME cells,
suggests that Wingless makes the DME cells competent to interpret a
pre-existing polarization signal (Morel, 2004).
In the case of DC, the permissive function of Wingless signalling
translates itself in the correct coordination of the different events, i.e.,
the cells have to elongate at the right time and the activity of their
cytoskeleton has to be properly linked to other events some of which are
transcriptional. Failure to do this will result in defects in dorsal closure.
These observations raise the question of the temporal requirements for Wg
signalling during DC (Morel, 2004).
ASGal4 (the amnioserosa specific driver, 332.3-Gal4) drives expression of Wingless from the elongation of the germband to
the end of DC. However, when driven by ASGal4, Wg can be detected only over the epidermal cells during the first phase of DC. This is probably due to the inability of Wingless to cross the deep fold existing between the AS and the epidermis during germband retraction and the zippering process. The provision of Wg from the amnioserosa rescues the defects of the DME cells of wingless mutants but not those of the more ventral cells. Although the DME cells, in contact with the AS, might have received Wg signal at the very onset of the overexpression (around stage 9-10), the more ventral epidermal cells seem to see the signal too late to elongate, suggesting that
Wg signalling is required before the beginning of DC for the cell shape and polarity changes. A hint at the timing of Wnt requirement for DC is provided
by experiments using a temperature sensitive allele of wingless. Removal of wingless function between 4 and 4.5
hours after egg laying, i.e., at stages 9-10, affects the shape of the dorsal
cuticle in a way similar to DC defects. This suggests that the polarizing
signal must occur very early, during germband elongation (Morel, 2004).
The notion of PCP has emerged from studies of the mechanism that determines the orientation of the hairs in the cells of the wing of Drosophila. A number of
studies have revealed the existence of protein complexes that mediate this
orientation by becoming asymmetrically distributed between the proximal and distal membranes of the epidermal cells. Thus, while Flamingo becomes
localised equally between the proximal and the distal sides of the cell, the distal side of the cell accumulates a complex composed of Frizzled and
Dishevelled and the proximal
side accumulates a complex formed by Strabismus and Prickled. Genetic
analysis of these complexes has led to the formulation of a model which
describes the propagation of the polarity from one cell to its neighbours, but which says nothing about the origin of the polarity that is being propagated.
In this model, Dsh, like Strabismus, Prickled or Frizzled, is an essential
component of the mechanism that propagates the polarity (Morel, 2004).
The observation of polarized distributions of Fmi, Dsh and Fz in the DME cells during dorsal closure has led to the suggestion of a link between the polarization of these cells and the process of PCP. However, no requirement has been found for elements of this pathway in dorsal closure. In particular, the PCP function of Dsh is not required for the polarization of the DME cells and the polarized localization of Fmi, which was quite unexpected considering the interdependence of Dsh and Fmi for their asymmetric localization in the
wing. This asymmetric distribution of Fmi is likely to play a
role in the polarized actin dynamics in response to the polarity signal.
Although this may appear surprising, it also invites a consideration of the notion of PCP (Morel, 2004).
The PCP pathway has been defined in a context of propagation of a polarity but not of its initial definition. In fact none of the experiments performed in the wing of Drosophila address the origin of the polarity that is being propagated. In DC, however, the process that was observed in the asymmetric distribution of proteins in the DME cells reflects the establishment of a polarity and not its propagation. From this perspective, the lack of a requirement for the PCP branch of Wnt signalling might not be that surprising as PCP Wnt signalling might be related to propagation or coordination of a polarity signal that has been generated in a different manner. However, the requirement for the ß-catenin-dependent Wg pathway might be significant and indicate the requirement for a transcriptional event in the establishment of PCP. This observation might also apply to the wing (Morel, 2004).
The ability to regenerate following stress is a hallmark of self-renewing tissues. However, little is known about how regeneration differs from homeostatic tissue maintenance. This study examined the role and regulation of Wingless (Wg)/Wnt signalling during intestinal regeneration using the Drosophila adult midgut. Wg was shown to be produced by the intestinal epithelial compartment upon damage or stress and it is exclusively required for intestinal stem cell (ISC) proliferation during tissue regeneration. Reducing Wg or downstream signalling components from the intestinal epithelium blocked tissue regeneration. Importantly, it was demonstrate that Wg from the undifferentiated progenitor cell, the enteroblast, is required for Myc-dependent ISC proliferation during regeneration. Similar to young regenerating tissues, aging intestines required Wg and Myc for ISC hyperproliferation. Unexpectedly, the results demonstrate that epithelial but not mesenchymal Wg is essential for ISC proliferation in response to damage, while neither source of the ligand is solely responsible for ISC maintenance and tissue self-renewal in unchallenged tissues. Therefore, fine-tuning Wnt results in optimal balance between the ability to respond to stress without negatively affecting organismal viability (Cordero, 2012).
