Wingless, neuroblast determination and brain development In the ventral nerve cord of Drosophila most axons are organized in a simple, ladder-like pattern. Two
segmental commissures connect the hemisegments along the mediolateral axis and two longitudinal connectives
connect individual neuromeres along the anterior-posterior axis. Cells located at the midline of the
developing CNS first guide commissural growth cones toward and across the midline.
four sequential steps involved in commissure development. Initially, single minded, jaywalker, Egf receptor and slit are involved in the first step in midline formation: the formation of the anlage of the CNS midline. Next the segment polarity genes hedgehog, engrailed, patched and wingless are involved in the specification of midline cell number. It is possible that midline and ectodermal pattern formations occur at the same time. In addition to the segment polarity genes other signaling mechanisms appear important. Notch, for example, is required to specify the different midline lineages.
The third step in commissure formation consists of the formation of commissures. Once the midline cells have been specified, they guide commissural growth cones toward and across the midline. Here, the Netrins, frazzled, commissureless, weniger, schizo, roundabout and karussel play an essential role. The fourth step in commissure development involves the separation of the commissures (Hummel, 1999).
wingless is required for neuroblast delamination from the neuroectoderm. wingless is regionally expressed in the neuroectoderm from which neuroblasts
develop. A conditional wingless mutation is used to inactivate CNS function without affecting
segmentation. The strip of wingless-expressing neuroectoderm generates apparently normal
neuroblasts after wingless inactivation; however, adjacent anterior and posterior neuroectoderm
requires wingless nonautonomously for subsequent neuroblast determination and formation. Loss of
wingless results in the absence or duplication of identified neuroblasts, highlighting its role in
generating neuroblast diversity in the CNS (Chu-LaGraff, 1993).
See Chris Doe's Hyper-Neuroblast map site for information on the expression of wingless in specific neuroblasts.
Mutations in Notch and wingless have been used to study the process of neurogenesis in the neuroectoderm. Patterns of delamination and mitosis are closely correlated: delamination occurs
either immediately after a cell has divided (in the case of microchaete precursors) or shortly before the
division (in the case of neuroblasts). In addition, cytoskeletal changes similar to those occurring
during mitosis can be seen in delaminating neuronal precursors. Thus, during both mitosis and
delamination, the discrete apicobasally oriented microfilament-tubulin bundles break down.
Microfilaments form a dense, diffuse cortical layer surrounding the entire cell body. Microtubules
are concentrated at the apically located centrosome. The relationship between mitosis and
delamination is supported by the finding that the neurogenic gene Notch and segment polarity gene
wingless affect both proliferation and delamination in the ventral neurectoderm. Thus, in
embryos expressing the truncated cytoplasmic domain of the neurogenic gene Notch under
heat-shock control, all ventral neurectodermal cells enter mitosis
prematurely, followed by the absence of neuroblast delamination. In wg loss-of-function mutants,
mitosis in the ventral neuroblast is irregular and generally postponed, accompanied by irregularities in the timing of
neuroblast delamination in general and the absence of a subset of neuroblasts (Hartenstein, 1994).
The Drosophila Frizzled (Fz) and Frizzled2 (DFz2) proteins function as receptors for Wingless (Wg)
in tissue culture cells. While previous results indicate that loss of function for fz results in tissue polarity
defects, the loss-of-function effects of Dfz2 are not known. The requirements of
fz and Dfz2 during neurogenesis have now been examined. Both Fz and DFz2 function in Wg signaling,
and loss of either of the two affects the same subset of neuroblasts as those affected by loss of wg.
While these defects are partially penetrant in embryos lacking either fz or Dfz2, the penetrance is
significantly enhanced in embryos lacking both. Since the penetrance of the CNS phenotypes is not
complete in double mutants, additional components that allow some degree of Wg signaling must exist
in vivo (Bhat, 1998).
In the ventral nerve cord of the Drosophila embryo, wg is expressed in row 5 cells within a segment.
It is nonautonomously required for the formation and specification of row 4 neuroblasts as well as for
the formation of a few neuroblasts in row 5 and most neuroblasts in row 6. Among those
neuroblasts that are affected in wg mutants, NB4-2, a row 4 neuroblast that gives rise to the RP2/sib
lineage, has
been one of the most studied neuroblasts in the CNS. The RP2 and its sibling cell are formed from the
first GMC of NB4-2 (this GMC is known as GMC-1 or GMC4-2a). In this lineage, wg is
required for both the formation and specification of this neuroblast. The elimination of maternal and zygotic fz gene products causes loss of NB4-2->GMC-1->RP2/sib lineage and failure in the formation of row 6 neuroblasts in the ventral nerve cord of the Drosophila embryo (Bhat, 1998).
