Wingless and segmentation In the trunk of the Drosophila embryo, the segment polarity genes are initially activated by the pair-rule
genes; later, the segment polarity genes maintain one another's expression through a complex network of cross-regulatory
interactions. These interactions, which are critical to cell fate specification, are similar in each of the
trunk segments. To determine whether segment polarity gene expression is established differently
outside the trunk, the regulation of the genes hedgehog (hh), wingless (wg), and engrailed
(en) was studied in each of the segments of the developing head. The cross-regulatory relationships
among these genes, as well as their initial mode of activation in the anterior head are significantly
different from those in the trunk. In addition, each head segment exhibits a unique network of segment
polarity gene interactions. It is proposed that these segment-specific interactions evolved to specify the
high degree of structural diversity required for head morphogenesis (Gallitano-Mendel, 1997).
The proposed interactions between hh, wg and en are described below.
1. The intercalary segment. In this cephalic segment, hh expression is en-independent. In addition, ptc mutations cause the loss of wg rather than ectopic wg expression The dependence of wg, en, and hh expression on ptc indicates a unique role for segment polarity genes in the intercalary segment. Unlike wg action in the trunk and gnathal segments, wg restricts rather than maintains en and hh expression in this segment. Finally, en expression, as it occurs in the trunk, depends on hh function. However, this dependence cannot be mediated through wg, since wg does not maintain en expression in the intercalary segment.
2. The antennal segment. As in the trunk, hh antennal expression depends on en, while wg expression requires hh. The requirement for hh is presumably mediated through ptc, which represses wg in this segment. Unlike in the trunk, wg restricts the expression domains of both en and hh. As in the intercalary segment, regulation of en by hh is wg- independent.
3. The ocular segment. In this segment, hh is en-independent and wg expression does not require hh. Although the wg domain (the head blob) does not expand in ptc mutant embryos, noncontiguous ectopic wg expression appears in its vicinity. Unlike its action in the trunk and the other head segments, wg is required to initiate en expression in the ocular segment. However, hh expression still expands in wg mutant embryos (as in the intercalary and antennal segment). As in the intercalary and antennal segments, regulation of en by hh does not depend on wg.
It is concluded that cross-regulatory interactions among the segment polarity genes in the anterior head are very different from those in the posterior head and trunk segments. The mode of patterning of the anterior head (the acron and cephalic segments) is thought to be more ancient than that of the posterior head (the gnathal segments). This distinction appears to be reflected in the segmentation mechanism used by certain present day short germ insects and primitive arthropods. In these organisms, the early germ band includes only the acron, cephalic segments, and tail. Gnathal and trunk segments are generated later in embryogenesis by a progressive budding process (Gallitano-Mendel, 1997).
A model for formation of the anterior and posterior compartments maintains that cells at the inter-compartment interface drive pattern formation by becoming the
source of a morphogen. Does this model apply to the ventral embryonic
epidermis of Drosophila? First, it is shown that interfaces between posterior (engrailed ON) and anterior
(engrailed OFF) cells are required for pattern formation. Second, evidence is provided that
Wingless could play the role of the morphogen, at least within part of the segmental pattern.
The cuticular structures are examined that develop after different levels of uniform Wingless
activity are added back to unsegmented embryos (wingless- engrailed-). Unsegmented embryos are small and spherical, carry a low number of unpolarised denticles and have no Keilin's organs, presumably because they have no functional parasegmental boundary. Because it is rich in
landmarks, the T1 segment is a good region to analyse. Here, the cuticle
formed depends on the amount of added Wingless activity. For example, a high
concentration of Wingless gives the cuticle elements normally found near the top of the
presumed gradient. Unsegmented embryos are much shorter than wild type. Alternation of cells expressing engrailed in the "on" and "off" state is necessary for segmentation. If Wingless is added in stripes, the embryos are longer than if it is added uniformly. The response to Wingless depends on whether engrailed is on or off. When en is on, Wingless determines an anterior segmental identity, and when en is off, WG determines a posterior identity. For example when en is off, the ventral abdomen makes naked cuticle instead of denticles. It is suggested that the Wingless
gradient landscape affects the size of the embryo, so that steep slopes would allow cells to
survive and divide, while an even distribution of morphogen would promote cell death.
Supporting the hypothesis that Wingless acts as a morphogen, it is found that at a distance these stripes
affect the type of cuticle formed and the planar polarity of the cells. There are two functions of engrailed: (1) the interface between en on and en off ensures sustained expression of the Wingless morphogen; (2) the presence or absence of en determines the cells as posterior or anterior and selects the responses to the morphogen (Lawrence, 1996).
Signaling by the Epidermal growth factor receptor (EGFR) plays a critical role in the
segmental patterning of the ventral larval cuticle in Drosophila: by expressing either a
dominant-negative EGFR molecule or Spitz, an activating ligand of EGFR, it is shown that EGFR signaling specifies the anterior denticles in each segment of the larval
abdomen. rhomboid, spitz and argos are expressed in denticle rows 2 and 3, just posterior to denticle row 1 in the engrailed expression "posterior" domain of larval ectoderm. These denticles derive from a segmental zone of
embryonic cells in which EGFR signaling activity is maximal. Expression of a dominant negative form of Egfr (DN-DER) leaves just two of the six rows of denticles, corresponding to rows 5 and 6. Expression of ectopic Egfr throughout the ventral epidermis results in larval cuticles showing considerably widened ventral denticle belts, with only narrow, naked stretches between them. Within these belts, the normal rows 1-4 are still recognisable by the morphology and orientation of their denticles. However, posterior to these, row 5 and 6 denticles are not apparent; these are replaced by a wide field of small denticles, apperently of the row 1-4 type. A similar phenotype of excessive denticles is seen after ubiquitous expression of an activated form of Ras (Stutz, 1997).
There is a competition between the denticle fate specified by EGFR signaling and the naked cuticle fate specified by Wingless signaling. The final pattern of the denticle belts is the product of this antagonism between the two signaling pathways. In the absence of Wg signaling, extra denticles form, instead of naked cuticle, whereas, ectopic activation of Wg causes naked cuticle to form, instead of denticles. Expressing DN-DER (that is removing Egfr) in wingless mutants (producing 'double-mutant' conditions) produces denticle belts with almost exclusively large denticles. Mostly based on their size, it is believed that the large denticles are of the row 5 type. This cuticle phenotype demonstrates that Egfr activity is responsible for the row 1-4 denticles that are seen in wingless mutants. There may be very little Egfr signaling in wingless loss of function mutants; the segmental stripes of rhomboid expression are weaker, and those of argos completely disappear. Thus, the segmental activation of Egfr signaling seems to be an indirect consequence of an early function of wingless. Egfr signaling activity can produce denticles in the complete absence of Wg signaling. Expressing Spitz ectopically produces denticles of the row 1-4 type. Egfr signaling does not require armadillo function to specify these small denticles. (Szuts, 1997).
How are precise boundaries formed between different cell types without early compartmentalization? One such boundary occurs between the wingless-expressing cells of the wing margin and the adjacent proneural cells, which give rise to margin sensory bristles. This boundary arises in part by a mechanism termed "self-refinement," by which Wingless protein represses wg expression in adjacent cells. Reduction or removal of WG activity, in this case accomplished using a temperature sensitive allele of wg, results in a failure to resolve this boundary properly. Dishevelled protein is required for the reception of WG signals. It is thought that DSH is activated by the WG signal. Expression of wg in dsh- clones results in expanded wg expression over as many as six cells from the normal wg boundary. Thus, reception of WG signaling is required to repress wg expression in a broad region of competence along the margin. Self-refinement is one of two identified signaling functions of margin Wingless. In the other function, WG signaling is necessary and sufficient for proneural gene expression and the subsequent formation of margin bristles in cells flanking the wg-expressing stripe. One might expect that the ectopic wg expressed within the clone might be able to induce ectopic proneural gene expression in wild-type cells surrounding the clone, leading to the formation of margin bristles abnormally distant from the margin. This phenotype has indeed been reported. One possible way that WG self-refinement might act is by repressing the Notch signaling pathway. It is suggested that heightened Notch activity along the dorsoventral boundary is responsible for the localized expression of boundary-specific genes, including wg. Recent evidence indicates that DSH-mediated WG signalling can inhibit Notch activity (Rulifson, 1996).
wingless (wg) and its vertebrate homologs, the Wnt genes, play critical roles in the
generation of embryonic pattern. In the developing Drosophila epidermis, wg is
expressed in a single row of cells in each segment, but it influences cell identities in all
rows of epidermal cells in the 10- to 12-cell-wide segment. Wg signaling promotes
specification of two distinct aspects of the wild-type intrasegmental pattern: the
diversity of denticle types present in the anterior denticle belt and the smooth or naked
cuticle constituting the posterior surface of the segment. The
expression of wild-type and mutant wg transgenes have been manipulated to explore the mechanism by which a single secreted signaling molecule can promote these distinctly different cell fates.
