wingless
Wnts, mesoderm and somitogenesis Axial structures (neural tube/notochord) and surface
ectoderm activate myogenesis in the mouse embryo; their
actions can be reproduced, at least in part, by several
molecules such as Sonic hedgehog and Wnts. Recently,
soluble Wnt antagonists have been identified. Among those
examined only Frzb1 has been found to be expressed in the
presomitic mesoderm and newly formed somites; thus,
its possible role in regulating myogenesis was investigated
in detail. When presomitic mesoderm or newly formed
somites are cultured with axial structures and surface
ectoderm on a feeder layer of C3H10T1/2 cells expressing
Frzb1, myogenesis is abolished or severely reduced in
presomitic mesoderm and the three most recently formed
somites. In contrast, no effect is observed on more
mature somites. Inhibition of myogenesis does not appear to
be associated with increased cell death since the final
number of cells in the explants grown in the presence of
Frzb1 is only slightly reduced in comparison with
controls. In order to examine the possible function of Frzb1 in
vivo, a method was developed based on the overexpression
of the soluble antagonist by transient transfection of WOP
cells with a Frzb1 expression vector and injection of
transfected cells into the placenta of pregnant females
before the onset of maternofetal circulation. Frzb1,
secreted by WOP cells, accumulates in the embryo and
causes a marked reduction in size of caudal structures.
Myogenesis is strongly reduced and, in the most severe
cases, abolished. This is not due to a generalized toxic
effect since only several genes downstream of the Wnt
signaling pathway such as En1, Noggin and Myf5 are
downregulated; in contrast, Pax3 and Mox1 expression
levels are not affected even in embryos exhibiting the most
severe phenotypes. Taken together, these results suggest
that Wnt signals may act by regulating both myogenic
commitment and expansion of committed cells in the mouse
mesoderm (Borello, 1999).
The spatial and temporal expression pattern of zebrafish wnt11 and the regulation
of the expression during zebrafish early development was examined, focusing on the interaction with the no tail (ntl) gene, a zebrafish ortholog of mouse Brachyury (T). Zygotic expression of wnt11 is first detected at the late blastula stage in the blastoderm margin, a presumptive mesoderm region. wnt11 expression coincides with mesoderm induction, and the expression is induced by mesoderm inducers such as the yolk cell or FGFs, indicating that, like ntl, wnt11 is one of the immediate-early
genes in mesoderm induction. Initial expression domains of wnt11 and ntl overlap, and these genes show a similar response to mesoderm inducers. However, analysis of the ntl mutant embryos
suggests that wnt11 and ntl are placed in distinct genetic pathways; the ntl mutation has no effect on wnt11 expression in the blastoderm margin. This is further supported by the result of RNA injection experiments showing that overexpression of Wnt11 does not affect ntl expression in the margin. Thus, wnt11 and ntl expression are induced and maintained independently in their initial phase of expression. In later stages, wnt11 is expressed in various organs, such as the somites, particularly in the developing notochord. Since no wnt gene has been reported to be expressed in the axial mesoderm, which is known to act as a signaling source that patterns the neural tube and somites, zebrafish wnt11 is the first wnt gene expressed in the notochord. Furthermore, in contrast to early expression, wnt11 expression in the notochord depends on Ntl activity. In the ntl mutant in which somite patterning is severely affected, wnt11 expression is completely lost, while another signaling molecule, sonic hedgehog, is expressed in the mutant notochord precursor cells. wnt11 expression in the somite also shows a characteristic pattern, correlated with the migration and differentiation of slow muscle precursors. These observations suggest a role for wnt11 in patterning the somites (Makita, 1998).
The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 (see Drosophila Wnt8) is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8, known to pattern ventro-lateral mesoderm, is the neural posteriorizing factor that acts upon neuroectoderm, Frizzled 8c and Frizzled 9 were first assigned to be functional receptors for Wnt8. Transplanted non-axial mesoderm was then transplanted into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induces the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of ß-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. It is thus concluded that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis (Momoi, 2003).
Xcadherin-11 is the Xenopus homolog to the mesenchymal cadherin-11. Similar
to epithelial and neural cadherins, overexpression of Xcadherin-11 leads to posteriorized phenotypes due to inhibition of
convergent extension movement. Because zygotic expression of Xcadherin-11 starts with gastrulation, the
ability of different growth factors involved in mesoderm differentiation to induce the expression of Xcadherin-11 was analyzed. Using
the animal cap assay, it was demonstrated that Xcadherin-11 is activated by Xwnt-8 or beta-catenin, but repressed by BMP-4.
Activin does not induce Xcadherin-11 but its synergistic function is required for the Xwnt-8/beta-catenin-mediated
activation of Xcadherin-11. Because Xcadherin-11 and Xenopus E- and N-cadherin are differentially regulated by growth
factors in the Xenopus animal cap, these results also reveal that this assay provides a helpful model system to elucidate the
molecular control mechanism of epithelial-mesenchymal conversion (Hadeball, 1998).
The beginning of mesodermal development involves the aggregation of newly
gastrulated cells into epithelial fields, as a prelude to organ formation. Wnt-11 gene is expressed by newly
gastrulated mesoderm cells within avian embryos. The expression pattern of
Wnt-11 also suggests that it may be involved in formation of the cardiogenic
fields and somites. Subsequently, the quail mesoderm cell line
QCE-6 was used as a culture model for examining the influence of Wnt-11 on early
mesoderm cell differentiation. This cell line has been shown to be
representative of early nondifferentiated mesoderm cells and has the
potential to differentiate into cardiomyocytes, endothelial or red blood cells.
Similar to early mesoderm cells, QCE-6 cells express Wnt-11. Wnt-11
regulates cellular interactions of QCE-6 cells, as demonstrated by alterations
in contact-inhibited growth, tight and gap junction formation and plakoglobin
expression related to Wnt-11 level of expression. Both the morphology and growth factor-induced differentiation of
QCE-6 cells are regulated in a cooperative fashion by Wnt-11 and fibronectin.
QCE-6 cells are the first cell line that has demonstrated: (1) Wnt-dependent
differentiation; (2) concentration-variable responses to Wnt protein, and (3)
altered cell phenotypes as a direct response to Wnt-5a class proteins (e.g.
Wnt-4 and Wnt-11) (Eisenberg, 1997).
Shortly after their formation, somites of vertebrate embryos differentiate along the
dorsoventral axis into sclerotome, myotome and dermomyotome. The dermomyotome
is then patterned along its mediolateral axis into medial, central and lateral
compartments, which contain progenitors of epaxial muscle, dermis and hypaxial
muscle, respectively. Wnt-11 was used as a molecular marker for the medial
compartment of dermomyotome (the 'medial lip') to demonstrate that BMP in the
dorsal neural tube indirectly induces formation of the medial lip by up-regulating Wnt-1
and Wnt-3a (but not Wnt-4) expression in the neural tube. Noggin in the dorsal somite
may inhibit the direct action of BMP on this tissue. Wnt-11 induction is antagonized by
Sonic Hedgehog, secreted by the notochord and the floor plate. Together, these results
show that the coordinated actions of the dorsal neural tube (via BMP and Wnts), the
ventral neural tube/notochord (via Shh) and the somite itself (via noggin) mediates
patterning of the dorsal compartment of the somite (Marcelle, 1997).
In the vertebrate embryo, the lateral compartment of the somite gives rise to muscles of the limb and
body wall and is patterned in response to lateral-plate-derived BMP4. Activation of the myogenic
program distinctive to the medial somite, i.e. relatively immediate development of the epaxial muscle
lineage, requires neutralization of this lateral signal. The properties of molecules
likely to play a role in opposing lateral somite specification by BMP4 were examined. It is proposed that the BMP4
antagonist Noggin plays an important role in promoting medial somite patterning in vivo. Noggin expression in the somite is under the control of a neural-tube-derived factor,
whose effect can be mimicked experimentally by Wnt1. Wnt1 is appropriately expressed in the neural
tube. It is shown that Sonic Hedgehog, expressed in both the notochord and neural tube is able to activate ectopic expression of Noggin
resulting in the blocking of BMP4 specification of the lateral somite. These results are consistent with a
model in which Noggin activation in the medial somite lies downstream of the SHH and Wnt pathways signaling from the notochord and neural tube (Hirsinger, 1997).