It has been argued that Fz might not function in the Wg signaling during wing and eye
development, and that in these tissues, while DFz2 functions to receive the Wg signal, Fz receives the
signal from some other Wnt. If DFz2 is
solely responsible for receiving the Wg signal in the CNS, elimination of Dfz2 should have eliminated
the NB4-2 lineage in a manner similar to loss of function for wg. Given that the wg-like CNS defects in
Dfz2 embryos are only partially penetrant, as is the case with fz mutants, the simplest explanation is
that there is a genetic redundancy between fz and Dfz2 and both function in the transduction of the Wg
signal. The observation that the penetrance of the RP2/sib lineage phenotype is significantly enhanced
in embryos lacking both fz and Dfz2 activities certainly reinforces this view. These results are also
consistent with the observation that during epidermal patterning, while the intracellular localization of
Arm is not significantly affected in embryos missing Dfz2, it is nearly lost in embryos missing both the
activities (Bhat, 1998 and references).
The role of the wingless gene has been studied in the embryonic brain development of Drosophila. wingless
is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller
domains in the anlagen of the deutocerebrum and tritocerebrum. In the protocerebral and deutocerebral anlagen, wg-expressing neuroblasts are first observed at late stage 10. At both stages 11 and 12, three groups of wg-immunoreactive cells are observed in the embryonic brain. These are referred to as wg-b1, wg-b2 and wg-b3 for cells in the protocerebrum, deutocerebrum and tritocerebrum respectively. At stage 12, the wg-b1 domain represents a significant part of the protocerebral anlage. The wg-b1 domain remains prominent throughout the rest of embryogenesis. At embryonic stage 15, the anterior end of the embryonic brain is Wg positive, with the wg-b1 domain forming a "cap" around the most anterior part of each hemisphere. The wg-b2 domain consists of markedly fewer cells than the wg-b1 domain. This small domain is formed of two groups of cells that are separated by a small distance. Wg immunoreactivity in the wg-b2 domain disappears at late stage 12. The wg-b3 domain comprises only a few cells and Wg immunoreactivity in the wg-b3 domain also ceases at late stage 12 (Richter, 1998).
Elimination of the wingless gene in
null mutants has dramatic effects on the developing protocerebrum; although initially generated,
approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death
at late embryonic stages. To characterize this deleted part of the brain in more detail, immunocytochemical labelling studies were carried out for the Brain specific homeobox (Bsh) protein and the Engrailed protein in wg mutant embryos. Bsh is expressed exclusively in the the developing brain; at embryonic stage 15, Bsh immunoreactivity is found in three small groups of cells in the developing protocerebrum. In the mutants, only two groups of Bsh-positive cells remain in the protocerebrum; the Bsh-immunoreactive cells, which are the most anterior cells in the wild-type protocerebrum, are missing in the mutant. En immunostaining in the wild-type embryonic brain labels three small cell clusters at borders of the protocerebral, deutocerebral and tritocerebral anlage, and also a small group of cells in the embryonic protocerebrum, which form the En secondary head spot. In stage 15 mutants, the most anterior group of En positive cells (En secondary head spot) is missing; however, the En cluster in the posterior protocerebrum is still present. Before stage 13, no obvious signs of structural defects in the protocerebrum are observed in wg mutants (Richter, 1998).
Using temperature sensitive mutants, a rescue of the mutant phenotype can
be achieved by stage-specific expression of functional Wingless protein during embryonic stages 9-10.
This time period correlates with that of neuroblast specification but preceeds the generation and
subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants
results in a dramatically oversized CNS. The oversized brains are patterned to a certain degree, in that the neuromeres can be identified and major axon tracts, like the longitudinal tract, are visible. To investigate the oversized brains further, the expression pattern of the brain specific homeobox (bsh) gene was studied. In the wild type, bsh is expressed in distinct domains of the protocerebrum. In the oversized brains, bsh expressing cells are scattered throughout the anterior brain and the region that expresses bsh is increased more than twofold. This indicates that the embryonic protocerebrum has increased that much in size. It is concluded that wingless is required for the development of
the anterior protocerebral brain region in Drosophila. It is proposed that an important role for wingless in
this part of the developing brain is the determination of neural cell fate. Interestingly, ectopic expression of Wnt-1 in the mouse spinal cord also causes massive overgrowth of the spinal cord tissue (Dickson, 1994). This overexpression is caused by increased mitosis (Richter, 1998).