Evidence is presented consistent with the idea that naked cuticle cell fate is specified
by a cellular pathway distinct from the denticle diversity-generating pathway. The protein encoded for by mutant allele wgPE2 is unable under any circumstances to promote the naked cuticle cell fate in the ventral epidermis, while it can promote denticle diversity (Hays, 1997).
Cells receiving the same level of Wg signaling respond
differently depending on their position within the segment: some cells are directed to secrete naked
cuticle, while others secrete diverse denticle types. This observation hints at a differential distribution of
molecules that interact with Wg to effect its signaling activity. All cells within the segment, however, are
competent to secrete naked cuticle even in a wg null
mutant background. When expression levels of either mutant allele wg PE4 or wg + are driven
at higher levels with a ubiquitous promoter, all cells in the ventral epidermis produce
naked cuticle with few or no denticles. This indicates that all cells in the segment can be directed into the
naked cuticle cell fate, and therefore presumably
would express a naked cuticle specifying Wg receptor. This cell fate pathway appears
to be triggered when Wg activity exceeds a certain threshold level. Dose sensitivity for naked cuticle specification is also observed under a variety of other circumstances (Hays, 1997).
When expressed uniformly, wg PE4 transgenes
rescue the wg mutant phenotype in a manner similar to the
wg + transgenes, promoting both denticle diversity
and naked cuticle specification. This contrasts with the
original wg PE4 mutant cuticle pattern, which shows little
denticle diversity. One potential explanation for these
disparate results is that the wg PE4 mutant protein may have a
limited range of action. To test this possibility, the transgenic protein was expressed in the native wg domain, and its distribution was examined. UAS-wg +and UAS-wg PE2 transgenic protein can be detected over several cell diameters surrounding the wg-expressing row of cells; UAS-wg PE4 cannot. Rather, this transgenic protein shows limited movement
away from the wg-expressing row of cells. The transgenic protein also appears to accumulate to higher levels within the wg-expressing domain, suggesting that it is not exported as efficiently as the full-length Wg molecule. Reduced secretion would contribute to the limited distribution of the mutant protein, but functional analyses indicate that at least some wg PE4 mutant protein reaches neighboring cell populations (Hays, 1997).
Since cuticle and denticle pathways are differentially activated by mutant Wg ligands, it is propose that at least two discrete classes of receptors for Wg may exist, each transducing a different cellular response. Broad Wg protein distribution across many cell diameters is required for the generation of denticle diversity, suggesting that intercellular transport of the Wg protein is an essential feature of pattern formation within the epidermal epithelium. An 85 amino acid region not conserved in vertebrate Wnts is dispensable for Wg function; structural features of the Wingless protein are discussed which are required for its distinct biological activities (Hays, 1997).
The basis for denticle diversity is still a mystery. Denticle type is not specified simply by level of Wg activity received, since uniform low levels of Wg can promote correct diverse
denticle identities. Previous work has indicated that other segment polarity genes, such as patched and engrailed, play a role in determining denticle identity. These gene activities may be involved in dictating the "pre-pattern" observed when uniform wg expression is provided to a wg mutant embryo. Indeed, when ectopic wild-type Wg is provided in a wg en double mutant background, naked cuticle is rescued but very little denticle diversity is observed (Hays, 1996 and Lawrence et al., 1996).
The dorsal median cells are unique mesodermal cells that reside on the surface of the ventral nerve cord in the Drosophila embryo. The Buttonless homeodomain protein is specifically expressed in these cells and is required for their differentiation. Proper buttonless gene expression and dorsal median cell differentiation require signals from underlying CNS midline cells. Thus, dorsal median cells fail to form in single-minded mutants and do not persist in slit mutants. Through analysis of rhomboid mutants and targeted rhomboid expression, it has been shown that the EGF signaling pathway regulates the number of dorsal median cells. wingless-patched double mutants exhibit defects in the restriction of dorsal median cells to segment boundaries and alterations in CNS midline cell fates (Zhou, 1997).
A key step in development is the establishment of cell type diversity across a cellular field. Segmental patterning within the Drosophila
embryonic epidermis is one paradigm for this process. At each parasegment boundary, cells expressing the Wnt family member
Wingless confront cells expressing the homeoprotein Engrailed. The Engrailed-expressing cells normally differentiate as one of two
alternative cell types. In investigating the generation of this cell type diversity among the 2-cell-wide Engrailed stripe, it has been shown that Wingless, expressed just anterior to the Engrailed cells, is essential for the specification of anterior Engrailed cell fate. In a
screen for additional mutations affecting Engrailed cell fate, anterior open (aop) (also known as yan) was identified, a gene encoding an inhibitory ETS-domain
transcription factor that is negatively regulated by the Ras-MAP kinase signaling cascade. Anterior open must be
inactivated for posterior Engrailed cells to adopt their correct fate. This is achieved by the EGF receptor (Egfr), which is required
autonomously in the Engrailed cells to trigger the Ras1-MAP kinase pathway. Localized activation of Egfr is accomplished by
restricted processing of the activating ligand, Spitz. Processing is confined to the cell row posterior to the Engrailed domain by the
restricted expression of Rhomboid. These cells also express the inhibitory ligand Argos, which attenuates the activation of Egfr in cell
rows more distant from the ligand source. Thus, distinct signals flank each border of the Engrailed domain, since Wingless is produced
anteriorly and Spitz posteriorly. Since En cells have the capacity to respond to either Wingless or Spitz, these cells
must choose their fate depending on the relative level of activation of the two pathways (O'Keefe, 1997).
The larval cuticle comprises a repeated array of precisely
patterned denticle belts interspersed with smooth cuticle. In abdominal segments, each of these belts is made up of
6 rows of denticles, where each row is of a characteristic size
and orientation reflecting fate decisions made by the underlying
cells. Using a lacZ reporter gene expressed in the
En cells, it has been demonstrated that the anterior En cells normally
produce smooth cuticle, while the
posterior En cells produce denticles and, thereby, form the first
row of each belt. Thus, cells in the En domain adopt either
a smooth or denticle fate depending on their position. To
identify genes involved in specifying En cell fates,
existing collections of mutants were screened for those in which anterior En
cells inappropriately produce denticles. Ectopic
denticles are observed immediately anterior to the denticle
belts in aop mutants. The extra denticles are
located at the lateral edges of denticle belts, and are more
commonly observed in the posterior segments.
To determine whether the En cell fates were altered in
these mutants, the En cells were visualized with a lacZ reporter
construct. Anterior En cells produce denticles
instead of the normal smooth cuticle.
Thus, aop function is required for some anterior En cells to
adopt the smooth cell fate (O'Keefe, 1997).
Spitz and Wingless signaling have been shown to have competing affects on En cell fate.
Anterior En cells assume a denticle fate when wg function is
eliminated at 8 hours AEL. Wg is expressed just anterior to the En
domain, in a region of smooth cuticle.
Thus, while Wg input instructs cells to adopt the smooth fate,
activation of Egfr instructs cells to adopt denticle fates. The
opposite response of En cells to these two signals raises the
question of what fate these cells would adopt in the absence of
both signals. To determine this, Egfr signaling was blocked by
expressing Aop Act in En cells while concomitantly removing
wg function using a conditional allele. When wg ts embryos
carrying both En-GAL4 and UAS-Aop Act are shifted to non-permissive
temperature at 8 hours AEL, the En cells adopt
smooth fates. This suggests that
smooth cuticle is the default cell fate. Wg signaling in this
context is required primarily for antagonizing the effect
of DER signaling in anterior En cells (O'Keefe, 1997).