Signals from the neural tube, notochord, and surface ectoderm
promote somitic myogenesis. Somitic myogenesis is under negative regulation as
well; BMP signaling serves to inhibit the activation of MyoD and Myf5 in Pax3-expressing cells. BMP-4 is highly expressed in both the dorsal-neural tube and lateral plate mesoderm; when ectopically expressed, between the axial (nerve cord) and paraxial (lateral plate mesoderm) tissues, BMP-4 can block somitic expression of MyoD. BMP antagonist Noggin is expressed within the dorsomedial lip of the
dermomyotome, where Pax3-expressing cells first initiate the expression of MyoD and Myf5 to give
rise to myotomal cells in the medial somite. Consistent with the expression of Noggin in dorsomedial
dermomyotomal cells that lie adjacent to the dorsal neural tube, coculture of
somites with fibroblasts programmed to secrete Wnt1 (which is expressed in dorsal neural tube) can
induce somitic Noggin expression. Ectopic expression of Noggin lateral to the somite dramatically
expands MyoD expression into the lateral regions of the somite, represses Pax3 expression in this
tissue, and induces formation of a lateral myotome. Together, these findings indicate that the timing and
location of myogenesis within the somite are controlled by relative levels of BMP activity and localized
expression of a BMP antagonist (Reshef, 1998).
In vertebrates, despite the evidence that extrinsic factors induce
myogenesis in naive mesoderm, other experiments argue that the initiation of
the myogenic program may take place independent of these factors. To resolve
this discrepancy, this issue was re-addressed using short-term in vivo
microsurgery and culture experiments in chick. The results show that the
initial expression of the muscle-specific markers Myf5 and
MyoD is regulated in a mesoderm-autonomous fashion. The reception of
a Wnt signal is required for MyoD, but not Myf5 expression;
however, the source of the Wnt signal is intrinsic to the
mesoderm. Gain- and loss-of-function experiments indicate that Wnt5b,
which is expressed in the presomitic mesoderm, represent the
MyoD-activating cue. Despite Wnt5b expression in the
presomitic mesoderm, MyoD is not expressed in this tissue: these
experiments demonstrate that this is due to a Bmp inhibitory signal that
prevents the premature expression of MyoD before somites form. These
results indicate that myogenesis is a multistep process that is initiated
prior to somite formation in a mesoderm-autonomous fashion: as somites form,
influences from adjacent tissues are likely to be required for maintenance and patterning of early muscles (Linker, 2003).
Activation of myogenesis in newly formed somites is dependent on signals derived from neighboring tissues, namely axial
structures (neural tube and notochord) and dorsal ectoderm. In explants of paraxial mesoderm from mouse embryos, axial
structures preferentially activate myogenesis through a Myf5-dependent pathway, while dorsal ectoderm is preferentially activated through a
MyoD-dependent pathway. Cells expressing Wnt1 will preferentially activate Myf5 while cells expressing
Wnt7a will preferentially activate MyoD. Wnt1 is expressed in the dorsal neural tube and Wnt7a in dorsal ectoderm in the early
embryo, therefore both can potentially act in vivo to activate Myf5 and MyoD, respectively. Wnt4, Wnt5a and Wnt6 exert an
intermediate effect activating both Myf5 and MyoD equivalently in paraxial mesoderm. Sonic Hedgehog synergises with both
Wnt1 and Wnt7a in explants from E8.5 paraxial mesoderm but not in explants from E9.5 embryos. Signaling through different
myogenic pathways may explain the rescue of muscle formation in Myf5 null embryos, which do not form an early myotome
but later develop both epaxial and hypaxial musculature. Explants of unsegmented paraxial mesoderm contain myogenic
precursors capable of expressing MyoD in response to signaling from a neural tube isolated from E10.5 embryos, the
developmental stage when MyoD is present throughout the embryo. Myogenic cells cannot activate MyoD in response to
signaling from a less mature neural tube. Together these data suggest that different Wnt molecules can activate myogenesis
through different pathways such that commitment of myogenic precursors is precisely regulated in space and time to achieve
the correct pattern of skeletal muscle development (Tajbakhsh, 1998).
Dorsoventral patterning of somites into sclerotome and dermomyotome involves the antagonistic actions of ventralizing and dorsalizing signals originating from tissues surrounding the somites. The notochord and
the floor plate of the neural tube provide a ventralizing signal(s) directing sclerotome development,
whereas the surface ectoderm and dorsal neural tube provide a dorsalizing signal(s) directing
dermomyotome development. Evidence has been provided that Sonic Hedgehog mediates the
ventralizing effects of notochord and floor plate, but the dorsalizing signal(s) that patterns the
dermomyotome has not been identified. The documented expression of Wnt1 and Wnt3a in the dorsal
neural tube, and Wnt4 and Wnt6 in the surface ectoderm at the time of dermomyotome specification,
prompted an investigation of the involvement of WNT proteins in patterning the dermomyotome. Tissue culture cells expressing these WNT family members can maintain and induce
dermomyotome marker expression in presomitic mesoderm explants, supporting the hypothesis that
WNT proteins mediate the dorsalizing effects of the surface ectoderm and dorsal neural tube on
somites (Fan, 1997).
Signals originating from tissues surrounding somites are involved in mediolateral and dorsoventral
patterning of somites and in the differentiation of the myotome. Wnt-1 and Wnt-3a, which encode
members of the Wnt family of cystein-rich secreted signaling molecules, are coexpressed at the dorsal
midline of the developing neural tube, an area adjacent to the dorsomedial portion of the somite.
Several lines of evidence indicate that Wnt-1 and Wnt-3a have the ability to induce the development of
the medial and dorsal portion of somites, as well as to induce myogenesis. To address whether these
Wnt signalings are really essential for the development of somites during normal embryogenesis, the development of somites was investigated in mouse embryos lacking both Wnt-1 and Wnt-3a. The medial compartment of the dermomyotome is not formed and the expression of a
lateral dermomyotome marker gene, Sim-1, is expanded more medially in the absence of these Wnt
signalings. In addition, the expression of a myogenic gene, Myf-5, is decreased at 9.5 days post coitum
whereas the level of expression of a number of myogenic genes, in particular MyoD, in the later stage appear normal.
These results indicate that Wnt-1 and Wnt-3a signalings actually regulate the formation of the medial
compartment of the dermomyotome and the early part of myogenesis. Differential regulation of Myf-5 and MyoD suggests that a second unknown signal from the surface ectoderm or from the dorsal neural tube in later stage embryos is sufficient for the activation of Myf-5 and MyoD expression and the progression of myotome development. Thus, there is a functional redundancy in myogenesis between inducing signals (Ikeya, 1998).
In vertebrates, the dorsoventral patterning of somitic mesoderm is controlled by factors expressed in
adjacent tissues. The ventral neural tube and the notochord function to promote the formation of the
sclerotome, a ventral somite derivative, while the dorsal neural tube and the surface ectoderm have been
shown to direct somite cells to a dorsal dermomyotomal fate. A number of signaling molecules are
expressed in these inducing tissues during times of active cell fate specification, including members of the
Hedgehog, Wnt, and BMP families. However, with the exception of the ventral determinant Sonic
hedgehog (expressed in the notochord and floor plate of the nerve cord), the functions of these signaling molecules with respect to dorsoventral somite patterning
have not been determined. The role of Wnt-1 (expressed in the dorsal neural tube), a candidate dorsalizing factor, has been investigated in the
regulation of sclerotome and dermomyotome formation. When ectopically expressed in the presomitic
mesoderm of chick embryos in ovo, Wnt-1 differentially affects the expression of dorsal and ventral
markers. Specifically, ectopic Wnt-1 is able to completely repress ventral (sclerotomal) markers and to
enhance and expand the expression of dorsal (dermomyotomal) markers. However, Wnt-1 appears to be
unable to convert all somitic mesoderm to a dermomyotomal fate. Delivery of an activated form of
beta-catenin to somitic mesoderm mimics the effects of Wnt-1, demonstrating that Wnt-1 likely acts directly
on somitic mesoderm, and not through adjacent tissues via an indirect signal relay mechanism. In response to Shh expression in dorsal somitic tissues, a marked diminution of BMP-4 expression is observed. This finding is consistent with the notion that Shh influences myotome formation through the elimination of BMP-4, which is a known repressor of MyoD transcription. Since MyoD expression is not significantly affected in response to Wnt signaling, it is concluded that Wnt-mediated up-regulation of BMP-4 message is not sufficient to down-regulate MyoD expression. Taken
together, these results support a model for somite patterning where sclerotome formation is controlled by
the antagonistic activities of Shh and Wnt signaling pathways. Shh is clearly required to suppress dorsal cell fates and promote ventral cell fates (Capdevila, 1998).