The Drosophila central nervous system derives from neural precursor cells, the neuroblasts (NBs), which are born from the neuroectoderm by the process of delamination. Each NB has a unique identity, which is revealed by the
production of a characteristic cell lineage and a specific set of molecular markers it expresses. These NBs delaminate
at different but reproducible time points during neurogenesis (S1-S5) and it has been shown for early delaminating NBs (S1/S2) that their identities depend on positional information conferred by segment polarity genes and
dorsoventral patterning genes. Mechanisms have been studied leading to the fate specification of a set of late delaminating neuroblasts, NB 6-4 and
NB 7-3, both of which arise from the engrailed (en) expression domain, with NB 6-4 delaminating first. No evidence is found for a direct role of hedgehog in the process of NB 7-3 specification. NB 7-3 normally requires Hh only for maintenance of Wg expression, which in turn leads to En maintenance.
Evidence is presented to show that the interplay of the segmentation genes naked cuticle (nkd) and gooseberry (gsb), both of which are targets of wingless (wg) activity, leads to differential commitment to NB 6-4 and NB 7-3 cell fate. In the absence of either nkd or gsb, one NB fate is replaced by the other. However, the temporal sequence of delamination is maintained, suggesting that formation and specification of these two NBs are under independent control (Deshpande, 2001).
In the En domain Wg plays a role both in NB formation and NB specification. The homeodomain transcription factor En is a prerequisite for the formation of the NBs 6-4 and 7-3, because in its absence both NBs fail to form. Since Wg signaling is necessary for maintaining En expression, it is also essential for the formation of these two NBs. Hh is co-expressed in the En domain and En maintains Hh expression in rows 6 and 7, and Hh in turn is essential for Wg expression in row 5, thereby constituting a maintenance loop. Thus, for late NBs in row 6 and 7, the expression of En is crucial and Hh is required to maintain En expression via Wg. However, for the separate specification of NB 6-4 and NB 7-3, differential regulation of two Wg targets, nkd and gsb, is essential (Deshpande, 2001).
Wg is a diffusible molecule expressed in row 5 and acts on neighboring rows, which include rows 6 and 7. However, row 6 differs from row 7 because it expresses gsb, which is, as stated above, a target of Wg signaling. The fact that row 7 does not express gsb, despite being under the influence of Wg raises the question of how this differential regulation is brought about. In this work it is shown that Nkd is essential for this regulation. Nkd is a negative regulator of the Wg signal transduction pathway, itself being a target of this pathway. In the absence of Nkd, Gsb is derepressed, owing to Wg hyperactivity in row 7, leading to the generation of an ectopic NB 6-4 like fate. Thus, the distinct identities of NB 6-4 and NB 7-3 are brought about by the interplay of Gsb and Nkd. For NB 6-4 specification, Gsb is an essential factor. In the absence of Gsb NB 6-4 fails to be specified and instead takes the identity of NB 7-3 fate. Conversely, for NB 7-3 specification, a Gsb-free environment, which is created by the activity of Nkd, is essential. In summary, NB 6-4 needs the expression of Gsb and En, whereas NB 7-3 needs En but the absence of Gsb (Deshpande, 2001).
However, the fact that gsb as well as nkd are targets of Wg signaling makes it difficult to explain why gsb is repressed by nkd only in the posterior region of the En stripe. The posterior En domain is further away from the Wg source than the anterior En domain and therefore should receive a lower signaling input when compared with the anterior region. As a consequence, this should lead to higher Nkd activity in the anterior En cells, leading to a stronger Gsb repression in this region -- the opposite of what was observed. A careful analysis of the expression pattern on the transcriptional level does not give any obvious clues to solve this apparent paradox. During early germ band extension (stage 8-9) nkd transcription is nearly ubiquitous with higher RNA levels in the two to four cell rows posterior to the En stripe. At late phase of germ band extension, nkd expression is most abundant anterior to the En stripe and lower just posterior to the En-stripe. No significant difference between the anterior and posterior En domain could be detected. One explanation for the differential regulation of gsb could be that, owing to earlier pair rule gene activity of paired, the level of Gsb protein at the time of NB 6-4 delamination in the anterior En region is high enough to override repression by Nkd activity. Alternatively, a direct differential regulation of the two Wg targets that is due to the different levels of Wg signaling could be responsible for the observed regulatory differences. It could be that the regulation is such that the amount of Wg signaling within the En stripe causes a relatively homogenous level of nkd expression in this region. At the same time, the transcriptional activation of gsb could be more sensitive to Wg signaling levels, resulting in a very strong activation, especially near to the Wg-expressing cells. As a result, the relatively low Nkd activity in the whole En stripe might be able to inhibit gsb expression in the region of low gsb activation only: the posterior En domain. A hint that a differential regulation of Wg targets indeed exists comes from the Wg-dependent En regulation: it seems that a lower Nkd activity is sufficient to repress gsb but not to inhibit en expression. This conclusion was drawn from the finding that overexpression of nkd within the En stripe using an EnGal4 driver line leads to a selective repression of gsb with no obvious effect on en expression itself. Clearly, additional work has to be carried out to clarify these points (Deshpande, 2001).