The posterior En cells, which adopt a denticle fate, either
cannot respond to Wg due to the absence of key signal transducers,
or they do not see effective concentrations of Wg. In
fact, it appears that the posterior
En cell does not receive Wg input. The presence of downstream signal transducers was tested in posterior En cells. Cells expressing either
an activated form of Armadillo or higher levels of wild-type
Disheveled respond as if they have received the Wg signal. In embryos carrying
both En-GAL4 and UAS-Arm S10, the expression of activated
Armadillo causes the posterior En cells to inappropriately
adopt the smooth cell fate. Identical results were obtained expressing Disheveled. Thus, Wg signal transducers downstream of
Disheveled are present in posterior En cells. During normal
patterning, these cells are probably not exposed to sufficient
Wg levels to antagonize the effects of Egfr in these cells (O'Keefe, 1997).
A model is presented for the cooperation between Wingless and Spitz in specifying cell fate in Engrailed expressing cells.
The En-expressing cells are flanked anteriorly by a cell row
producing Wg and posteriorly by a cell row expressing
Rhomboid, which produces secreted Spitz. The En cell
nearest the Spi source receives a higher concentration of Spi, and
thus activates the Egfr pathway sufficiently to specify a denticle fate.
Reciprocally, the En cell nearest the Wg source receives a higher
concentration of Wg and adopts a smooth fate. Spi also activates the
Egfr pathway in the Rho-expressing cell, which therefore produces
and secretes Argos. Argos can inhibit Spi activation of the
Egfr pathway at a distance. As a
consequence, the Egfr pathway is not sufficiently activated in the
anterior En cell to out compete Wg signaling in this cell, and it
adopts a smooth fate. In fact, the specific targets of Egfr signaling responsible for conferring the denticle fate are unknown (O'Keefe, 1997).
In cell culture assays, Frizzled and Dfrizzled2, two
members of the Frizzled family of integral membrane
proteins, are able to bind Wingless and transduce the
Wingless signal. To address the role of these proteins in the
intact organism and to explore the question of specificity of
ligand-receptor interactions in vivo, a
genetic analysis of frizzled and Dfrizzled2 in the embryo has been conducted.
These experiments utilize a small gamma-ray-induced
deficiency that uncovers Dfrizzled2.
Dfz2-deficiency homozygotes die shortly after hatching
and exhibit a subtle disorganization of denticle patterning
with occasional ectopic denticles in posterior compartments.
These data suggest that Dfz2 and/or other genes removed by
the 469-2 deficiency play a minor or largely redundant role
in cuticle patterning during embryogenesis.
Mutants lacking
maternal frizzled and zygotic frizzled and Dfrizzled2 exhibit
defects in the embryonic epidermis, CNS, heart and midgut
that are indistinguishable from those observed in wingless
mutants. Epidermal patterning defects in the frizzled,
Dfrizzled2 double-mutant embryos can be rescued by
ectopic expression of either gene. In frizzled;Dfrizzled2 double mutant embryos, ectopic production of Wingless does not
detectably alter the epidermal patterning defect, but
ectopic production of an activated form of Armadillo
produces a naked cuticle phenotype indistinguishable from
that produced by ectopic production of activated Armadillo
in wild-type embryos. These experiments indicate that
frizzled and Dfrizzled2 function downstream of wingless and
upstream of armadillo, consistent with their proposed roles
as Wingless receptors. The lack of an effect on epidermal
patterning of ectopic Wingless in a frizzled;Dfrizzled2
double mutant argues against the existence of additional
Wingless receptors in the embryo or a model in which
Frizzled and Dfrizzled2 act simply to present the ligand to
its bona fide receptor. These data lead to the conclusion that
Frizzled and Dfrizzled2 function as redundant Wingless
receptors in multiple embryonic tissues and that this role
is accurately reflected in tissue culture experiments. The
redundancy of Frizzled and Dfrizzled2 explains why
Wingless receptors were not identified in earlier genetic
screens for mutants defective in embryonic patterning (Bhanot, 1999).
In the wild-type epidermis, wg functions in an autocrine
pathway to maintain its own expression and in a paracrine regulatory loop to maintain
expression of en in adjacent cells. In the epidermis at gastrulation,
when wg function is first detected, a stripe of cells in the
anterior half of each parasegment expresses wg and an adjacent
stripe of cells in the posterior half express en. This pattern is initiated by
pair-rule and gap genes, but its maintenance requires paracrine
signaling by Wg to the en expressing cells and both paracrine
signaling by Hh and autocrine signaling by Wg to the wg
expressing cells. Thus, in wg mutant embryos the pattern of wg
and en gene expression is initiated correctly but is not
maintained.
In fz;Dfz2 double-mutant embryos, the En stripes begin to
fade at stage 9/10 and are completely absent from the epidermis
by mid stage 10, similar to wg mutants. By
contrast, en expression within the CNS is maintained
as it is in wg mutants. Consistent with a defect
in Wg signaling, Wg expression is greatly reduced in fz;Dfz2 mutants (Bhanot, 1999).
At the end of gastrulation, wg participates in the
morphogenesis of various embryonic structures. In the
embryonic central nervous system, wg is expressed by row 5
neuroblasts (NBs) and its function is required to specify NBs
in rows 4 and 6. Null mutants of
wg show a loss or duplication of several NBs, the most
extensively studied being NB-4. The NB-4 lineage gives rise
to two RP2 motoneurons per segment that innervate the dorsal
musculature and are missing in wg mutant embryos.
RP2 neurons are marked by their expression of even-skipped (eve). Mutant embryos missing maternal fz and
zygotic fz and Dfz2 or missing only zygotic Dfz2 were
examined using an antibody against Eve. fz;Dfz2 double-mutant
embryos show a
complete loss of RP2 neurons in all hemisegments. As
observed in the epidermis, the fzR52 allele shows residual
activity: in fz;Dfz2 double mutants carrying the fzR52 allele,
approximately 26% of the double-mutant embryos show
Eve-positive RP2 staining in 1-3 hemisegments. Interestingly,
469-2 homozygous embryos also show a weakly penetrant RP2
phenotype. In approximately 21% of the 469-2 homozygous
embryos, an RP2 neuron is either missing or misplaced in 1-3
hemisegments. It is concluded that fz and
Dfz2 are largely but not entirely redundant in specifying RP2
identity (Bhanot, 1999).
Wnt genes are often expressed in overlapping
patterns, where they affect a wide array of developmental
processes. To address the way in which various Wnt
signals elicit distinct effects, the activities
of two Wnt genes in Drosophila, DWnt-4, and wingless, were compared.
These Wnt signals produce distinct responses
in cells of the dorsal embryonic epidermis.
Whereas wingless acts independently of hedgehog signaling
in these cells, DWnt-4 requires Hh
to elicit its effects. Expression of Wg
signal transduction components does not mimic expression
of DWnt-4, suggesting that DWnt-4 signaling proceeds
through a distinct pathway. The dorsal epidermis
may therefore be useful in the identification of novel
Wnt signaling components (Buratovich, 2000).
DWnt-4 and wg are expressed in many of the same cells
during Drosophila embryogenesis, including the ventral epidermis. However, in
the cells of the dorsal epidermis each gene is expressed
in distinct groups of cells. Whereas wg is expressed in
the most posterior row of cells in each parasegment
throughout most of embryogenesis, DWnt-4 is expressed
in the anterior region of the parasegment. This expression is transient, beginning at stage 10
and fading by the end of stage 12. The wg-like
ventral expression of DWnt-4 is dependent on hh, which may be
due to shared regulatory elements between the two
genes. However, since the dorsal expression of the two
genes is nonoverlapping, this aspect of DWnt-4 expression
appears to be regulated differently.
Since the dorsal stripes of DWnt-4 lie in between the
hh and wg stripes, the effect these genes
have on dorsal DWnt-4 expression was examined. In wg mutants
DWnt-4 is expressed normally, indicating that its expression
in the epidermis is not dependent on the activity of
wg. However, in hh mutants, both dorsal and
ventral expression is eliminated. The regulation
of DWnt-4 by hh within the anterior half of the dorsal
parasegment suggests that it acts in concert with hh
to pattern these cells (Buratovich, 2000).
Unlike the ventral epidermis, where hh and wg cooperate
in the specification of naked cuticle, the dorsal epidermis
is patterned through complementary activities of wg and
hh. The anterior half of each
parasegment consists of three cells types (1o, 2o, and 3o) that are all dependent on the activity of hh.