Establishment of the dorsoventral axis is central to animal embryonic organization. In Xenopus two
different classes of signaling molecules function in the dorsoventral patterning of the mesoderm. Both
the TGF-beta-related products of the BMP-2 and BMP-4 genes and the Wnt molecule encoded by
Xenopus Wnt-8 specify ventral fate and appear to inhibit dorsal mesodermal development. The similar
functions of these molecularly very different classes of signaling molecules prompted a study of their
mutual regulation and a comparison of their roles in mesoderm patterning. Wnt-8 and
BMP-4 are indistinguishable in their abilities to induce expression of ventral genes. Although BMP-2/-4
signaling regulates Wnt-8 expression, these genes do not function in a linear pathway because Wnt-8
overexpression cannot compensate for an inhibition of BMP-2/-4 function; rather, BMP-4
overexpression rescues ventral gene expression in embryos with inhibited Wnt-8 function. Wnt-8 and BMP-2/-4 differ in their abilities to regulate dorsal gene expression. While BMP-4
appears to generally inhibit the expression of dorsal genes, Xenopus Wnt-8 only inhibits the expression
of the notochord marker Xnot. Whereas the inhibitory effect of BMP-2/-4 localizes dorsal mesodermal
fate, these results suggest that Xenopus Wnt-8 functions in the further patterning of the dorsal mesoderm
into the most dorsal sector (from where the notochord develops) and the dorsolateral sector (from where
the somites differentiate) (Hoppler, 1998).
Several Wnt genes are expressed in the developing limbs
and head, implying roles in skeletal development. To explore these functions, retroviral
gene transfer was used to express Wnt-1 ectopically in the limb buds and craniofacial region of chick embryos.
Infection of wing buds at stage 17 and of tissues in the head at stage 10 results in skeletal abnormalities
whose most consistent defects suggested a localized failure of cartilage formation. To test this
hypothesis, micromass cultures of prechondrogenic mesenchyme were infected in vitro. Expression of Wnt-1 causes a severe block in chondrogenesis. Wnt-7a, a gene endogenously
expressed in the limb and facial ectoderm, has a similar inhibitory effect. Further analysis of this
phenomenon in vitro showed that Wnt-1 and Wnt-7a have mitogenic effects only in early
prechondrogenic mesenchyme, that cell aggregation and formation of the prechondrogenic blastema
occurs normally, and that the block to differentiation is at the late-blastema/early-chondroblast
stage. These results indicate that Wnt signals can have specific inhibitory effects on cytodifferentiation
and suggest that one function of endogenous Wnt proteins in the limbs and face may be to influence
skeletal morphology by localized inhibition of chondrogenesis (Rudnicki, 1997).
Long bones of the appendicular skeleton are formed from
a cartilage template in a process known as endochondral
bone development. Chondrocytes within this template
undergo a progressive program of differentiation
from proliferating to postmitotic prehypertrophic to
hypertrophic chondrocytes, while mesenchymal cells
immediately surrounding the early cartilage template form
the perichondrium. Members of the Wnt family of secreted signaling molecules have been implicated in regulating chondrocyte differentiation. Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb: Wnt-5a is expressed in the perichondrium; Wnt-5b is expressed in a subpopulation of
prehypertrophic chondrocytes and in the outermost cell layer of the perichondrium, and Wnt-4 is expressed in cells of the joint region. Misexpression experiments demonstrate that two of these Wnt molecules, Wnt-5a and Wnt-4, have
opposing effects on the differentiation of chondrocytes and
that these effects are mediated through divergent signaling
pathways. Specifically, Wnt-5a misexpression delays the
maturation of chondrocytes and the onset of bone collar
formation, while Wnt-4 misexpression accelerates these two
processes. Misexpression of a stabilized form of beta-catenin
also results in accelerated chondrogenesis, suggesting that
a beta-catenin/TCF-LEF complex is involved in mediating the
positive regulatory effect of Wnt-4. A number of the genes
involved in Wnt signal tranduction, including two members
of the Frizzled gene family, which are believed to encode
Wnt-receptors, show very dynamic and distinct expression
patterns in cartilaginous elements of developing chicken
limbs. Misexpression of putative dominant-negative forms
of the two Frizzled proteins results in severe shortening of
the infected cartilage elements due to a delay in chondrocyte maturation, indicating that an endogenous Wnt signal does indeed function to promote chondrogenic differentiation (Hartmann, 2000).
The perichondrium flanking the diaphysis of the
cartilage element is involved in negatively regulating the
maturation of hypertrophic chondrocytes. Wnt-5a, which is
expressed in the perichondrium/periosteum and has been
shown to delay maturation of the hypertrophic chondrocytes, could be one of the
factors involved in this regulatory role of the perichondrium.
The periarticular expression of PTHrP
is also involved in a negative regulation of chondrocyte
regulation. A negative feedback loop
between Ihh and PTHrP regulates the progression of
chondrocytes from the proliferative to the prehypertrophic state. Since Wnt-5a misexpression does not influence the Ihh/PTHrP loop and seems to control a different checkpoint in the chondrocyte maturation process, it is proposed that Wnt-5a
secreted by the perichondrium acts as a negative regulator,
independent of the PTHrP-signal. Significantly, the
Wnt-5a signaling pathway in the cartilage does not appear to
be mediated by beta-catenin, since misexpressing a stabilized
form of beta-catenin results in a phenotype that is the opposite of
that produced by Wnt-5a. Wnt-5a has been shown to signal
through a G-protein-linked phosphatidylinositol pathway in
zebrafish, suggesting that this might be
the pathway involved in negatively regulating the maturation
of hypertrophic chondrocytes by the perichondrial Wnt-5a
signal (Hartmann, 2000).
A novel mouse member of the Wnt family, Wnt13, has been identified. Among mouse
Wnt genes, Wnt13 is most closely related to Wnt2. Sequence comparisons and
chromosomal localization strongly suggest that Wnt13, rather than Wnt2, is the mouse
ortholog of both the human WNT13 and Xenopus XWnt2 genes. Wnt13 is expressed in
the embryonic mesoderm during gastrulation. At later stages, transcripts are detected in
the dorsal midline of the diencephalon and mesencephalon, the heart primordia, the
periphery of the lung bud and the otic and optic vesicles. These data suggest that Wnt13
function might partially overlap with those of other Wnt genes in the cell signaling
mechanisms controlling mesoderm specification during gastrulation and some aspects of
brain, heart and lung formation (Zakin, 1998).
The regulation of the Gli genes during somite formation has
been investigated in quail embryos. The Gli genes are a
family encoding three related zinc finger transcription
factors, Gli1, Gli2 and Gli3, which are effectors of Shh
signaling in responding cells. A quail Gli3 cDNA has been
cloned and its expression compared with Gli1 and Gli2.
These studies show that Gli1, Gli2 and Gli3 are co-activated
at the time of somite formation, thus providing a
mechanism for regulating the initiation of Shh signaling in
somites. Embryo surgery and paraxial mesoderm explant
experiments show that each of the Gli genes is regulated by
distinct signaling mechanisms. Gli1 is activated in response
to Shh produced by the notochord, which also controls the
dorsalization of Gli2 and Gli3 following their activation by
Wnt signaling from the surface ectoderm and neural tube.
This surface ectoderm/neural tube Wnt signaling has both
negative and positive functions in Gli2 and Gli3 regulation:
these signals repress Gli3 in segmental plate mesoderm
prior to somite formation and then promote somite
formation and the somite-specific activation of Gli2 and
Gli3. These studies, therefore, establish a role for Wnt
signaling in the control of Shh signal transduction through
the regulation of Gli2 and Gli3, and provide a mechanistic
basis for the known synergistic actions of surface
ectoderm/neural tube and notochord signaling in somite
cell specification (Borycki, 2000).
A model is presented for Wnt and Shh signaling in the control of Gli gene
activation during somite formation. In this model, in the segmental plate mesoderm, Gli3 is maintained in a repressed state by Wnt signaling through beta-catenin.
When anteriormost segmental plate mesoderm initiates somite
formation, Wnt/beta-catenin signaling undergoes a negative to positive
switch, leading to derepression of Gli3, to the initiation of somite
formation, and to activation of the somite-specific expression of Gli2
and Gli3. It is suggested that this switch in Wnt/beta-catenin
function might be mediated by transcription cofactors such as
Groucho, NLK and CtBP, factors that are known to control the transcription activities of beta-catenin/LEF1/tcf complexes in segmental plate mesoderm. The process of somite formation and the regulated expression of beta-catenin cofactors might be be under the control of the segmentation genes. Quantitative changes in Wnt signaling at the time of somite formation, resulting from the
activation of Wnt expression in the neural tube and loss of Wnt inhibitors in newly forming somites, would then mediate increased levels of beta-catenin. This high level of beta-catenin would participate in both the cytoplasmic
cell adhesion processes to initiate somite formation as well as in new beta-catenin/LEF1/tcf transcription complexes for Gli2 and Gli3 activation.
The Gli2 and Gli3 proteins produced in newly formed somites would then become activated as nuclear transcription factors in response to the Shh
that is produced by the notochord, leading to their participation in the activation of Shh response genes, including Gli1 and Ptc1 (Borycki, 2000).
The developmental signals that govern cell specification
and differentiation in vertebrate somites are well
understood. However, little is known about the downstream
signaling pathways involved. A combination of Shh protein and Wnt1 or Wnt3a-expressing fibroblasts is sufficient to activate skeletal
muscle-specific gene expression in somite explants. The molecular mechanisms by which the Wnt-mediated signal acts on myogenic precursor cells has been examined. Chick frizzled 1 (Fz1), beta-catenin and Lef1 are
expressed during somitogenesis. Lef1 and beta-catenin
transcripts become restricted to the developing myotome.