This study used the posterior adult Drosophila midgut to address the role of Wg and its downstream signalling pathway during the proliferative response of ISCs to acute damage of the intestinal epithelium. The results suggest that, in response to stress or damage, Wg production is induced in enteroblasts (EBs), which stimulates ISC proliferation and subsequent midgut regeneration in a Myc-dependent manner. The results place Wg induction downstream of the damage/stress activated kinase JNK (Cordero, 2012).
The visceral mesoderm (VM) that surrounds the Drosophila midgut was proposed to constitute the Wg niche and be the sole source of the ligand required for ISC maintenance and homeostatic tissue self-renewal. The current results suggest that Wg produced within the intestinal epithelium and not the VM is essential for intestinal regeneration in response to acute damage. Furthermore, neither source of the ligand seems solely responsible for homeostatic self-renewal. Apparent discrepancies with the previous study could have different explanations. One possibility is that the ISC phenotype of whole wg mutants may be the result of combined Wg loss in VM and midgut epithelium. Alternatively, minimal levels of wg may be sufficient to maintain ISCs during homeostasis while a higher threshold of ligand production and subsequent signalling activation may be required for regeneration. In such a scenario, wg knockdown by RNA interference may still leave enough Wg to maintain the tissue under homeostatic conditions. The latter possibility is favored over the former since combined wg knockdown in esg;how>wg-IR midguts did not show significant loss of ISCs. Dose-dependent roles of Wnt signalling have been previously reported to regulate haematopoietic stem cells and tumourigenesis. Another possible explanation to the mild or absent role of Wg in homeostatic self-renewal may be compensation by other Wnt ligands. Therefore, while Wg is the main ligand required for regeneration, different ligands may compensate for each other during homeostatic maintenance of the tissue. Recent work in the murine small intestine has shown that one of the differentiated progeny of ISCs, the Paneth cell, expresses multiple growth factors such as EGFs and Wnt3 and are sufficient to drive the formation of 'crypt-like' structures from single Lgr5-expressing ISCs in vitro. Paneth cells have therefore been proposed as an important component of the Wnt ISC niche. In vivo work involving ablation of Paneth cells suggests the presence of potential compensatory mechanisms during homeostasis, while their role in intestinal regeneration has been suggested. The current work demonstrates that Wg from the transient daughter of the ISC, the EBs, is essential for efficient ISC proliferation during regeneration of damaged midgut epithelium. Therefore, Drosophila EBs could be seen as a functional homologue of the vertebrate Paneth cell and represent an essential component of the ISC niche. In the particular case of Wg, it is proposed that EBs represent a 'regeneration-specific ISC niche'. Growth factors such as IL-6/Upds and EGF-like ligands are important components of the Drosophila ISC niche. Intriguingly, the EGF-like ligand Spitz has been shown to be expressed in the small progenitor cells (ISCs/EBs) in the midgut, which is characterized by the expression of escargot. Even though direct functional assessment of the role of Spitz and other growth factor from EBs remains to be performed the current results strongly point to these cells as a potential general source of factors essential for ISC proliferation (Cordero, 2012).
Previous work suggests that midgut regeneration involves an intricate crosstalk between multiple signalling pathways. JAK/Stat signalling seems to be a central component of this response. Current work group suggests that JAK/Stat signalling is an important mediator of the hyperplastic phenotype resulting from loss of Apc in the Drosophila midgut. The results presented in this study suggest that damage to the midgut results in parallel activation of Wg/Myc and JAK/Stat. Knockdown of either pathway does not affect upregulation of the other pathway in response to damage even though midguts are still unable to regenerate. A similar scenario has been reported in the interplay between EGFR and JAK/Stat signalling. Therefore, activation of multiple pathways is a necessary condition for proper midgut regeneration. Likewise, midgut hyperproliferation in response to ectopic Wg signalling requires Myc and involves concerted activation of EGFR and JAK/Stat (Cordero, 2012, under review). Consistently, forced overexpression of ectopic Myc only is not sufficient to drive ISC proliferation and cannot overcome the absence of other proliferating signals such as JAK/Stat or Wg during midgut regeneration (Cordero, 2012).
One important question in the stem cell arena is whether tissue homeostasis and regeneration are controlled by similar mechanisms. Work in the mammalian intestine has shown examples where genes redundant for normal homeostasis are required for intestinal regeneration and Apc-driven intestinal hyperplasia. Therefore, the regenerative process cannot be interpreted as a simple acceleration of tissue self-renewal. Consistent with this concept, the curren work shows that partial reduction of the levels of Wg and Myc prevents ISC hyperproliferation during regeneration and aging but does not lead to long-term loss of ISCs. Furthermore, components of Wnt signalling such as Pygo are required for intestinal regeneration upon damage but dispensable for ISC proliferation in homeostatic conditions. Therefore, modulating Wg levels could lead to controlled ISC proliferation in conditions of hyperplasia without crossing a threshold that affects tissue integrity (Cordero, 2012).