The position of the DWnt-4 expressing cells relative to
the wg-expressing cells indicates that DWnt-4 is expressed in
the presumptive 3o cells. The posterior half of the parasegment
consists of a single cell type, 4o, and is dependent
on wg activity. The dorsal epidermis is patterned beginning
at 6 h of development, the time at which the expression
of DWnt-4 is initiated in these cells. Prior to this
point, between 3 h and 6 h, the expression of wg and hh
are mutually dependent (Buratovich, 2000).
To determine whether DWnt-4 is able to modulate the
patterning of the dorsal epidermis, and whether it mimics
or otherwise regulates wg signaling in these cells, it was ubiquitously expressed using the GAL4 system. The results of ubiquitous
expression of wg or DWnt-4 were compared. Ubiquitous expression
of wg driven by a GAL4 insertion under the control of a
daughterless enhancer (daGAL4) results
in a uniform lawn of 4o cells. Thus the
hh-dependent cell types are deleted or transformed to 4o
fates.
Ubiquitous expression of DWnt-4 elicits a distinct response
in the hh-dependent cells, while having no effect
on the wg-dependent cells. The phenotype
produced by ectopic DWnt-4 is variable and dependent
on levels of ectopic expression. With one copy of
ectopic DWnt-4 expressed at 29oC, 21% (23/108) of the
segments exhibit a 2o-3o-4o pattern, in which 1o cells are
missing and 3o cells are expanded. In
contrast, 62% of the segments exhibit either a 1o-3o-4o or
a 3o-4o pattern; it was found that 1o and 3o cells are
difficult to distinguish. Lower levels of expression produced
by rearing the flies at a lower temperature produces
a higher percentage of embryos with a pattern that is
more clearly 1o-3o-4o along the dorsal midline, since the
2o cell fate is still apparent laterally. Nevertheless,
the 2o-3o-4o phenotype shows that DWnt-4 can
abolish 1o cells, and indicates that the primary effect of
DWnt-4 is to expand 3o cells at the expense of the other
two cell types (Buratovich, 2000).
These data show that cells in the anterior half of each
parasegment have the ability to respond to both Wnt
genes, but that each gene elicits a distinct response.
Whereas Wg transforms these cells to 4o cells or deletes
them, DWnt-4 appears to modulate the specification of
cell fate within the hh-dependent domain but has no effect
on cell fate specification by wg. The phenotypes
produced by ectopic DWnt-4 and wg therefore appear to
be qualitatively distinct, in that each gene induces ectopic
specification of different cell types (Buratovich, 2000).
The alteration in pattern by DWnt-4 suggests three possible
interactions with hh. (1) DWnt-4 might affect the
anterior half of the parasegment through modification of
hh expression. However, analysis of hh transcripts following
ectopic DWnt-4 expression has revealed that hh expression
is not affected. (2) Since DWnt-4 expression requires hh activity, it could be a downstream effector of hh in pattern specification.
(3) DWnt-4 could act in concert with hh to alter pattern.
To address these possibilities,
DWnt-4 was ectopically expressed in a hh temperature sensitive mutant
shifted to the restrictive temperature at 6 h. Under these
conditions the entire anterior half of the parasegment is
missing in hh mutants. When DWnt-4
is ectopically produced in this background, anterior cell
fates still fail to be specified, indicating that
DWnt-4 does not simply act downstream of hh but requires
hh for its activity after 6 h of development. If hh ts
mutants are shifted to the restrictive temperature at 7 h,
one row of 3o denticles typically forms, while 1o and 2o fates are still missing. If DWnt-4 is ectopically
expressed under these conditions, the number of 3o rows increases, supporting the conclusion that DWnt-4 acts in concert with hh to specify 3o cell fates (Buratovich, 2000).
The formation of segmental grooves during mid embryogenesis in the Drosophila epidermis depends on the specification of a single row of groove cells posteriorly adjacent to cells that express the Hedgehog signal. However, the mechanism of groove formation and the role of the parasegmental organizer, which consists of adjacent rows of hedgehog- and wingless-expressing cells, are not well understood. This study reports that although groove cells originate from a population of Odd skipped-expressing cells, this pair-rule transcription factor is not required for their specification. It was further found that Hedgehog is sufficient to specify groove fate in cells of different origin as late as stage 10, suggesting that Hedgehog induces groove cell fate rather than maintaining a pre-established state. Wingless activity is continuously required in the posterior part of parasegments to antagonize segmental groove formation. These data support an instructive role for the Wingless/Hedgehog organizer in cellular patterning (Mulinari, 2009).
It has been reported that segmental groove formation requires the activity of engrailed (en) and hh and that en has a function that is independent of its role in hh activation. More recently, it was been found that en is not expressed in groove cells, thus creating a non-cell-autonomous requirement for en. To address this issue, the role of hh and en in segmental groove formation was reinvestigated (Mulinari, 2009).
It was found that segmental grooves do not form in hh mutants. When hh was overexpressed, the four to five cell rows posterior to the Hh source constricted apically, elongated their apical-basal axis and took on a shape characteristic of segmental groove cells. Very similar cell behavior was observed in patched (ptc) mutants or when activated Ci, which mediates hh activity, was expressed. These observations suggest that Hh can organize segmental groove formation. No cell constrictions were observed in the ventral epidermis, indicating that a different mechanism might regulate cell shape there (Mulinari, 2009).
To address the proposed hh-independent function of en, en, invected (inv) double mutants were investigated in which hh expression was maintained using prd-Gal4. Segmental grooves were rescued in these mutants, suggesting that en is not required for segmental groove formation independent of its role in hh activation. By contrast, it was found that en represses groove cell behavior when ectopically expressed together with hh. A previous study that reported a requirement of en in groove formation was based on the analysis of en, inv, wg triple mutants, in which hh expression was maintained but did not rescue groove formation. This result was confirmed, but it is proposed that wg may be required in en mutants to allow the morphological differentiation of grooves (Mulinari, 2009).
Analysis of ptc mutants, or embryos overexpressing hh, reveals that a broad region of cells posterior to the en expression domain are specified as groove cells. However, groove-like invaginations form only at the edges of these regions. This is even more obvious in double mutants of ptc and the segment polarity gene sloppy paired 1 (slp1), which is required for maintained wg expression. In slp1, ptc mutants, wg expression fades prematurely and Hh signaling is constitutively active. This results in a substantial expansion of the number of groove cells. However, furrows differentiate only at the edges of groove cell populations. It is proposed that the morphological differentiation of segmental grooves can occur only at the interface between groove and non-groove cells (Mulinari, 2009).
To test this, wg, ptc double mutants were used in which Hh signaling is active throughout the epidermis and all cells take on a groove fate. Interestingly, these embryos did not differentiate grooves. A similar observation has been reported in en, inv, wg mutants, in which hh expression is sustained, leading to the suggestion that en might be required for groove specification (Mulinari, 2009).
Analysis of cell behavior in wg, ptc mutants showed, however, that cells throughout the tissue constrict their apices but fail to form invaginating furrows. The failure of wg, ptc mutants and en, inv, wg; UAS-hh embryos to differentiate grooves might be due to the absence of non-groove cells in the epidermis and the concomitant absence of an interface with groove cells (Mulinari, 2009).
The pair-rule gene odd is initially expressed in 4- to 5-cell wide stripes in even-numbered parasegments. At early gastrulation, odd expression expands to segmental periodicity and is subsequently refined to a single row of prospective groove cells located posterior to en. Continued expression of odd in these cells requires hh. In odd5 mutant embryos, grooves are unaffected in odd-numbered parasegments, but partially missing in even-numbered parasegments, and residual grooves coincide with regions in which odd expression is detectable (Vincent, 2008). These observations have been interpreted as indicating that groove fate might be specified prior to the requirement of Hh and differentiation of the groove. Thus, the later activity of Hh might not induce, but merely maintain, groove cell identity that has been pre-established in the odd-expressing cell population (Vincent, 2008). However, this hypothesis is based on the presumption that odd has a function in groove cell specification and this has not been demonstrated (Mulinari, 2009).
Residual grooves in odd5 mutants have been attributed to the hypomorphic nature of the odd5 allele; however, the molecular lesion in odd5 is unknown. Therefore the nucleotide sequence of odd5 was determined and a substitution was found that mutates codon 84 from CAG to a TAG stop codon. The resulting truncated peptide, which lacks all four putative zinc fingers encoded by wild-type odd, is no longer restricted to the nucleus but uniformly distributed in the cell. Thus, odd5 is likely to be a null allele (Mulinari, 2009).