Furthermore, beta-catenin is expressed prior to the time at
which MyoD transcripts can be detected. Expression of beta-catenin mRNA is regulated by positive and negative signals
derived from neural tube, notochord and lateral plate
mesoderm. These signals include Bmp4, Shh and
Wnt1/Wnt3a itself. In somite explants, Fz1, beta-catenin and
Lef1 are expressed prior to activation of myogenesis in
response to Shh and Wnt signals. Thus, these data show that
a combination of Shh and Wnt1 upregulates expression of
Wnt pathway components in developing somites prior to
myogenesis (Schmidt, 2000).
Muscles of the body and bones of the axial skeleton derive from specialized regions of somites. Somite development is influenced by adjacent structures. In particular, the dorsal neural tube and the overlying ectoderm have been shown to be necessary for the induction of myogenic precursor cells in the dermomyotome. Members of the Wnt family of signaling molecules, which are expressed in the dorsal neural tube and the ectoderm, are postulated to be responsible for this process.
It is shown in this study that ectopically implanted Wnt-1-, -3a-, and -4-expressing cells alter the process of somite compartmentalization
in vivo. An enlarged dorsal compartment results from the implantation of Wnt-expressing cells ventrally between the neural tube/notochord and epithelial somites, at the expense of the ventral compartment, the sclerotome. Thus, ectopic
Wnt expression is able to override the influence of ventralizing signals arising from notochord and floor plate. This shift of
the border between the two compartments was identified by an increase in the domain of Pax-3 expression and a complete
loss of Pax-1 expression in somites close to the ectopic Wnt signal. The expanded expression of MyoD and desmin provides
evidence that it is the myotome that increases as a result of Wnt signaling. Paraxis expression is also drastically amplified
after implantation of Wnt-expressing cells, indicating that Wnts are involved in the formation and maintenance of somite
epithelium and suggesting that Paraxis is activated through Wnt signaling pathways. Taken together these results suggest
that ectopic Wnts disturb the normal balance of signaling molecules within the somite, resulting in an enhanced
recruitment of somitic cells into the myogenic lineage (Wagner, 2000).
In vertebrates, the dermis is well known to initiate the
formation of cutaneous appendages in the overlying epidermis. In avian embryos, the origin of the
dermis from different body regions has been investigated by
the heterospecific chick/quail cell marking technique. Facial
and cranial dermis originates from the neural crest, except in
the occipital and otic regions where it derives from cephalic
mesoderm. In
the trunk, the ventral and lateral dermis, as well as that of the
limb, are each derived from the somatopleure, whereas the dorsal
dermis derives from somites. Somites
are segmental units of the paraxial mesoderm, which appear
in a cephalo-caudal sequence. The ventral part of a somite
becomes mesenchymal, forming the sclerotome which gives
rise to the vertebrae and at least part of the ribs. Its dorsal portion remains epithelial, forming the so-called dermomyotome. The latter's medial half
gives rise to the dorsal dermis and to the epaxial muscles, while its lateral cells
migrate to form the limb and hypaxial musculature (Olivera-Martinez, 2001 and references therein).
To investigate the origin and nature of the signals responsible for specification of the dermatomal lineage, excised axial organs in 2-day-old chick embryos were replaced by grafts of the dorsal neural tube, or the ventral neural tube plus the notochord, or aggregates of cells engineered to produce Sonic hedgehog (Shh), Noggin, BMP-2, Wnt-1, or Wnt-3a. By E10, grafts of the ventral neural tube plus notochord or grafts of cells producing Shh lead to differentiation of cartilage and muscles, and an impaired dermis derived from already segmented somites. In
contrast, grafts of the dorsal neural tube, or of cells producing Wnt-1, trigger the formation of a feather-inducing dermis. These results
show that the dermatome inducer is produced by the dorsal neural tube. The signal can be Wnt-1 itself, or can be mediated, or at least
mimicked by Wnt-1 (Olivera-Martinez, 2001).
Proper longitudinal growth of long bones relies on the regulation of specific spatial patterns of chondrocyte proliferation and differentiation. The roles of two members of the Wnt family (Wnt5a and Wnt5b) in long bone development have been examined. Wnt5a is required for longitudinal skeletal outgrowth and both Wnt5a and Wnt5b regulate the transition between different chondrocyte zones independently of the Indian hedgehog (Ihh)/parathyroid hormone-related peptide (PTHrP) negative feedback loop. Important cell cycle regulators such as cyclin D1 and p130, a member of the retinoblastoma family, exhibit complimentary expression patterns that correlate with the distinct proliferation and differentiation states of chondrocyte zones. Furthermore, Wnt5a and Wnt5b appear to coordinate chondrocyte proliferation and differentiation by differentially regulating cyclin D1 and p130 expression, as well as chondrocyte-specific Col2a1 expression. These data indicate that Wnt5a and Wnt5b control the pace of transitions between different chondrocyte zones (Yang, 2003).
The functional difference of Wnt5a and Wnt5b may rely on which of the Wnt receptors, namely Frizzleds, are expressed in chondrocytes. The vertebrate Wnt family is comprised of at least 18 different members and there are at least 10 different Frizzleds in the human genome (http://www.stanford.edu/~rnusse/wntgenes/mousewnt.html). So far, three distinct Wnt signaling pathways have been identified. It has been shown that the canonical Wnt pathway, which is mediated by ß-catenin-LEF/TCF transcription complex, positively regulates cyclin D1 expression. The data indicate that Wnt5b may signal through the canonic pathway, whereas Wnt5a antagonizes it. This is supported by recent studies that show a Wnt/Ca2+ pathway mediated by the Xenopus Wnt5a decreases ß-catenin protein level. It is interesting to note that Wnt1, a canonic Wnt in other systems, acts similarly to Wnt5a in regulating Col2a1 reporter in the primary chondrocytes. It is likely that a single Wnt, for example Wnt5a or Wnt1, can elicit different responses depending on which Frizzled is expressed. When human Frizzled 5 is expressed together with either Wnt5a or Wnt5b in the early Xenopus embryos, a secondary axis is induced, indicating Wnt5a and Wnt5b both act as Wnt1 when Frizzled 5 is available. Since Frizzled 5 shows little specificity with different Wnts, it is unlikely that Frizzled 5 is a major Frizzled involved in transducing Wnt5a/Wnt5b signals in chondrocytes. Since several different Frizzled genes are expressed in the developing cartilage, Wnt5a and Wnt5b signaling in chondrocytes may be very complex, and involve both canonical and non-canonical pathways at the same time (Yang, 2003).
The vertebral column derives from somites generated by segmentation of presomitic mesoderm (PSM). Somitogenesis involves a molecular oscillator, the segmentation clock, controlling periodic Notch signaling in the PSM. A novel link has been established between Wnt/ß-catenin signaling and the segmentation clock. Axin2, a negative regulator of the Wnt pathway, is directly controlled by Wnt/ß-catenin and shows oscillating expression in the PSM, even when Notch signaling is impaired, alternating with Lfng expression. Moreover, Wnt3a is required for oscillating Notch signaling activity in the PSM. It is proposed that the segmentation clock is established by Wnt/ß-catenin signaling via a negative-feedback mechanism and that Wnt3a controls the segmentation process in vertebrates (Aulehla, 2003).
Axin2 mRNA expression oscillates alternately with Lfng expression, raising the question, how are the cyclic signaling activities of Wnt and Notch intercalated? The data show that Notch signaling in the posterior PSM is downregulated when Wnt3a activity and Axin2 are lacking but is ectopically upregulated when Axin2 is overexpressed. These observations suggest that Notch signaling acts downstream of Axin2 and, thus, downstream of Wnt3a. How could this tight link between Wnt and Notch signaling be achieved? (Aulehla, 2003).
A direct link between both signaling cascades has been suggested previously. In Drosophila, the intracellular domain of Notch, NICD, has been shown to bind to the PDZ domain of Dishevelled (Dsh). Dsh interacts antagonistically with Notch, and, therefore, it has been suggested that Dsh blocks Notch signaling directly through binding of NICD. Axin also binds to the PDZ domain of Dvl, the vertebrate homolog of Dsh, and the Axin homolog Axin2 is likely to act in a similar manner. Therefore, in the PSM, Dvl might inhibit Notch signaling through binding of NICD, whereas Axin2 binding to the PDZ domain of Dvl might release NICD and thus trigger Notch target gene activation (Aulehla, 2003).