Besides row 6 neuroectoderm, row 3 neuroectoderm also has the potential to generate an ectopic NB 7-3. It has been shown previously that in embryos mutant for ptc, neuroectodermal cells in the area of row 3 begin to express En and additional serotonergic neurons can be found in these mutant embryos, which suggests the presence of an ectopic NB 7-3 like fate. Additionally, when En is ubiquitously expressed, only row 3 has the ability to give rise to an ectopic NB 7-3 fate. In all cases, this occurs at the cost of row 3 NBs such as NB 3-3. It is thought that this might reflect that row 3 neuroectoderm, which is right in the middle of the segment, represents something like a 'ground state' in the neuroectoderm: in this area neither Hh nor Wg signaling may take place. Therefore the decision to specify late row 3 or late row 7 NBs seems to be only dependent on the absence or presence of En, respectively (Deshpande, 2001).
Previous work has indicated that genes expressed in proneural clusters are involved in specifying the individual fates of NBs that develop from these clusters. The finding that NB 6-4 and NB 7-3 can be mutually transformed while the sequence of birth does not change suggests that the mechanism for the timing of late NB delamination is independent from mechanisms that regulate NB identity. This might be reminiscent of early NBs. Initiation of S1 NB formation requires the activity of proneural genes that have been shown to be dependent on pair-rule genes. The identity of the NBs delaminating from these clusters, however, is dictated by the activity of segment polarity genes. Thus, the control of proneural gene expression that enables NB formation and the control of segmentation genes conferring NB identity occurs in parallel. At later stages, pair-rule gene expression vanishes and can no longer be responsible for NB formation. How is NB formation regulated in the following segregation waves? One possibility is that after the first segregation wave, NB formation and identity are more tightly linked; the finding that specific NBs like NB 4-2 are sometimes not transformed but missing in wg mutant embryos seems to support this idea. However, the finding that the transformed NB 6-4 and NB 7-3 are delaminating according to the 'old identity' shows that, at least in these cases, NB formation and specification is independent. The results favour the idea that the timing of the formation of proneural clusters within the neuroectoderm is generally independent of the segment polarity genes investigated here. This does not exclude permissive functions, such as those of En, which enable the proneural cluster formation as such. According to this hypothesis, intrinsic or extrinsic factors present in the position of the proneural cluster at the time of delamination govern the identities of the NBs. This might be not only true for the positional regulation of NB identity but also for the determination of NB identity along the temporal axis. Indeed, heterochronic transplantation experiments strongly support the possibility that one or more extrinsic factors exist that lead to stage specific NB identities. It will be a challenge for the future to identify these factors, and to investigate whether similar mechanisms exist in higher organisms (Deshpande, 2001).
The hindgut and wingless During Drosophila embryogenesis, the development of the
midgut endoderm depends on interactions with the
overlying visceral mesoderm. The
development of the hindgut also depends on cellular
interactions, in this case between the inner ectoderm (hindgut ectoderm) and
outer hindgut visceral mesoderm (HVM). In this section of the gut, the
ectoderm is essential for the proper specification and
differentiation of the mesoderm, whereas the mesoderm is
not required for the normal development of the ectoderm.
Wingless and the fibroblast growth factor receptor
Heartless act over sequential but interdependent phases of
hindgut visceral mesoderm development. Wingless is
required to establish the primordium and to enhance
Heartless expression. Later, Heartless is required to
promote the proper differentiation of the hindgut visceral
mesoderm itself (Martin, 2001).
The caudal mesoderm gives rise to two populations of visceral
mesoderm, the HVM and the LVM (longitudial visceral mesoderm that surrounds the endodermal midgut). The HVM
primordium is distinguishable at stage 10 both by the expression
of bagpipe (bap) and by virtue of its relatively higher
levels of Twist. It includes all mesodermal cells caudal
to the 15th Wg stripe, which, unlike mesoderm cells
in the trunk, are organised in multiple layers soon after gastrulation. In contrast,
the LVM primordium arises from cells adjacent and egg-posterior
to the HVM (Martin, 2001).
The hindgut ectoderm is derived from a ring of cells that lies
just anterior to the posterior midgut primordium at the cellular
blastoderm stage. At stage
7, these cells invaginate into the embryo and, by stage 10, form
a hollow tube that extends by cell division and rearrangement.
HVM cells become closely associated with the invaginated
hindgut ectoderm at stage 11. Later in
this stage, all HVM cells begin to express Connectin and this together
with Twist expression, can be used to follow the cells as they
move over the hindgut ectoderm tube (Martin, 2001).