To exclude the possibility that groove formation may be rescued by read-through of the stop codon in odd5 mutants, or that odd may be required redundantly, segmental grooves were investigated in Df(2L)drmP2 mutants, in which odd and its sister genes drumstick (drm) and sister of odd and bowl (sob) are entirely deleted. In these embryos, normal grooves formed in odd-numbered parasegments in the complete absence of odd function (Mulinari, 2009).
Next even-numbered parasegments were investigated in which grooves are partially missing. odd encodes a transcriptional repressor that regulates the expression of other segmentation genes in the early embryo. In odd mutants, derepression of the en activator fushi-tarazu in even-numbered parasegments results in the formation of an ectopic en stripe posterior to the normal stripe. Simultaneously, wg expands anteriorly and becomes expressed adjacent to the ectopic en-expressing cells. This results in the formation of an ectopic parasegment boundary with reversed polarity. Thus, the outward-facing edges of both en stripes are genetically anterior and lined by wg-expressing cells that do not form grooves. The inward-facing edges of the normal and ectopic en stripes fuse in some areas, and these corresponded to areas in which grooves were missing, as cells that were genetically posterior to en and could respond to the Hh signal had been replaced by en-expressing cells. The fusion of normal and ectopic en stripes was more severe in Df(2L)drmP2 mutants; however, islands of invaginating groove cells could still be observed, demonstrating that groove fate is specified in the absence of odd, drm and sob function in all parasegments. It is concluded that all cells that are genetically posterior to en are specified as groove cells in the absence of odd function and the partial absence of grooves in even-numbered parasegments in odd mutants is a secondary consequence of the pair-rule phenotype of these embryos. The slightly more severe pair-rule phenotype seen in Df(2L)drmP2 mutants might be due to a contribution from one of the odd sister genes, most likely sob, to pair-rule function, or could be caused by low-level read-through of the stop codon in the odd5 allele (Mulinari, 2009).
Finally, to investigate whether odd is sufficient to trigger cell shape changes, a UAS-odd transgene was expressed either alone or together with hh in the epidermis. No induction of groove cell behavior other than that triggered by hh was observed. Together, the data show that odd plays no essential role in groove cell specification and that odd paralogs are unlikely to act redundantly in this process (Mulinari, 2009).
The identification of odd as a groove cell marker led Vincent to suggest that groove fate might be specified prior to Hh requirement and that Hh may merely maintain groove fate instead of having an inducing role (Vincent, 2008). This study demonstrate that grooves are specified in the absence of odd function; however, this could be due to an odd-independent, early-acting mechanism present in the cells from which grooves arise (Mulinari, 2009).
In order to address whether groove fate is pre-established in the odd-expressing cell population, it was asked if groove fate could be induced in cells of a different origin at a later point in time. lines (lin) mutants were used in which late wg expression is altered resulting in the formation of an ectopic segment boundary at the anterior edge of the en domain in the dorsal epidermis. Importantly, the early expression of pair-rule or segment polarity genes is not affected (Mulinari, 2009).
In lin mutants, ectopic expression of the groove marker odd was initiated at stage 12 in a single row of groove-forming cells anterior to en that are derived from a previously non-odd-expressing cell population that does not contribute to grooves in the wild type. Ectopic grooves require hh as they were not induced in hh, lin double mutants, and ectopic odd expression was not induced in this background. An increase in hh levels in lin mutants resulted in the specification of groove fate in all cells except those expressing en. These results suggest that hh is sufficient, late in development, to specify groove cell fate in cell populations of different origins and that earlier-acting factors present in the population of odd-expressing cells posterior to en are not required. Very similar results have been reported by Piepenburg (2000), who showed that segment border cells form solely in response to the Hh signal that emanates from the en domain (Mulinari, 2009).
The findings are consistent with the role of Hh in the regulation of cell shape in other systems. Thus, during Drosophila eye development, Hh has been shown to control cell shape in the morphogenetic furrow, and Hh activation in other tissues is sufficient to induce apical constriction and groove formation. It is likely that Hh plays a similar role in tissue morphogenesis in other organisms. During neural tube closure in vertebrates, cells undergo similar shape changes involving apical-basal elongation and apical constriction, which is likely to be in response to Hh sources in the notochord and floor plate. Accordingly, knockout of sonic hedgehog is associated with defects in neural tube closure in mice. These observations suggest that Hh might be a principal inducer of cell shape across species (Mulinari, 2009).
It has previously been established that wg antagonizes the activity of hh in the specification of segment border cells (Piepenburg, 2000). However, it is not clear whether wg has a similar role in segmental groove formation, and a late requirement of wg to antagonize Hh-mediated groove specification has been questioned (Vincent, 2008). To investigate a direct role of wg in groove specification, a dominant-negative form of the transcription factor pan (panDN), which suppresses Wg signaling, was expressed. For this, pnr-Gal4, which initiates expression in the dorsal epidermis at stage 10-11 and thus does not affect early wg function, was used. Embryos that express panDN formed a single row of ectopic groove cells anterior to the en domain, confirming the results in lin mutants. Strikingly, inactivating Wg signaling and increasing Hh levels at the same time by co-expression of panDN and hh resulted in the expansion of groove fate to all cells except those expressing en. These results show that Wg signaling is required after stage 10 to repress groove specification anterior to en, thus making the activity of Hh asymmetric. These results also confirm observations that Hh is sufficient to induce groove fate in cells from different positions along the anterior-posterior axis and suggest that groove fate is not determined before stage 10 (Mulinari, 2009).
To confirm the ability of wg to repress groove fate, wg was expressed posterior to en in cells that normally take on groove fate. This resulted in the loss of Odd from many cells, suggesting that wg indeed antagonizes hh activity. Interestingly, these cells still formed grooves. However, these grooves appeared much earlier than segmental grooves, suggesting that they are ectopic parasegmental grooves caused by ectopic wg expression, as recently suggested (Larsen, 2008). Together, these data therefore support the contention that Wg signaling is required to repress Hh-mediated induction of groove fate after stage 10, thus permitting the formation of segmental grooves posterior, but not anterior, to en in the wild type (Mulinari, 2009).
Wnt signaling specifies cell fates in many tissues during vertebrate and
invertebrate embryogenesis. To understand better how Wnt signaling is
regulated during development, genetic screens were performed to isolate
mutations that suppress or enhance mutations in the fly Wnt homolog,
wingless (wg). This study finds that loss-of-function mutations in
the neural determinant SoxNeuro (also known as Sox-neuro,
SoxN) partially suppress wg mutant pattern defects.
SoxN encodes a HMG-box-containing protein related to the vertebrate
Sox1, Sox2 and Sox3 proteins, which have been implicated in patterning events
in the early mouse embryo. In Drosophila, SoxN has been
shown to specify neural progenitors in the embryonic central nervous system.
This study shows that SoxN negatively regulates Wg pathway activity in the
embryonic epidermis. Loss of SoxN function hyperactivates the Wg
pathway, whereas its overexpression represses pathway activity. Epistasis
analysis with other components of the Wg pathway places SoxN at the level of
the transcription factor Pan (also known as Lef, Tcf) in regulating target
gene expression. In human cell culture assays, SoxN represses Tcf-responsive
reporter expression, indicating that the fly gene product can interact with
mammalian Wnt pathway components. In both flies and in human cells, SoxN repression is potentiated by adding ectopic Tcf, suggesting that SoxN interacts with the repressor form of Tcf to influence Wg/Wnt target gene transcription (Chao, 2007).
SoxN downregulates the Wg/Wnt pathway to reduce target gene expression. Downregulation is a crucial process because it sensitizes the signal response to allow rapid on/off switching and also keeps the system off in cells that are not actively responding to signal. Many genes have been shown to negatively regulate Wg/Wnt pathway activity through the destabilization of Arm/beta-catenin. Far fewer are known to exert negative
regulatory effects downstream of Arm. The vertebrate Sox proteins -- Sox9, XSox3,
XSox17α and XSox17ß -- as well as Chibby, a conserved nuclear factor,
antagonize Wg/Wnt signaling by binding to Arm/beta-catenin and preventing it
from partnering with Tcf to activate target gene expression. SoxN, however,
does not bind beta-catenin in cell-culture assays, and does not share strong
homology with the C-terminal sequences through which vertebrate Sox proteins
bind this protein. Furthermore, SoxN function is not influenced
by Arm levels. No difference was observed in SoxN-mediated TOPflash repression
when cells were induced by co-transfection with a constitutively stabilized beta-catenin versus with Wnt-induced medium. Instead, both TOPflash and genetic experiments indicate that SoxN function depends on Tcf and Gro, its co-repressor (Chao, 2007).