In summary, negative-feedback inhibition of Wnt/β-catenin signaling via Axin2 might trigger Notch target gene activation and link both pathways. Destabilization of Axin2 would then reestablish Wnt signaling and lead to inhibition of Notch. Such a mechanism would explain the alternating waves of Wnt/β-catenin and Notch target activation observed in the PSM. However, the Notch signaling cascade also appears to have a modulating effect on the oscillations of Wnt signaling, as indicated by the data on Axin2 expression in Dll1-/- embryos (Aulehla, 2003).
Axin2 expression in the posterior PSM and tail bud depends on Wnt3a. This conclusion is based on two arguments. First, Axin2 transcription is not detectable in the PSM and tail bud of 10.25 dpc vt/vt embryos, well before axial development is arrested. Second, transgenic embryos expressing the lacZ reporter under control of a mutated Axin2 promoter, deficient in Lef/Tcf binding sites, do not show reporter activity in the tail bud and PSM. In addition, Axin2 mRNA expression is graded along the PSM, with the highest level in the tail bud, during phase 1 of the cycle and can be detected up to presomite I. (Aulehla, 2003).
The combined data suggest that Wnt/β-catenin signaling reaches up to the anterior PSM and occurs in a graded manner. However, Wnt3a transcription is restricted to the tail bud. These observations would be easily explained by the assumption that Wnt3a protein, following translation in the tail bud, is not rapidly degraded but subjected to slow decay in the extracellular environment. A gradient of Wnt3a protein and signaling activity would thus be established along the PSM, while the embryo elongates caudally. In agreement with this interpretation is the finding that other components of the Wnt/β-catenin pathway, such as the regulatory proteins Lef1 and Tcf1, are also expressed in the entire. This could be coincidental but makes perfect sense in the light of these data. Whatever the mechanism may be, the data provide indirect evidence for a gradient of Wnt/β-catenin signaling along the PSM (Aulehla, 2003).
It is proposed that Wnt3a controls intracellular oscillations of Wnt/β-catenin (Wnt) and Notch (NICD) signaling activity in the PSM. At the onset of the clock cycle, Wnt activates Axin2 transcription through Dvl, which may inhibit Notch signaling. Axin2 protein accumulates and inhibits Wnt signaling downstream of Dvl, through a negative feedback mechanism, and may also remove the inhibition of Notch through interaction with Dvl. Notch then upregulates Lfng. In parallel, Axin2 mRNA and protein are degraded, probably triggered by constant Wnt signaling upstream of Dvl. Thus, Wnt/β-catenin signaling downstream of Dvl is reestablished, and a new cycle begins. (Aulehla, 2003).
The limb musculature arises by delamination of premyogenic cells from the lateral dermomyotome. Initially the cells express Pax3 but, upon entering the limb bud, they switch on the expression of MyoD and Myf5 and undergo terminal differentiation into slow or fast fibers, which have distinct contractile properties that determine how a muscle will function. In the chick, the premyogenic cells express the Wnt antagonist Sfrp2, which is downregulated as the cells differentiate, suggesting that Wnts might regulate myogenic differentiation. The role of Wnt signalling during myogenic differentiation has been investigated in the developing chick wing bud by gain- and loss-of-function studies in vitro and in vivo. Wnt signalling changes the number of fast and/or slow fibers. For example, in vivo, Wnt11 decreases and increases the number of slow and fast fibers, respectively, whereas overexpression of Wnt5a or a dominant-negative Wnt11 protein has the opposite effect. The latter shows that endogenous Wnt11 signalling determines the number of fast and slow myocytes. The distinct effects of Wnt5a and Wnt11 are consistent with their different expression patterns, which correlate with the ultimate distribution of slow and fast fibers in the wing. Overexpression of activated calmodulin kinase II mimics the effect of Wnt5a, suggesting that it uses this pathway. Overexpression of the Wnt antagonist Sfrp2 and DeltaLef1 reduces the number of myocytes. In Sfrp2-infected limbs, the number of Pax3 expressing cells is increased, suggesting that Sfrp2 blocks myogenic differentiation. Therefore, Wnt signalling modulates both the number of terminally differentiated myogenic cells and the intricate slow/fast patterning of the limb musculature (Anakwe, 2003).
WNT signaling plays a major role in patterning the dermomyotome of the somitic mesoderm. However, knowledge of downstream target genes and their regulation is limited. To identify new genes involved in the development and early patterning of the somite, a comparison was performed of gene expression by microarray between the presomitic mesoderm and the 5 most recently formed somites of the mouse at embryonic day 9.5; this approach identified 207 genes upregulated and 120 genes downregulated in somite formation. Expression analysis and functional categorization of these genes demonstrate this to be a diverse pool that provides a valuable resource for studying somite development. Thus far, three genes expressed in the dermomyotome of the early somite were found. Consistent with their expression patterns, these genes are transcriptional targets of WNT signals, but display differential activation by different WNTs. One of these genes, Troy, is a direct target of canonical WNT signaling, while the other 2 genes, Selp and Arl4, are not. Thus, microarray study using microdissected tissues not only provides global information on gene expression during somite development, it also provides novel targets to study the inductive signaling pathways that direct somite patterning (Buttitta, 2003).
A critical step in skeletal morphogenesis is the formation of synovial joints, which define the relative size of discrete skeletal elements and are required for the mobility of vertebrates. Several Wnt genes, including Wnt4, Wnt14, and Wnt16, are expressed in overlapping and complementary patterns in the developing synovial joints, where ß-catenin protein levels and transcription activity were up-regulated. Removal of ß-catenin early in mesenchymal progenitor cells promoted chondrocyte differentiation and blocked the activity of Wnt14 in joint formation. Ectopic expression of an activated form of ß-catenin or Wnt14 in early differentiating chondrocytes induced ectopic joint formation both morphologically and molecularly. In contrast, genetic removal of ß-catenin in chondrocytes led to joint fusion. These results demonstrate that the Wnt/ß-catenin signaling pathway is necessary and sufficient to induce early steps of synovial joint formation. Wnt4, Wnt14, and Wnt16 may play redundant roles in synovial joint induction by signaling through the ß-catenin-mediated canonical Wnt pathway (Guo, 2004).
Signals that govern development of the osteoblast lineage are not well understood. Indian hedgehog (Ihh), a member of the hedgehog (Hh) family of proteins, is essential for osteogenesis in the endochondral skeleton during embryogenesis. The canonical pathway of Wnt signaling has been implicated by studies of Lrp5, a co-receptor for Wnt proteins, in postnatal bone mass homeostasis. In the present study it is demonstrated that beta-catenin, a central player in the canonical Wnt pathway, is indispensable for osteoblast differentiation in the mouse embryo. Moreover, evidence is presented that Wnt signaling functions downstream of Ihh in development of the osteoblast lineage. Finally Wnt7b is identified as a potential endogenous ligand regulating osteogenesis. These data support a model that integrates Hh and Wnt signaling in the regulation of osteoblast development (Hu, 2005).
The regulation of cell adhesion in epithelia is a fundamental process governing morphogenesis in embryos and a key step in the progression of invasive cancers. The molecular pathways controlling the epithelial organisation of somites have been examined. Somites are mesodermal epithelial structures of vertebrate embryos that undergo several changes in cell adhesion during early embryonic life. Wnt6 in the ectoderm overlaying the somites, but not Wnt1 in the neighbouring neural tube, is the most likely candidate molecule responsible for the maintenance of the epithelial structure of the dorsal compartment of the somite: the dermomyotome. The signalling pathway that mediates Wnt6 activity have been analyzed. The experiments suggest that the Wnt receptor molecule Frizzled7 probably transduces the Wnt6 signal. Intracellularly, this leads to the activation of the ß-catenin/LEF1-dependent pathway. Finally, it is demonstrated that the bHLH transcription factor paraxis, which has been shown to be a major player in the epithelial organisation of somites, is a target of the ß-catenin signal. It is concluded that ß-catenin activity, initiated by Wnt6 and mediated by paraxis, is required for the maintenance of the epithelial structure of somites (Linker, 2005).
During embryonic development in amniotes, the extraembryonic mesoderm, where the earliest hematopoiesis and vasculogenesis take place, also generates smooth muscle cells (SMCs). It is not well understood how the differentiation of SMCs is linked to that of blood (BCs) and endothelial (ECs) cells. This study shows that, in the chick embryo, the SMC lineage is marked by the expression of a bHLH transcription factor, dHand. Notch activity in nascent ventral mesoderm cells promotes SMC progenitor formation and mediates the separation of SMC and BC/EC common progenitors marked by another bHLH factor, Scl. This is achieved by crosstalk with the BMP and Wnt pathways, which are involved in mesoderm ventralization and SMC lineage induction, respectively. These findings reveal a novel role of the Notch pathway in early ventral mesoderm differentiation, and suggest a stepwise separation among its three main lineages, first between SMC progenitors and BC/EC common progenitors, and then between BCs and ECs (Shin, 2009).