The Wnt signalling pathway is a central regulator of homeostasis in the mammalian intestine. Inactivating mutations in Wnt pathway components lead to a very rapid loss of intestinal tissue. The current results show that, with the exception of Tcf, knocking down Wnt/Wg signalling has a rather mild and component-dependent role in homeostatic self-renewal of the Drosophila midgut. This is indeed consistent with previous reports. Although this scenario may appear at odds with that of the mammalian intestine there are indeed many similarities. In contrast, to genetic ablation of β-Catenin or Tcf4, the impact of Wnt inhibitors such as LRP6 blocking antibodies or Frizzled traps, which partially decrease Wnt signalling is much more subtle with little impact on intestinal homeostasis. In addition, it is also possible that β-Catenin signalling that is independent of ligand may explain the strong phenotype of TcfDN midguts. For example, it is known that the phosphorylation of β-Catenin by AKT/PKB has important roles in mammalian intestinal homeostasis (Cordero, 2012).
Although the similarities between the fly gut and the intestinal systems are often highlighted, intrinsic differences in the rates of homeostatic proliferation are observed between the two systems. The mouse intestine shows a high rate of homeostatic proliferation and undergoes complete self-renewal in 3-4 days, while the homeostatic fly midgut is a much more quiescent tissue. Basal proliferation rates in young, undamaged Drosophila midguts are very low and essentially undetectable by simple pH3 staining. Lineage-tracing experiments show it takes almost a month to achieve complete self-renewal of the midgut epithelium. Therefore, the homeostatic vertebrate intestine could be more comparable to the regenerating fly midgut. On the other hand, the homeostatic Drosophila midgut resembles a lowly proliferative epithelium like that of the mammalian urinary bladder or liver, which both show remarkable regenerative potential and shift from an almost quiescent to a hyperproliferative state in response to injury. Recent work uncovering an inducible role of Wg in regeneration of the bladder epitheliu suggests potential general implications of the current work. Additionally, one can expect that the intestinal epithelium of flies living in the wild, which is subject to constant challenges, might show higher basal proliferation than that of laboratory animals. Therefore, similarities between the mammalian and Drosophila intestine are likely to outweight their differences (Cordero, 2012).
Wnt signaling regulates synaptic plasticity and neurogenesis in the adult nervous system, suggesting a potential role in behavioral processes. This study probed the requirement for Wnt signaling during olfactory memory formation in Drosophila using an inducible RNAi approach. Interfering with β-catenin expression in adult mushroom body neurons specifically impairs long-term memory (LTM) without altering short-term memory. The impairment is reversible, being rescued by expression of a wild-type β-catenin transgene, and correlates with disruption of a cellular LTM trace. Inhibition of wingless, a Wnt ligand, and arrow, a Wnt coreceptor, also impairs LTM. Wingless expression in wild-type flies is transiently elevated in the brain after LTM conditioning. Thus, inhibiting three key components of the Wnt signaling pathway in adult mushroom bodies impairs LTM, indicating that this pathway mechanistically underlies this specific form of memory (Tan, 2013).
This study was prompted by a previous discovery that a
casein kinase Iγ homolog (CkIγ), gilgamesh (gish), is required
for STM in Drosophila (Tan, 2010). CkIgγmediated phosphorylation of the cytoplasmic tail of
Lrp5/6 (Arr) is crucial for Wnt/β-catenin signaling (Davidson, 2005), and it was predicted that disruption of the Wnt signaling pathway would perturb STM. Surprisingly, however, it was found
that knockdown of the four Wnt signaling components leaves
STM intact. The likely explanation for this discrepancy is that
Gish serves other important functions in STM formation
besides its role in LTM through phosphorylation of the Arr
receptor (Tan, 2013).
How does Wnt signaling in the MB neurons mediate the
formation of LTM? Since the normal expression of β-catenin,
Wg, and Arr is required in the set of MB neurons defined by
P{MB-GeneSwitch}12-1, and Wg is a short-range ligand, a model is favored in which the Wnt ligand, Wg, participates in an
autocrine fashion in the MB neurons. Spaced conditioning,
which produces long-term behavioral memory, but not massed
or single-cycle conditioning, leads to a transient increase in wg
expression in the MB neurons, perhaps as a step downstream
of Creb. The subsequent secretion of Wg by the MB neurons
activates the Fz/Arr receptor, leading to the accumulation
of β-catenin in the MB neurons. β-catenin, in turn,
orchestrates transcriptional changes in the MB neurons that
are required for LTM, as well as the breaking and remaking
of cell contacts through N-cadherin function, which is necessary
for the reorganization of synapses for LTM storage.
Recently, ribonucleoprotein particles containing synaptic protein
transcripts were shown to exit the nucleus through a
nuclear envelope budding process in response to Wnt signaling
at the Drosophila neuromuscular junction (Speese, 2012).
Wnt-dependent nuclear budding could provide the initial step
for transporting RNAs to synapses for local protein synthesis
and LTM formation (Tan, 2013).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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wingless
continued:
Biological Overview
| Evolutionary Homologs
| Transcriptional regulation
|Targets of Activity
| Protein Interactions
| mRNA Transport
| Developmental Biology
| References
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