As the germband retracts, the hindgut tube undergoes
considerable morphological rearrangements. By stage 13, it
lies longitudinally from the anus and bends
at right angles to join the posterior midgut. Over time, the
bend flattens laterally and the hindgut lengthens, revealing
morphological subdivisions. HVM cells continue
to cover the ectodermal tube during these stages and as they
mature, they begin to express Myosin (Martin, 2001).
To determine whether hindgut ectoderm requires interactions
with mesoderm to form normally, its development
was followed in twist mutant embryos that lack mesoderm. In these embryos,
cells of the hindgut ectoderm can form a tube that bends
anteriorly, lengthens partially at stage 13 and expresses Wg and
Dichaete in restricted domains, similar to wild-type embryos. Thus, characteristic features of ectodermal gut
development occur in the complete absence of surrounding
visceral mesoderm (Martin, 2001).
To test whether hindgut ectoderm is needed for the proper
development of the HVM, hindgut ectoderm cells were
selectively killed early in their embryogenesis using the
GAL4/UAS system. 455.2GAL4, which is expressed in the
primordium of the hindgut ectoderm from stage 9 onward but
not in the caudal mesoderm, was used to drive expression of
reaper, a gene whose protein product promotes death of those
cells in which it is expressed (Martin, 2001).
As in wild-type embryos, Twist is expressed strongly in the
prospective HVM at early stage 11 in embryos carrying
455.2GAL4;UASreaper constructs.
However, even though the morphology of the hindgut ectoderm
appears normal at this stage, the bulk of the HVM
is not closely associated with the hindgut ectoderm.
By late stage 11, the number of HVM cells expressing
Connectin or Twist is clearly reduced.
This is concomitant with the severe disruption in the
morphology of the hindgut ectoderm. During stage 13, many
HVM cells die. The surviving cells are those that
attach to any remaining hindgut ectoderm; these cells
persist, differentiating to form a variable amount of visceral
muscle. Thus it is concluded that the hindgut
ectoderm acts as a template to promote the development and
differentiation of the HVM (Martin, 2001).
The hindgut ectoderm might be required simply because it is
an essential substrate for the development of the HVM. To test
whether it is also the source of essential signals,
the formation of the HVM was examined in embryos with mutations in dpp, hh and wg, which are known to be expressed in the hindgut
ectoderm during embryogenesis.
Of these, only Wg is essential for the differentiation of the
HVM. In embryos mutant for wg, bap expression is reduced
and Twist expression fails to be enhanced in the HVM at stage
10. By stage 11, even fewer cells express
Connectin and Twist. By mid stage 12, some
embryos completely lack Connectin and Twist expression in
the caudal mesoderm, while in other embryos only
a few cells maintain their expression and these cells are unable
to cover the whole of the hindgut ectoderm. The few remaining cells may eventually
express Myosin, but this is difficult to assess because
some syncitial somatic muscle cells appear to attach
ectopically to the hindgut ectoderm in these embryos.
In wg embryos, proctodeal cells fail to divide after gastrulation. Thus, the hindgut ectoderm
is very small and this reduction in the size could indirectly cause
the defects observed in the development of surrounding visceral
mesoderm. To test this, the development of the
HVM was compared in string (stg) embryos, where all cells fail to divide after
the cellular blastoderm stage (cycle 13). As in wg embryos, a very small ectodermal hindgut develops, but many more visceral mesoderm cells maintain Connectin expression. These cells eventually envelop the whole of the ectodermal tube and
differentiate to express Myosin. Thus, the defects
in wg embryos cannot solely be explained by the reduced size
of the hindgut ectoderm, suggesting that Wg signaling is
required directly for the normal development of the HVM (Martin, 2001).
If Wg signals to the surrounding mesoderm and promotes the
development of visceral mesoderm, then the Wg signaling
pathway should be activated in the caudal mesoderm.
Consequently, in wg embryos, the development of the HVM
might be rescued if the Wg signaling pathway was activated
autonomously throughout the mesoderm. One of the
components needed to transduce the Wg/Wnt signaling
pathway is Armadillo (Arm). A constitutively active form of arm
(arm S10C) under the control of the UAS promoter and two mesodermal GAL4 drivers, twistGAL4 and 24BGAL4, were used to activate the Wg pathway in the mesoderm. twistGAL4 activates expression throughout the mesoderm
from stage 6 onwards and is maintained in the HVM
throughout embryogenesis. In contrast, 24BGAL4 drives weak and patchy
expression at stage 9, which is reinforced by late stage 10, and spreads
to most mesoderm cells, including those of the HVM.