The precise function of the Notch pathway in the process of muscle and BC/EC lineage separation remains to be elucidated. The data suggest that, during chick ventral mesoderm differentiation, the Notch pathway acts together with the BMP and Wnt pathways, and that it plays a 'permissive', rather than an 'instructive', role in mediating the separation of SMCs and BC/ECs. The Notch pathway does not control the induction of but rather the balance between these two populations. Evidence is provided that the induction of these lineages is controlled by the activities of both the BMP pathway, as a general ventral mesoderm inducer, and the canonical Wnt pathway, as a strong SMC lineage inducer. Ectopic activation of the BMP pathway can induce both SMC and BC/EC lineages, with the balance of SMCs and BC/ECs being regulated by Notch activity. It is not clear whether the induction of SMCs by the BMP pathway is a direct or indirect process, or whether it requires an active Wnt pathway. In this analysis, a stronger and wider ectopic dHand induction was observed by CA-β-Catenin than by CA-ALK6 around the anterior primitive streak where BMP antagonists are highly expressed, suggesting that the induction of SMCs by the Wnt pathway does not require active BMP signaling. A recent in vitro study suggested that Notch activity promotes the degradation of Scl by facilitating its ubiquitination, and that this process requires the transcriptional regulation of Notch pathway activity through Suppressor of Hairless. Although there is no direct evidence in support of a similar phenomenon in the current system, it could in principle act as a possible mechanism for the Notch activity-mediated segregation of SMCs and BC/ECs. Furthermore, Nrarp (an ankyrin-repeat protein that is transcriptionally regulated by the Notch signaling pathway), in addition to serving as a Notch-activity readout and a feedback regulator of the Notch pathway, has also been shown to positively regulate the canonical Wnt pathway by blocking the ubiquitination and increasing the stability of Lef1 in zebrafish. This might also serve as a possible mechanism for the Notch and Wnt pathway-mediated SMC specification observed in this system (Shin, 2009).
How signaling gradients supply positional information in a field of moving cells is an unsolved question in patterning and morphogenesis. This study asks how a Wnt signaling gradient regulates the dynamics of a wavefront of cellular change in a flow of cells during somitogenesis. Using time-controlled perturbations of Wnt signaling in the zebrafish embryo, segment length was changed without altering the rate of somite formation or embryonic elongation. This result implies specific Wnt regulation of the wavefront velocity. The observed Wnt signaling gradient dynamics and timing of downstream events support a model for wavefront regulation in which cell flow plays a dominant role in transporting positional information (Bajard, 2014).
Wnt homologs and heart development During early chick heart development the expression pattern of N-cadherin (see Drosophila Cadherin-N), a calcium-dependent cell
adhesion molecule, suggests its involvement in morphoregulation and the stabilization of cardiomyocyte
differentiation. In Drosophila, contractile vessel development requires wingless. Elimination of wingless function for a short time period after gastrulation in Drosophila results in the selective loss of heart precursors. The data presented are consistent with the likelihood that Wnt signaling may be involved in the regulation of the N-cadherin/ß-catenin-mediated events associated with vertebrate heart development. The Wnt2 gene is expressed in the early mouse heart field of 7.5-8 days of gestation. This coincides with the period of events described here (Linask, 1997).
Wingless is known to be required for induction of cardiac mesoderm in Drosophila, but the function of Wnt family proteins, vertebrate homologs of wingless, in cardiac myocytes remains unknown. When medium conditioned by HEK293 cells overexpressing Wnt-3a or -5a is applied to cultured neonatal cardiac myocytes, Wnt proteins induce myocyte aggregation in the presence of fibroblasts, concomitant with increases in ß-catenin and N-cadherin in the myocytes and with E- and M-cadherins in the fibroblasts. The aggregation is inhibited by anti-N-cadherin antibody and induced by constitutively active ß-catenin. Thus, increased stabilization of complexed cadherin-ß-catenin in both cell types appears crucial for the morphological effect of Wnt on cardiac myocytes. Furthermore, myocytes overexpressing a dominant negative frizzled-2, but not a dominant negative frizzled-4, fail to aggregate in response to Wnt, indicating frizzled-2 to be the predominant receptor mediating aggregation. By contrast, analysis of bromodeoxyuridine incorporation and transcription of various cardiogenetic markers show Wnt to have little or no impact on cell proliferation or differentiation. These findings suggest that a Wnt-frizzled-2 signaling pathway is centrally involved in the morphological arrangement of cardiac myocytes in neonatal heart through stabilization of complexed cadherin-ß-catenin (Toyofuku, 2000).
In vertebrates it has long been observed that repressive signals from the neural tube block cardiogenesis. A signal
from the neural tube that blocks cardiogenesis in the adjacent anterior paraxial mesoderm of stage 8-9 chick embryos can be mimicked
by ectopic expression of either Wnt-3a or Wnt-1, both of which are expressed in the dorsal neural tube. Repression of cardiogenesis by
the neural tube can be overcome by ectopic expression of a secreted Wnt antagonist. On the basis of both in vitro and in vivo results,
it is proposed that Wnt signals from the neural tube normally act to block cardiogenesis in the adjacent anterior paraxial mesendoderm (Tzahor, 2001).
In Drosophila, the BMP family member Dpp and the Wnt family member Wingless are required for the maintained expression of the NK homeobox gene tinman and for subsequent cardiogenesis. Although in vertebrates BMP signals play a positive role in promoting the expression of the NK homeobox gene, Nkx-2.5, and subsequent heart formation, Wnt signals paradoxically repress heart formation in vertebrates. A simple explanation for this discrepancy is that heart precursors in flies are generated in the dorsal mesoderm, adjacent to the wingless expression domain in the ectoderm, while in vertebrates, cardiac progenitors arise in regions of low or absent Wnt signaling. This redeployment of signals to control heart development may reflect a fundamental difference
between the metameric origin of the Drosophila heart precursors versus the induction of a heart field in the anterior domain of vertebrate embryos. It is proposed that newly invaginated mesodermal cells in the anterior region of the chick embryo are uniformly exposed to a cardiac-inducing signal from the anterior endoderm. In gastrula stage embryos, Wnt antagonists promote heart formation in the anterior lateral mesoderm, while Wnt signaling in the posterior of the embryo blocks ectopic heart formation in posterior lateral mesoderm. In neurula stage embryos, progression of cells within the cardiac field to the cardiac fate is subsequently repressed in the dorsomedial region of this field by both Wnt signals and anti-BMPs secreted by the axial tissues. Conversely, cardiogenesis is promoted in the ventrolateral region of the heart field by the presence of BMPs and the absence of Wnt signals (Tzahor, 2001).
Heart induction in Xenopus occurs in paired regions of the dorsoanterior mesoderm in response to signals from the Spemann organizer
and underlying dorsoanterior endoderm. These tissues together are sufficient to induce heart formation in noncardiogenic ventral marginal
zone mesoderm. Similarly, in avians the underlying definitive endoderm induces cardiogenesis in precardiac mesoderm. Heart-inducing
factors in amphibians are not known, and although certain BMPs and FGFs can mimic aspects of cardiogenesis in avians, neither can induce the full range of activities elicited by the inducing tissues. The Wnt antagonists Dkk-1 and Crescent can induce heart formation in explants of ventral marginal zone mesoderm. Other Wnt antagonists, including the frizzled domain-containing proteins Frzb and Szl, lack this
activity. Unlike Wnt antagonism, inhibition of BMP signaling does not promote cardiogenesis. Ectopic expression of GSK3beta, which inhibits
beta-catenin-mediated Wnt signaling, also induces cardiogenesis in ventral mesoderm. Analysis of Wnt proteins expressed during gastrulation reveals that
Wnt3A and Wnt8, but not Wnt5A or Wnt11, inhibit endogenous heart induction. These results indicate that diffusion of Dkk-1 and Crescent from the organizer
initiate cardiogenesis in adjacent mesoderm by establishing a zone of low Wnt3A and Wnt8 activity (Schneider, 2001).
In the chick, heart mesoderm is induced by signals from the anterior endoderm. Although BMP-2 is expressed in the anterior endoderm,
BMP activity is necessary but not sufficient for heart formation: one or more additional factors from anterior endoderm are required. Crescent is a Frizzled-related protein that inhibits Wnt-8c and is expressed in anterior
endoderm during gastrulation. At the same stages, expression of Wnt-3a and Wnt-8c is restricted to the primitive streak and posterior
lateral plate, and is absent from the anterior region where crescent is expressed. Posterior lateral plate mesoderm normally forms blood,
but co-culture of this tissue with anterior endoderm, or infection with RCAS virus encoding either crescent, induces formation of beating heart muscle and represses formation of blood. Dkk-1, a Wnt inhibitor of a different protein family, similarly induces heart-specific gene expression in posterior lateral plate mesoderm. Furthermore, ectopic Wnt signals can repress heart formation from anterior mesoderm in vitro and in vivo: forced expression of either Wnt-3a or Wnt-8c can promote
development of primitive erythrocytes from the precardiac region. It is concluded that inhibition of Wnt signaling promotes heart formation in the anterior lateral
mesoderm, whereas active Wnt signaling in the posterior lateral mesoderm promotes blood development. A model is proposed in which two orthogonal gradients,
one of Wnt activity along the anterior-posterior axis and the other of BMP signals along the dorsal-ventral axis, intersect in the heart-forming region to induce
cardiogenesis in a region of high BMP and low Wnt activity (Marvin, 2001).