One way to explain these observations is that SoxN contributes to the
assembly or stability of the Tcf repressor complex on DNA. The
consensus-sequence recognition for HMG domains in the Sox and Tcf families is
reported to be similar, although XSox3 and XSox17ß fail to bind a consensus
Tcf DNA sequence. It is shown that SoxN does not compete for Tcf-binding sites as
a means of repressing target gene transcription, but the data support a model
in which SoxN might bind DNA elsewhere or might bind Tcf sites transiently to
initiate or stabilize the assembly of a repressor complex (Chao, 2007).
A similar model may explain the results from Xenopus that showed
that XSox3-mediated repression does not require interaction between XSox3 and
beta-catenin. XSox3 strongly interferes with dorsal fate specification in
Xenopus embryos and represses TOPflash-reporter activity in vitro.
HMG-domain mutations render XSox3 inactive in embryos without affecting its
interaction with beta-catenin or its repression in TOPflash assays. Thus, it
is the DNA-binding domain, not the beta-catenin-interacting C-terminus, that
is relevant to its in vivo function in dorsal determination in
Xenopus. XSox3 represses the expression of the dorsal-specific
Nodal-related gene Xnr5 through optimal core binding sequences
adjacent to and partially overlapping with Tcf sites in the Xnr5
promoter (Zhang, 2003). By contrast, the fly SoxN shows no discrepancy between its behavior in TOPflash assays and its in vivo effects. This suggests that the synthetic
Tcf-binding sites arranged in the TOPflash-reporter plasmid are sufficient to support SoxN repressor function (Chao, 2007).
Because adding Tcf-site competitor DNA does not diminish the repressive
capacity of limiting amounts of SoxN, the role of SoxN in repression does not appear to be stoichiometric. Therefore, the idea is favored that Sox proteins may act in a
catalytic fashion during repressor-complex assembly at Wnt target gene
promoters, rather than forming a structural part of the repressor complex
itself. It was not possible to detect direct binding of SoxN with either Tcf,
Gro or Arm, raising the possibility that SoxN interacts with some as yet
unidentified protein that chaperones assembly of the repressor complex. A
SoxN-binding cofactor, SNCF, has been identified in Drosophila (Bonneaud, 2003), but this gene is expressed only in pre-gastrulation embryos. Because Wg signaling occurs exclusively post-gastrulation, and specification of naked cuticle begins more than 4 hours after gastrulation, it is not thought that SNCF is a likely candidate for mediating this aspect of SoxN function. Rather, it is likely to play a role in the neuronal specification events promoted by SoxN at earlier stages of embryogenesis (Chao, 2007).
It is curious that uniformly overexpressed SoxN represses Wg
signal transduction in dorsal epidermal cells more severely than in ventral
cells. This effect is evident in both cuticle pattern elements and in
en expression, and is reminiscent of defects observed in the
'transport-defective' class of wg mutant alleles, which includes
wgNE2. These mutations restrict Wg-ligand movement
ventrally to promote only local signaling response while simultaneously
abolishing all dorsal signaling, suggesting a fundamental difference in
ventral and dorsal cell response. Perhaps it is not a coincidence that the
NC14 mutation was isolated in the wgNE2 mutant
background. Further analysis of SoxN function may help to determine the molecular basis for dorsoventral differences in Wg signal transduction (Chao, 2007).
The Drosophila tracheal tree consists of a tubular network
of epithelial branches that constitutes the respiratory
system. Groups of tracheal cells migrate towards
stereotyped directions while they acquire specific tracheal
fates. This work shows that the wingless/WNT signaling
pathway is needed within the tracheal cells for the
formation of the dorsal trunk (DT) and for fusion of the
branches. These functions are achieved through the
regulation of target genes, such as spalt in the dorsal trunk
and escargot in the fusion cells. The pathway also aids
tracheal invagination and helps guide the ganglionic
branch. Moreover the wingless/WNT pathway displays
antagonistic interactions with the Dpp
pathway, which regulates branching along the dorsoventral
axis. Remarkably, the wingless gene itself, acting through
its canonical pathway, seems not to be absolutely required
for all these tracheal functions. However, the artificial
overexpression of wingless in tracheal cells mimics the
overexpression of a constitutively activated Armadillo
protein. The results suggest that another gene product,
possibly a WNT, could help to trigger the wingless cascade
in the developing tracheae (Llimargas, 2000).
Null wg
mutants show a tracheal phenotype that is significantly weaker
than is found when other members of the wingless pathway
are removed: in wg mutants, fragments of DT are formed and
fused, yet, in other mutants of the pathway, the DT is
eliminated. This suggests, in acting during DT development,
the wingless cascade does more than just respond to wg. But
whereas loss of function shows that wg is not absolutely
required for DT formation, its misexpression in tracheal cells
indicates that it is sufficient to confer DT identity. These
unconventional results are difficult to reconcile with a simple
model.
A possible explanation is that an additional factor or factors
might help to trigger the Wg cascade to form the DT,
the other Drosophila Wg genes being the most likely
candidates. Intriguingly, in porcupine mutants, the DT also fails to
form, suggesting that por mutants lack this factor/s. Therefore,
if the activation of the pathway depends, at least in part, on
other Wnt genes, the results indicate that por would be required
for their secretion too. The Wg cascade could be
activated by a particular Wnt or Wnt combination or
alternatively, Wnt genes could be all functionally redundant.
Wnt4 maps near wg and both genes are deficient in Df(2R)RF.
However, the tracheal phenotype of this deficiency is almost
identical to that of wgCX4, ruling out the possibility that zygotic Wnt4 might
act on its own or in combination with wg in forming the DT.
Wnt2 mutants are homozygous viable, suggesting that Wnt2 is
dispensable in DT formation. However, it might contribute to
DT formation in combination with wg or other Wnts. So far,
there are no point mutations for Wnt3, and thus any possible
tracheal phenotype could not be assessed. Other Wnt genes
have recently been identified by the genome sequence project, and
these also remain candidates (Llimargas, 2000).
One can envisage a model where the Wg
pathway has a basal activity stimulated upon binding of any
Wnt or Wnt gene combination to the receptor complex. These
low levels of Wg pathway activity would be sufficient
to allow DT formation and DT fusion (presumably through
activation of some fusion genes). Tracheal invagination,
activation of other fusion genes, and guidance of GBs would
require higher levels of activity of the pathway -- these would
depend on the binding of wg itself, sourced from nearby tissues (Llimargas, 2000).
arm has been shown to play dual but separable roles: one in
Wg signal affecting gene expression and the other in cell
adhesion. Null arm mutants have a tracheal phenotype that
is, in part, due to loss of its adhesive function. This phenotype can not be simply explained by
a decrease in cell adhesion.
The results of this work provide several indications that at least part of arm
function in the trachea is indeed due to its action in Wg
signaling: (1) an arm allele that specifically impairs
Wg signal produces a similar phenotype to that of an
arm null allele; (2) some tracheal defects are related to a
direct or indirect regulation of tracheal target genes; (3)
other members of the Wg pathway participate in the
same tracheal events (Llimargas, 2000).
Which part of the arm tracheal phenotype is due to its
adhesive role? arm has a role in cell adhesion by interacting
with shotgun, which encodes DE-cadherin. shg and arm
mutants are both defective in branch fusion. However, the cause of these two phenotypes does not
appear to be the same, since esg (a marker of fusion fate) is
normally expressed in shg mutants but
not in arm mutants. This indicates that the fusion cells are
normally specified in shg mutants, whereas arm is required
within the fusion cells themselves to regulate fusion markers.
Therefore, arm might have a dual function during branch
fusion: it is first required to activate the fusion fate through the
Wg signaling and later, through its interaction with shg,
it is required for the cell reorganizations that lead to branch
fusion (Llimargas, 2000).