Expression of armS10C under the control of twistGAL4, but
not 24BGAL4, partly rescues the development of the HVM in
wg embryos as seen by an increase in the expression of
Connectin at stage 12. One explanation for the
difference between the two drivers is that expression of
armS10C driven by 24BGAL4 is too late to rescue HVM
development. However, twistGAL4 also drives transient and
weak expression in the hindgut ectoderm, and it could be this expression that
provides the rescuing effect (Martin, 2001).
To clarify the results of the rescue experiments,
the Wg signaling pathway was activated solely in the hindgut ectoderm of
wg embryos using 455.2GAL4. In these embryos,
more HVM cells express Connectin at stage 12 than in wg
embryos and these cells eventually cover the whole of the hindgut ectodermal template. Since the size of the hindgut ectoderm does not appear
to be significantly rescued in these embryos, it is suggested that
this tissue acquires the necessary properties to support visceral
mesodermal development throughout its length. Importantly
however, despite the continuous expression of armS10C
throughout the hindgut ectoderm, the rescue observed is not
as strong as when using the twistGAL4 driver. Thus,
in wg embryos, both hindgut ectoderm and mesoderm respond
to the activation of the Wg signaling pathway and promote
the development of the HVM (Martin, 2001).
Although wg is expressed in the primordium of the hindgut
ectoderm, it is also expressed in epidermal stripes and transiently
in the mesoderm. Any of these sources of Wg might
promote the development of the HVM. Indeed, in fork head (fkh)
embryos, which lack Wg expression in the hindgut ectoderm many more caudal mesoderm cells begin to express Connectin than in wg embryos. By
stage 12 however, Connectin expression begins to diminish quite
rapidly in fkh embryos, but this is most likely due to the loss of
hindgut ectoderm at later stages. This suggests that at
least for the early stages of HVM development, the source of
Wg need not be the hindgut ectoderm alone. It has been shown that loss of Wg signaling after stage 11 does not affect HVM development (Martin, 2001).
This analysis shows that the establishment of the HVM
primordium at stage 10 relies on Wg but not on htl.
Consequently, there is an early htl-independent role for Wg.
However, in this first phase of HVM development, Wg is also
required for the normal development of htl expression in the
HVM and in wg embryos, expression of htl in the cells that
normally give rise to the HVM does not become enhanced at
stages 9-10. At stages 11 and 12, Wg and htl partly function
independent of one another in controlling Connectin
expression in the HVM. Thus, the loss of Connectin
expression in the HVM is more severe in embryos mutant for
both wg and htl than in either wg or htl mutant embryos. All wg;htl double mutant embryos lose expression of Connectin
in the HVM by stage 12, whereas at the same stage,
some wg embryos maintain Connectin expression in a few
cells and there is weak expression of Connectin in
htl embryos. If Wg can act in parallel with htl to
promote Connectin expression in the HVM, this explains why
misexpression of Wg throughout the hindgut ectoderm of a
htl mutant embryo directs strong expression of Connectin in
HVM cells at stage 12. Differentiation of the HVM requires htl. Wg is not
required past stage 11 for the continued development of the
HVM, and the HVM fails to differentiate and express
Myosin when Wg is misexpressed in the hindgut ectoderm of
a htl mutant embryo (Martin, 2001).
The Drosophila embryonic hindgut is a robust system for the study of patterning and morphogenesis of epithelial organs. In a period of about 10 h, and in the absence of significant cell division or apoptosis, the hindgut epithelium undergoes morphogenesis by changes in cell shape and size and by cell rearrangement. The epithelium concomitantly becomes surrounded by visceral mesoderm and is characterized by distinct gene expression patterns that forecast the development of three morphological subdomains: small intestine, large intestine, and rectum. At least three genes encoding putative transcriptional regulators, drumstick (drm), bowl, and lines (lin), are required to establish normal hindgut morphology. The defect in hindgut elongation in drm, bowl, and lin mutants is due, in large part, to the requirement of these genes in the process of cell rearrangement. Further, drm, bowl, and lin are required for patterning of the hindgut, i.e., for correct expression in the prospective small intestine, large intestine, and rectum of genes encoding cell signals (wingless, hedgehog, unpaired, Serrate, dpp) and transcription factors (engrailed, dead ringer). The close association of both cell rearrangement and patterning defects in all three mutants suggest that proper patterning of the hindgut into small intestine and large intestine is likely required for its correct morphogenesis (Iwaki, 2001).