Zygotic Wnt signaling has been shown to be involved in dorsoventral mesodermal patterning in Xenopus embryos, but how
it regulates different myogenic gene expression in the lateral mesodermal domains is not clear. Transient exposure of embryos or explants to lithium, which mimics Wnt/ß-catenin signaling, has been used as a tool to regulate the activation of this pathway at different times and places during early development. Activation of Wnt/ß-catenin signaling at the early gastrula stage rapidly induces ectopic expression of XMyf5 in both the dorsal and ventral mesoderm. In situ hybridization analysis reveals that the induction of ectopic XMyf5 expression in the dorsal mesoderm occurs within 45 min
and is not blocked by the protein synthesis inhibitor cycloheximide. By contrast, the induction of XMyoD is observed after
2 h of lithium treatment and the normal expression pattern of XMyoD is blocked by cycloheximide. Analysis by RT-PCR of ectodermal explants isolated soon after midblastula transition indicates that lithium also specifically induces XMyf5 expression, which takes place 30 min following lithium treatment and is not blocked by cycloheximide, arguing strongly
for an immediate-early response. In the early gastrula, inhibition of Wnt/ß-catenin signaling blocks the expression of XMyf5
and XMyoD, but not of Xbra. Zygotic Wnt/ß-catenin signaling interacts specifically with bFGF and eFGF to promote XMyf5 expression in ectodermal cells. These results suggest that Wnt/ß-catenin pathway is required for regulating myogenic gene expression in the presumptive mesoderm. In particular, it may directly activate the expression of the XMyf5 gene in the muscle precursor cells (Shi, 2002).
Formation of the vertebrate heart requires a complex interplay of several temporally regulated signalling cascades. In Xenopus laevis, cardiac specification occurs during gastrulation and requires signals from the dorsal lip and underlying endoderm. Among known Xenopus Wnt genes, only Wnt-11 shows a spatiotemporal pattern of expression that correlates with cardiac specification: this indicates that Wnt-11 may be involved in heart development. Loss- and gain-of-function experiments show that XWnt-11 is required for heart formation in Xenopus embryos and is sufficient to induce a contractile phenotype in embryonic explants. Treating the mouse embryonic carcinoma stem cell line P19 with murine Wnt-11 conditioned medium triggers cardiogenesis, which indicates that the function of Wnt-11 in heart development has been conserved in higher vertebrates. XWnt-11 mediates this effect by non-canonical Wnt signalling, which is independent of ß-catenin and involves protein kinase C and Jun amino-terminal kinase. These results indicate that the cardiac developmental program requires non-canonical Wnt signal transduction (Pandur, 2002).
Because XWnt-11 has been implicated to act through non-canonical Wnt-pathways, whether activation of these pathways stimulates cardiogenesis was examined in animal caps. The known mediators of non-canonical Wnt signalling are protein kinase C (PKC), Ca2+/calmodulin-dependent kinase II (CamKII) and JNK. In contrast to overexpression of XWnt-11, neither injections of constitutively active CamKII nor a dishevelled construct that specifically activates JNK signalling (dshDeltaDIX) nor incubation with the PKC activator phorbol-12-myristate-13-acetate (PMA) induced cardiac gene expression in animal caps. But activation of both PKC and JNK (PMA plus dshDeltaDIX) results in robust cardiac gene expression (Pandur, 2002).
Whether components of non-canonical Wnt pathways can override dnXWnt-11 inhibition of cardiac gene expression was tested in DMZ explants. It was first verified that dnXWnt-11 expression in DMZ explants downregulate cardiac gene transcription. Then tests were performed to see whether inhibitors of PKC or CamKII can phenocopy the effect of dnXWnt-11. Whereas treatment of uninjected DMZ explants with a PKC inhibitor (bisindolylmalmeimide I; BIM) mimics the effect of dnXWnt-11 on cardiac gene expression, specific inhibition of CamKII by KN-93 has no effect. In conjunction with the animal cap experimentation, these data implicate the involvement of PKC but not CamKII in XWnt-11-induced cardiogenesis. Consistent with these findings, the expression of cardiac genes in DMZ explants can be rescued from dnXWnt-11 inhibition by treatment with PMA. To determine whether JNK activation can compensate for the inhibitory effects of dnXWnt-11, dshDeltaDIX RNA was injected with dnXWnt-11 into the dorsolateral region; cardiac gene expression was restored to normal amounts. Thus, a block in XWnt-11 signalling can be overcome by activation of JNK. XWnt-11 overexpression in animal halves increased JNK-1 phosphorylation without altering the total amount of JNK-1 present, which indicated that JNK-1 was activated by XWnt-11. Activation of JNK-1 requires PKC activity; the stimulating effect of XWnt-11 is blocked by BIM. XWnt-8, which acts through ß-catenin, failed to activate JNK-1.
These results clearly show that signalling cascades activated by Wnt-11 are crucial for initiating a cardiac developmental program. The data also show that both PKC and JNK are essential for the cardiogenic activity of Wnt-11 and provide further evidence on the primary importance of non-canonical Wnt signalling pathways in understanding Wnt functional activity (Pandur, 2002).
Wnt11 is a secreted protein that signals through the non-canonical planar cell polarity pathway and is a potent modulator of cell behavior and movement. In human, mouse, and chicken, there is a single Wnt11 gene, but in zebrafish and Xenopus, there are two genes related to Wnt11. The originally characterized Xenopus Wnt11 gene is expressed during early embryonic development and has a critical role in regulation of gastrulation movements. A second Xenopus Wnt11-Related gene (Wnt11-R) has been identified that is expressed after gastrulation. Sequence comparison suggests that Xenopus Wnt11-R, not Wnt11, is the ortholog of mammalian and chicken Wnt11. Xenopus Wnt11-R is expressed in neural tissue, dorsal mesenchyme derived from the dermatome region of the somites, the brachial arches, and the muscle layer of the heart, similar to the expression patterns reported for mouse and chicken Wnt11. Xenopus Wnt11-R exhibits biological properties similar to those previously described for Xenopus Wnt11, in particular the ability to activate Jun-N-terminal kinase (JNK) and to induce myocardial marker expression in ventral marginal zone (VMZ) explants. Morpholino inhibition experiments demonstrate, however, that Wnt11-R is not required for cardiac differentiation, but functions in regulation of cardiac morphogenesis. Embryos with reduced Wnt11-R activity exhibit aberrant cell-cell contacts within the myocardial wall and defects in fusion of the nascent heart tube (Garriock, 2005).
Normal development of the cardiac atrioventricular (AV) endocardial cushions is essential for proper ventricular septation and morphogenesis of the mature mitral and tricuspid valves. This study demonstrates spatially restricted expression of both Wnt-9a (formerly Wnt-14) and the secreted Wnt antagonist Frzb in AV endocardial cushions of the developing chicken heart. Wnt-9a expression is detected only in AV canal endocardial cells, while Frzb expression is detected in both endocardial and transformed mesenchymal cells of the developing AV cardiac cushions. Evidence that Wnt-9a promotes cell proliferation in the AV canal and overexpression of Wnt-9a in ovo results in enlarged endocardial cushions and AV inlet obstruction. Wnt-9a stimulates ß-catenin-responsive transcription in AV canal cells, duplicates the embryonic axis upon ventral injections in Xenopus embryos and appears to regulate cell proliferation by activating a Wnt/ß-catenin signaling pathway. Additional functional studies reveal that Frzb inhibits Wnt-9a-mediated cell proliferation in cardiac cushions. Together, these data argue that Wnt-9a and Frzb regulate mesenchymal cell proliferation leading to proper AV canal cushion outgrowth and remodeling in the developing avian heart (Person, 2005).
A complex regulatory network of morphogens and transcription factors is essential for normal cardiac development. Nkx2-5 is among the earliest known markers of cardiac mesoderm that is central to the regulatory pathways mediating second heart field (SHF) development. This study has examined the specific requirements for Nkx2-5 in the SHF progenitors. Nkx2-5 was found to potentiate Wnt signaling by regulating the expression of the R-spondin3 (Rspo3) gene during cardiogenesis. R-spondins are secreted factors and potent Wnt agonists that in part regulate stem cell proliferation. The data show that Rspo3 is markedly downregulated in Nkx2-5 mutants and that Rspo3 expression is regulated by Nkx2-5. Conditional inactivation of Rspo3 in the Isl1 lineage resulted in embryonic lethality secondary to impaired development of SHF. More importantly, it was found that Wnt signaling is significantly attenuated in Nkx2-5 mutants and that enhancing Wnt/beta-catenin signaling by pharmacological treatment or by transgenic expression of Rspo3 rescues the SHF defects in the conditional Nkx2-5(+/-) mutants. A previously unrecognized genetic link between Nkx2-5 and Wnt signaling was uncovered that supports continued cardiac growth and proliferation during development. Identification of Rspo3 in cardiac development provides a new paradigm in temporal regulation of Wnt signaling by cardiac-specific transcription factors (Cambier, 2014).