The results indicate that the Wg pathway acts in DT
formation by regulating sal expression. Although this
regulation could be direct, it is also possible that the Wg
pathway acts by regulating the number of cells that will later
express sal under a Wg-independent control. Two pieces
of evidence show that normally the Wg pathway is
active only in the DT during primary branching. (1) The
constitutive activation of Arm cause integration of the visceral branches (VBs)
into DT. Such transformation would not be expected if the
pathway were normally active in the VBs. (2) In the
absence of tkv, the pathway must be constitutively activated to
observe sal expression in every tracheal cell. If the pathway
were normally active in all tracheal cells, the mere absence of
tkv should be sufficient to allow sal expression. Moreover, the
pathway seems to be required in the DT cells themselves. The
expression of dominant negative Pangolin in all tracheal cells impairs DT
formation, indicating that there is an autonomous requirement
within the tracheal cells. When the expression of dominant negative Pangolin in
the tracheae is maintained only in the DT cells, similar DT
defects are observed (Llimargas, 2000).
The Dpp pathway is required to form the dorsal and ventral
branches while the Wg pathway
is required for DT formation. Accordingly, only the VBs and
transverse connectives (TCs), which are independent of these two pathways, form in
their absence. The Wg and the Dpp pathways are known to
act antagonistically in several structures, such as the wing or
the leg disc. Similarly, the Wg and the Dpp
pathways have opposing activities during primary branching.
Only when the Dpp pathway is not active is the Wg
pathway able to confer DT identity to the tracheal cells. This
antagonism is likely to be mediated through the negative
regulation of sal by kni. sal is positively
regulated by the Wg pathway and kni is activated by the
Dpp pathway. However, when the
Wg signal is constitutive in a wild type background, the
VBs (which express kni) seem to adopt a DT identity.
Remarkably, in the VBs, the Dpp pathway is neither active nor
does it control kni expression. This indicates
that kni expression is not sufficient to prevent sal regulation by
the Wg pathway and that other targets of Dpp might
also antagonize the WG/WNT pathway (Llimargas, 2000).
Sequencing of the Drosophila genome has revealed that there are 'silent' homologs of many important gene family
members that were not detected by classic genetic approaches. Why have so many homologs been conserved during evolution?
Perhaps each one has a different but important function in every system. Perhaps each one works independently in a different part of the
body. Or, perhaps some are redundant. This study takes one well known gene family and analyzes how the individual members contribute
to the making of one system, the tracheae. There are seven DWnt genes in the Drosophila genome, including wingless. The wg gene helps to pattern the developing trachea but is not responsible for all Wnt functions there. Each one of the seven DWnts was tested in several ways and evidence was found that wg and DWnt2
can function in the developing trachea: when both genes are removed together, the phenotype is identical or very similar to that observed when the Wnt pathway is
shut down. DWnt2 is expressed near the tracheal cells in the embryo in a pattern different from wg's, but is also transduced through the canonical Wnt pathway. The seven DWnt genes vary in their effectiveness in specific tissues, such as the tracheae; moreover, the epidermis and the tracheae respond to DWnt2 and Wg differently. It is suggested that the main advantage of retaining a number of similar genes is that it allows more subtle forms of control and more flexibility during evolution (Llimargas, 2001).
DWnt proteins bind as ligands to a family of receptor proteins --
four Frizzled (Fz) homologs in Drosophila, of which Fz
and Fz2 are the most important and act through a cascade of
genes [e.g., disheveled, armadillo
(arm), pangolin] on the nucleus. If, therefore, Wg is the only ligand acting from the outside of the cell on the receptors, the wg- phenotype should be identical to the phenotype when fz and fz2 are removed -- in some organs, this is so. However, in the trachea, although removal of the two receptor proteins or one of the
intracellular proteins in the cascade eliminates all dorsal trunk (DT), removing only Wg leaves some DT intact. Therefore, it seems that another molecule, presumably a DWnt, acts through the canonical Wnt pathway to build DT. This study asks which DWnt is responsible (Llimargas, 2001).
Overexpression of wg or other downstream elements of
the Wnt pathway in the tracheal cells results in increased DT at the expense of the ventral branch. To
investigate further, each one of the seven
DWnts was overexpressed locally in the embryonic trachea in a normal
background. Overexpression of five DWnt genes
(DWnt5, -4, -6, -8, and
-10) had no detectable effects; indeed, the flies were
viable, fertile, and seemed normal. This experiment suggests that the
tracheae are not particularly sensitive to these five proteins. To
check whether these proteins are made properly and can function, they
were tested in other assays. DWnt6 and DWnt8 are
able to affect tracheal development in a sensitized background. DWnt5 produces a phenotype in the ventral nerve cord
when expressed with the neural specific driver 1407Gal4, in
agreement with the phenotype produced by a HS-DWnt5. Moreover, protein expression is detected in the tracheae when DWnt5 is expressed in tracheal cells. DWnt4 produces ectopic denticles in the ventral epidermis when overexpressed with armGal4, and the flies die as
pharate adults, showing several defects in the wings when crossed to
ptcGal4. No noticeable phenotype was found when overexpressing
DWnt10 in several structures, and, thus, the activity of
this line awaits confirmation. However, DWnt10
together with three other DWnts (DWnt4,
-6, and wg) were removed in Df(2L)RF embryos and a
phenotype similar to wg- was found, because there still
was some DT. This experiment argues
that at least zygotic DWnt 4, -6 and
-10 do not have a significant function in the trachea under
normal conditions. However, overexpression of DWnt2 locally
in the tracheal cells does affect its development in a way similar to
that of wg, producing an excess of DT cells and DT material
at the expense of the ventral branch. These tracheae
are defective; they fail to fill with air and the flies die as
embryos and young larvae. This result suggests that both wg
and DWnt2 act or can act in the developing trachea (Llimargas, 2001).
The tracheal placodes are specified by stage 10 in a
specific part of the dorsal ectoderm and express several markers such
as trachealess. The expression of DWnt2 is suggestive: it is expressed close to and dorsal to the tracheal placode by stage 10 and early stage 11 but later disappears (Llimargas, 2001).
The spalt (sal) gene (coding for a transcription
factor) is expressed in the dorsal ectoderm, including some tracheal
cells, during stage 10 and persists later in those tracheal cells that form the DT. sal is absolutely required for DT formation and is thus a good marker for DT cell identity. The most dorsal cells that express
sal also coexpress DWnt2. The pattern of wg expression differs strikingly from that of DWnt2, although both gene products are made near the tracheal cells. In arm mutants, Sal is not expressed in
tracheal cells and no DT is formed, suggesting that
sal expression in tracheal cells depends on activation of
the Wnt pathway. Thus, sal can be induced in the tracheal
cells wherever either Wg or DWnt2 proteins are received (Llimargas, 2001).
The above results suggest that wg and DWnt2, made
near the tracheal cells, together sponsor DT formation. In
wg- embryos, some DT is still formed. However, the tracheal phenotype of wg-DWnt2-
embryos is significantly different from that of
wg- embryos: in 40%-45% of
hemisegments, the DT is completely missing, and in the remaining
55%-60%, only some reduced and thin DT forms.
Interestingly, in practically all hemisegments of Df(2L)RF
DWnt2- embryos, the DT is completely missing, indicating that other DWnts account for these traces of DT. Nevertheless, wg-DWnt2-
double-mutant embryos are very similar to or indistinguishable from fz-fz2- embryos, suggesting that wg and
DWnt2 sponsor virtually all DT formation (Llimargas, 2001).
Removal of Wg and DWnt2 proteins (in
wg-DWnt2-
embryos) eliminates detectable expression of sal in the
presumptive tracheal cells of the DT, whereas in wg-
embryos, very low levels of sal still can be detected in
some embryos. The early expression of sal in the dorsal ectoderm still is observed in both wg- and wg-DWnt2- embryos. In wg-DWnt2-
embryos, late kni expression in tracheal cells is normal, as is the case in
arm mutants. In addition, dpp expression
also is normal -- Dpp has been shown to inhibit sal expression by activating kni in tracheal cells. Thus, the lack of sal must be caused
by the absence of direct or indirect stimulation by the DWnt pathway
and not due to repression by the Dpp pathway (Llimargas, 2001).
Does DWnt2 act through the canonical Wnt pathway? It seems so, because
the ectopic effects of DWnt2 protein are blocked in embryos that lack
the arm gene. Moreover, in wg-DWnt2-
embryos, the DT can be substantially rescued by expressing a constitutively active form of Arm in the tracheal cells. Also, the tracheal phenotype of porcupine (por) mutants is very similar to that of
wg-DWnt2-
embryos, indicating that por also might be required for
DWnt2 secretion (Llimargas, 2001).