This study shows that drm, bowl, and lin are required for the gene expression patterns that distinguish these three domains: lin is required for expression characteristic of large intestine (dpp, dri, and en) and rectum (Ser, hh, and wg); drm and bowl are required for expression characteristic of small intestine (hh and upd). By both morphological criteria (cell shape, presence or absence of boundary cells) and gene expression patterns (expanded expression of genes expressed in the small intestine), lin hindguts appear to consist of a greatly expanded small intestine and to lack the large intestine and rectum. In contrast, both morphological and gene expression characteristics of drm and bowl hindguts indicate that they lack most or all of the small intestine, and consist only of large intestine (which remains unelongated) and rectum. A model consistent with these data is that lin functions in the hindgut to repress small intestine fate and to promote large intestine and rectum fate, while establishment of the small intestine requires the activity of drm and bowl. The requirement for drm (but not bowl) for wg expression at the most anterior of the hindgut could be explained if the domain of bowl function in the small intestine does not extend to the most anterior of the hindgut (consistent with the expression of bowl). Since they have opposite effects on Ser expression, bowl and drm may function in different ways, possibly in different pathways, to promote small intestine fate (Iwaki, 2001).
The function of lin as both an activator and repressor of gene activity in the developing hindgut is consistent with molecular and genetic characterization of its function in other embryonic tissues. In the developing dorsal epidermis, lin is required for transcriptional regulation (both activation and repression) of targets downstream of wg signaling. In the developing posterior spiracles, lin is required for the activation by Abd-B of its transcriptional targets. lin encodes a novel protein that is expressed globally throughout the embryo, including the developing hindgut. When ectopically expressed, Lin protein is detected in nuclei of cells signaled by Wg. The early expression of wg throughout the hindgut primordium, starting at the blastoderm stage and continuing to stage 10, might, analogous to its effect in the dorsal epidermis, activate Lin. This might be required for Lin to carry out its function, demonstrated here, of promoting expression of genes characteristic of large intestine identity (otp, dpp, en, and dri), and repressing expression of genes characteristic of small intestine identity (hh, upd, and Ser).
The phenotypes described for wg, hh, dpp, dri, Ser, and en do not suggest a role for these genes in hindgut elongation by cell rearrangement. Mutations in Ser, dri, and en do not appear to affect overall hindgut morphology. The hindgut in wg mutants is extremely small, suggesting that the critical function of wg in hindgut development is in establishing and maintaining the primordium, but not in elongation. dpp mutant hindguts are shorter, consistent with the role of dpp in endoreplication in the large intestine; nevertheless, dpp hindguts have a roughly normal diameter. In hh mutant embryos, the rectum degenerates and hindgut length is reduced, but the overall morphology, particularly the narrowing of the large intestine, appears normal (Iwaki, 2001).
Only in upd embryos is a defect in both elongation and narrowing of the hindgut observed; significantly, upd is expressed only in the small intestine, the same domain that is largely missing from drm and bowl mutants. Shorter and wider hindguts are seen in younger upd embryos, but the majority of hindguts in mature upd embryos appear normal. Thus, while upd may at least partially mediate drm and bowl function in the hindgut, there must be other targets of these genes that are required for cell rearrangement in the hindgut (Iwaki, 2001).
In a genetic screen to identify mutations that suppress or enhance the mutant
phenotype of the wg temperature-sensitive allele
wgIL114, two EMS-induced mutations were isolated that subtly
modify the wgIL114 mutant cuticle pattern. These modifier
mutations, AR2 and DH15, fail to complement each other and thus
identify a single complementation group, linked to wg on the second
chromosome. These nonsense mutations are shown to disrupting the
RacGAP50C locus. The two alleles produce identical phenotypes. Both mutations show no increase in severity when placed in trans to a deficiency for the region and so are likely to represent
loss-of-function alleles (Jones, 2005).
wgIL114 embryos at the restrictive temperature are much
smaller than wild-type embryos and secrete the 'lawn of denticles' cuticle
pattern typical of low pathway activity. The
modifier lines AR2 and DH15 were found to alter the
wgIL114 phenotype in a similar way, increasing the spacing
between denticles and the overall body size of the doubly mutant embryos.
This phenotype indicates that the modifier
mutations considerably reduce the severity of the wg loss-of-function
phenotype, resulting in a larger, healthier embryo even though the cuticle
pattern is only subtly altered. Both AR2 and DH15 also modify
other wg alleles, including wgCX4, an RNA null allele
of wg. The cuticle pattern defects of weak wg alleles
are more dramatically suppressed by the AR2 and DH15 mutations.
The wgPE2 hypomorphic allele produces a protein with a lower
affinity for the receptor complex. wgPE2 homozygous
mutants show segmental denticle diversity, but little or no naked cuticle
separating the belts. The wgPE2
AR2 doubly homozygous mutants show an increase in the amount of naked
cuticle, indicating that some Wg signaling is restored (Jones, 2005).
In an otherwise wild-type background, the AR2 and DH15 mutations
are recessive lethal, and homozygous embryos show an excess specification of
naked cuticle at the expense of denticles. Wild-type embryos secrete an average of 5.95 ± 0.15 rows of
denticles/belt in abdominal segments 3-6. In contrast,
AR2 mutant embryos secrete an average of only 3.95 ± 0.64 rows of
denticles/belt. The mutant
denticles also differ morphologically from the wild type. Wild-type denticles
show a wide range in size, with those in row 5 being largest. The AR2 and DH15 mutant denticle belts contain fewer of
these large denticles: most denticles observed are similar in size to the
smaller denticles normally found in rows 1-4 of the wild-type belt pattern.
The slight suppression of wg mutant phenotypes and the ectopic
specification of naked cuticle are consistent with an increase in Wg pathway
activity, suggesting that the AR2 and DH15 mutations might disrupt
a negative regulator of the pathway (Jones, 2005).
RacGap50C interacts genetically
with nkd and appears to act at the same level or downstream of Axin in
the control of Arm stabilization. The data indicate that RacGap50C probably does
not act through Rac1 to negatively regulate Wg activity, nor are other GTPases
likely to be involved in this aspect of epidermal patterning since the cuticle
defects of mutant embryos can be rescued by a form of RacGap50C that lacks
catalytic residues in the GTPase-activating domain. Moreover, previous work
shows that other Rho family members are unlikely to be involved in Wg-mediated
patterning. Overexpressing either constitutively active or dominant-negative
Rho, Rac, or cdc42 transgenes disrupts dorsal closure but
does not appear to affect ventral patterning. Loss of maternal Rho
activity has been found to alter embryonic segmental pattern, but this is due to
an early effect on establishing segmentation gene expression patterns. Ras activation
through the EGF signaling cascade has been found to affect epidermal patterning,
but in a way that counteracts Wg signaling. Thus a GTPase-activating protein
would be expected to positively influence Wg-mediated patterning if it acted
through Ras, rather than the negative influence observed for RacGap50C (Jones, 2005).
For these reasons, it is believed that the role of RacGap50C in Wg signaling
may instead parallel its role in cytokinesis, where it seems to function
primarily as an adaptor molecule. RacGap50C was identified by the Saint
laboratory through a yeast two-hybrid screen for molecules that interact with
pebble, a RhoGEF that is essential for cytokinesis (Somers, 2003).
Subsequently, it was shown that RacGap50C protein also binds to
Pavarotti, a kinesin-like molecule, thus forming a bridge between the
microtubule and actin cytoskeletons. This adaptor function appears critical for
the proper positioning of the acto-myosin contractile ring at the end of mitosis (Jones, 2005).
One could imagine that a structural role for RacGap50C in linking the
microtubule and actin cytoskeletons might be relevant to its regulation of Wg
pathway activity. Apc2, a scaffolding molecule that is an essential component of
the destruction complex, is known to interact with both microtubules and the
cortical actin cytoskeleton.
Mutations that disrupt the cortical localization of Apc2 compromise function of
the destruction complex, suggesting that subcellular localization of the complex
may be critical. Furthermore, recent work demonstrates that Axin, another scaffolding component in the
complex, changes its subcellular localization in response to Wg signaling and is
consequently degraded. Thus the positioning of the destruction complex and/or some
of its subunits may play a critical role in regulating its Arm-degrading
activity. RacGap50C may be involved directly in linking the destruction complex
to the cell cortex to promote its proper activity or may restrict movement of
Axin to the cortex for its Wg-mediated destruction. In either case, loss of
RacGap50C function would reduce the normal degradation of Arm and thereby
cause ectopic Wg pathway activity (Jones, 2005).
Another possible explanation for RacGap50C's effects on Wg pathway activity
would not require direct interaction with any pathway component. Rather, some
coordination between the microtubule and actin cytoskeletons may be generally
required for many different cellular processes. The loss of this adaptor
molecule would 'loosen' the connection between these two filamentous networks
and compromise many cellular events indirectly. It may be that cytokinesis and
Wg signal transduction are particularly sensitive to such perturbations or that
they simply produce the earliest or most easily detected phenotypes in response
to them. A general requirement for microtubule and actin network coordination in
destruction complex function could be of great importance in understanding
oncogenic Wnt pathway activity. Current work on this problem focuses on
distinguishing whether RacGap50C interacts specifically with the destruction
complex or acts more generally by controlling cellular architecture (Jones, 2005).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
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continued:
Biological Overview
| Evolutionary Homologs
| Transcriptional regulation
|Targets of Activity
| Protein Interactions
| mRNA Transport
| Developmental Biology
| References
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