If DWnt2 sponsors at least part of DT formation, one might
expect that loss of DWnt2 alone would affect trachea in some
noticeable way. Surprisingly, DWnt2-
embryos and larvae have normal trachea and normal
expression of sal. However, the flies have
reduced viability and the males are sterile (Llimargas, 2001).
In normal embryos, the wg gene is expressed in a row
of cells at the rear of the A compartment, whereas DWnt2 is
expressed at the front. Wg protein spreads to make a gradient that
patterns the anterior compartment. DWnt2 protein is expressed where the concentration of Wg is low or absent; that is where the
tracheal placodes form and where the cuticle secretes denticles.
Thus, in wg- embryos, where there is
no Wg protein and the denticles are continuous, one might expect
the tracheal placodes and DWnt2 expression to form one continuous stripe and, indeed, they do (Llimargas, 2001).
This adventitious expression of DWnt2 in a broad domain in
wg- embryos could compensate at least
in part for the lack of wg itself. Indeed, in these embryos,
it must be mainly DWnt2 that activates some sal and
determines most or all of the DT found. No change could be detected
in the pattern of wg RNA or protein distribution in
DWnt2 mutants (Llimargas, 2001).
The potency of DWnt2 and Wg in the tracheae was assayed:
DWnt2-wg-
double mutants were taken and each of the two missing proteins were added back in the normal pattern of expression for the wg gene.
DWnt2 and Wg were found to both rescue some DT in the trachea;
however, only Wg can partially rescue the various embryonic defects in
morphology found in wg- embryos. When
either DWnt2 or Wg is expressed locally in the tracheal cells, each gives strong rescue, and more DT is made (Llimargas, 2001).
The DWnt2 gene was also expressed in wild-type embryos either
universally and strongly (arm VP16 Gal4) or in stripes
(ptcGal4), and in both cases, the tracheae are altered to
the same extent as when DWnt2 is expressed in the tracheal
cells alone. However, DWnt2 fails to alter the cuticle pattern, whereas wg produces a naked cuticle phenotype. This lack of effect of DWnt2
on the epidermis is remarkable since both the drivers used are strong and,
when wg is driven, are more than adequate to make a naked
cuticle. Interestingly, when DWnt2 is missexpressed in the
eye, it also does not emulate the phenotype produced by missexpression
of wg. Moreover, the effects of overexpressing DWnt2 in the ovary are stronger than when overexpressing wg. All these results argue that the tracheal cells and other tissues, including the epidermis, the eye, and the ovary are differentially sensitive to the two DWnt molecules, the trachea and the ovary being particularly responsive to DWnt2 (Llimargas, 2001).
There are several ways this difference could be achieved. Perhaps DWnt2
does not act through the canonical Wnt pathway in some tissues, such as
the ectoderm or the eye. Perhaps DWnt2, on its way to the tracheal
cells, could be secreted or processed differently. Perhaps the tracheal
cells have something that allows efficient presentation of the ligand
to the Fz receptors, or they lack a component that, in other tissues,
impedes DWnt2 binding or transduction. One possibility is that
glucosaminoglycans help breathless (btl, an FGF
receptor expressed in tracheal cells) and are needed for
Wnt signaling. Maybe Btl helps to gather or modify the heparan
sulfate glucosaminoglycans, thereby altering the presentation of DWnt2
to the two receptors, Fz and Dfz2. Whatever the explanation, the
tracheal cells are more responsive than other tissues to the DWnt2 signal (Llimargas, 2001).
The other DWnts were examined.
DWnts5, -6, -8, and -10 were drived in
the epidermis of wild-type embryos with one copy of ptcGal4;
none of these affected the cuticle pattern in a noticeable way. Are
these DWnts able to affect tracheal development in the
wg-DWnt2-
double mutants? Each of these five DWnts were added back to either the
tracheal cells themselves or in the pattern of normal wg
expression. DWnts6 and -8 are each able to rescue DT partially,
whereas -4, -5 and -10 did not. Note that DWnt6
and DWnt8 are not able to produce a tracheal phenotype when
expressed in tracheal cells of normal embryos, but they can do so in a
sensitized background (Llimargas, 2001).
The results indicate that wg and DWnt2 make the
main contribution to DT formation, as the absence of both genes
completely eliminates DT in many cases. However, traces of DT still are
formed in about half the hemisegments of
wg-DWnt2-
embryos, indicating that contributions of other genes might help. Also,
rescue experiments show that some other DWnts are able to activate the pathway. In agreement with this result, in
most Df(2L)RF DWnt2- embryos, all DT is
missing, indicating that DWnt6 and/or -4
and/or -10 can compensate weakly for the absence of
wg and DWnt2. However, expression of
DWnt6 and DWnt10 does not suggest that they act in tracheal development in the wild type. DWnt4 is
expressed in a similar pattern to that of wg but does
not seem to assist wg during embryogenesis. In
addition, none of DWnt4, -6, or
-10 affected tracheal development when expressed in tracheal
cells of wild-type embryos. Most likely, they produce traces of DT in
the wg-DWnt2-
embryos, because those embryos offer a very sensitive test of stimulation of the Wnt pathway. It remains unclear whether these DWnts make any residual contribution to DT in the wild type (Llimargas, 2001).
However, several observations suggest that DWnt2 contributes
to tracheal development in the wild-type fly. Notably, DWnt2 is expressed near the tracheal cells at the appropriate stage, and when
overexpressed in tracheal cells, it mimics the effects of
overexpressing wg or a constitutively activated Arm. But,
most importantly, the phenotype of
wg-DWnt2-
embryos indicates that wg and DWnt2 together are
responsible for virtually all DT formation. Thus, DWnt2 probably
cooperates with Wg or reinforces its main action (Llimargas, 2001).
Nevertheless, DWnt2- embryos do not
show a visible tracheal phenotype, indicating, at first sight, that the
gene does not normally contribute to DT formation. This lack of
abnormality suggests that wg alone (or with some help from
different DWnts) is sufficient to sponsor normal development
in these mutant flies. Nevertheless, it remains possible that
DWnt2 could act in the wild-type. There are at least two
alternative hypotheses that could explain the lack of tracheal
phenotype in DWnt2- embryos (Llimargas, 2001).
(1) The loss of DWnt2 could induce compensatory changes
in the amount, distribution, or activity of the other DWnts.
As in the case of DWnt6, -4, and -10, the expression of DWnt5 and
DWnt8 (expression is detected only in the CNS at early stages) does not suggest that they act in tracheal development, although contributions under the level of detection cannot be discarded. Moreover, although some small changes have been detected in the expression of some DWnts in
wg- and wg-DWnt2-
embryos (e.g., the loss of DWnt5 expression in the labial segment at stage 10 as well as loss of expression in lateral clusters of the thoracic segments at stage 11), no changes have been detected in the pattern of
expression that might account for any strong tracheal rescue of
DWnt2- embryos.
Therefore, it is not clear how other DWnts could contribute
to the complete DT formation in DWnt2- embryos (Llimargas, 2001).
(2) It is supposed that all DWnts bind the receptor with different
affinities, with Wg binding most strongly. In the wild type, the DWnts
could compete, but Wg would be most effective: the contribution of
DWnt2 to DT formation would be minor. However, in embryos lacking Wg,
mainly DWnt2 (which is expressed in a broader domain in
wg-
embryos and is not now competing with
Wg) could bind and partially substitute for Wg. In the absence
of DWnt2, Wg (and maybe other DWnts) would have no competition from
DWnt2 and would become even more efficient, compensating for the
contribution to DT formation that DWnt2 has in the wild type. Finally,
in the absence of both Wg and DWnt2, other DWnts, even if they did not
act in the wild type, could now bind to the unoccupied receptors and
have some tiny effect on DT formation (Llimargas, 2001).
Complications of this kind may bedevil attempts to analyze the precise
wild type contributions of individual members of other gene families (Llimargas, 2001).
Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.
The Interactive Fly resides on the
wingless
continued:
Biological Overview
| Evolutionary Homologs
| Transcriptional regulation
|Targets of Activity
| Protein Interactions
| mRNA Transport
| Developmental Biology
| References
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