Wnts and kidney, liver, and pancreas development Members of the bone morphogenetic protein (BMP) family exhibit overlapping and dynamic expression
patterns throughout embryogenesis. However, little is known about the upstream regulators of these
important signaling molecules. There is some evidence that BMP signaling may be autoregulative as
demonstrated for BMP4 during tooth development. Analysis of BMP7 expression during kidney
development, in conjunction with studies analyzing the effect of recombinant BMP7 on isolated kidney
mesenchyme, suggest that a similar mechanism may operate for BMP7. A beta-gal-expressing reporter allele has been generated at the BMP7 locus to closely monitor expression of BMP7 during embryonic
kidney development. In contrast to other studies, the current analysis of BMP7/lacZ homozygous mutant embryos shows that BMP7 expression is not subject to autoregulation in any tissue. In addition, this
reporter allele was used to analyze the expression of BMP7 in response to several known survival factors (EGF,
bFGF) and inducers of metanephric mesenchyme, including the ureteric bud, spinal cord and LiCl. These
studies show that treatment of isolated mesenchyme with EGF or bFGF allows survival of the mesenchyme
but neither factor is sufficient to maintain BMP7 expression in this population of cells. Rather, BMP7
expression in the mesenchyme is contingent on an inductive signal. Thus, the reporter allele provides a
convenient marker for the induced mesenchyme. Interestingly LiCl has been shown to activate the Wnt
signaling pathway, suggesting that BMP7 expression in the mesenchyme is regulated by a Wnt signal.
Treatment of whole kidneys with sodium chlorate to disrupt proteoglycan synthesis results in the loss of
BMP7 expression in the mesenchyme, whereas expression in the epithelial components of the kidney are
unaffected. Heterologous recombinations of ureteric bud with either limb or lung mesenchyme demonstrate
that expression of BMP7 is maintained in this epithelial structure. Taken together, these data indicate that
BMP7 expression in the epithelial components of the kidney is not dependent on cell-cell or cell-ECM
interactions with the metanephric mesenchyme. By contrast, BMP7 expression in the metanephric
mesenchyme is dependent on proteoglycans and possibly Wnt signaling. Treatment of kidney explants with sodium chlorate inhibits proteoglycan synthesis and arrests branching of the ureteric bud. As Wnt-11 is expressed in the tips of the ureter and expression of both Wnt-11 and BMP7 in the mesenchyme are lost in chlorate-treated kidneys, these data are consistent with Wnt 11 functioning as an inductive signal emanating from the ureteric bud to initiate BMP7 expression in the mesenchyme. These data also suggest that expression of BMP7 in the condensed mesenchyme requires proteoglycans. In contrast, expression in the pre-tubular aggregates or in the epithelial components is not dependent on proteoglycans (Godin, 1998).
Development of the mammalian kidney is initiated by ingrowth of the ureteric bud into the metanephric blastema. In response to
signal(s) from the ureter, mesenchymal cells condense, aggregate into pretubular clusters, and undergo epithelialization to form
simple epithelial tubules. Subsequent morphogenesis and differentiation of the tubular epithelium lead to the establishment of a
functional nephron. Wnt-4, a secreted glycoprotein that is required for tubule formation, is
sufficient to trigger tubulogenesis in isolated metanephric mesenchyme; in contrast, Wnt-11, which is expressed in the tip of the
growing ureter, is not sufficient. Wnt-4 signaling depends on cell contact and sulphated glycosaminoglycans and is only required for
triggering tubulogenesis but not for later events. The Wnt-4 signal can be replaced by other members of the Wnt gene family
including Wnt-1, Wnt-3a, Wnt-7a and Wnt-7b. Further, dorsal spinal cord, which has been thought to mimic ureteric signaling
in tubule induction induces Wnt-4 mutant as well as wild-type mesenchyme suggesting that spinal cord derived signal(s) most
likely act by mimicking the normal mesenchymal action of Wnt-4. These results lend additional support to the notion that
Wnt-4 is a key auto-regulator of the mesenchymal to epithelial transformation that underpins nephrogenesis adding another
level of complexity in the hierarchy of molecular events mediating tubulogenesis (Kispert, 1998).
In the vertebrate embryo, development of the excretory system is characterized by the successive formation of three distinct
kidneys: the pronephros, mesonephros, and metanephros. While tubulogenesis in the metanephric kidney is critically dependent on the signaling molecule Wnt-4, it is unknown whether Wnt signaling is equally required for the formation of renal epithelia in the other embryonic kidney forms. The expression of Wnt genes was investigated during the pronephric kidney development in Xenopus. Wnt4 was found to be associated with developing pronephric tubules, but is absent from the pronephric duct. Onset of pronephric Wnt-4 expression coincides with mesenchyme-to-epithelium transformation. To investigate Wnt-4 gene function, gain- and loss-of-function experiments were performed. Misexpression of Wnt4 in the intermediate and lateral mesoderm causes abnormal morphogenesis of the pronephric tubules, but is not sufficient to initiate ectopic tubule formation. A morpholino antisense oligonucleotide-based gene knockdown strategy was used to disrupt Wnt-4 gene function. Xenopus embryos injected with antisense Wnt-4 morpholinos develop normally, but marker gene and morphological analysis reveal a complete absence of pronephric tubules. Pronephric duct development is largely unaffected, indicating that ductogenesis may occur normally in the absence of pronephric tubules. These results show that, as in the metanephric kidney, Wnt-4 is critically required for tubulogenesis in the pronephric kidney, indicating that a common, evolutionary conserved gene regulatory network may control tubulogenesis in different vertebrate excretory organs (Saulnier, 2002).
Reciprocal cell-cell interactions between the ureteric epithelium and the metanephric mesenchyme are needed to drive growth and differentiation of the embryonic kidney to completion. Branching morphogenesis of the Wolffian duct derived ureteric bud is integral in the generation of ureteric tips and the elaboration of the collecting duct system. Wnt11, a member of the Wnt superfamily of secreted glycoproteins, which have important regulatory functions during vertebrate embryonic development, is specifically expressed in the tips of the branching ureteric epithelium. The role of Wnt11 in ureteric branching has been explored, and a targeted mutation of the Wnt11 locus was used as an entrance point into investigating the genetic control of collecting duct morphogenesis. Mutation of the Wnt11 gene results in ureteric branching morphogenesis defects and consequent kidney hypoplasia in newborn mice. Wnt11 functions, in part, by maintaining normal expression levels of the gene encoding glial cell-derived neurotrophic factor (Gdnf). Gdnf encodes a mesenchymally produced ligand for the Ret tyrosine kinase receptor that is crucial for normal ureteric branching. Conversely, Wnt11 expression is reduced in the absence of Ret/Gdnf signaling. Consistent with the idea that reciprocal interaction between Wnt11 and Ret/Gdnf regulates the branching process, Wnt11 and Ret mutations synergistically interact in ureteric branching morphogenesis. Based on these observations, it is
concluded that Wnt11 and Ret/Gdnf cooperate in a positive
autoregulatory feedback loop to coordinate ureteric branching by maintaining an appropriate balance of Wnt11-expressing ureteric epithelium and Gdnf-expressing mesenchyme to ensure continued metanephric development (Majumdar, 2003).
To bypass the essential gastrulation function of Fgf8 and study its role in lineages of the primitive streak, a new mouse line, T-Cre, was used to generate mouse embryos with pan-mesodermal loss of Fgf8 expression. Surprisingly, despite previous models in which Fgf8 has been assigned a pivotal role in segmentation/somite differentiation, Fgf8 is not required for these processes. However, mutant neonates display severe renal hypoplasia with deficient nephron formation. In mutant kidneys, aberrant cell death occurs within the metanephric mesenchyme (MM), particularly in the cortical nephrogenic zone, which provides the progenitors for recurring rounds of nephron formation. Prior to mutant morphological changes, Wnt4 and Lim1 expression, which is essential for nephrogenesis, is absent in MM. Furthermore, comparative analysis of Wnt4-null homozygotes reveals concomitant downregulation of Lim1 and diminished tubule formation. These data support a model whereby FGF8 and WNT4 function in concert to induce the expression of Lim1 for MM survival and tubulogenesis (Berantoni, 2005).
Mammalian nephrons form as a result of a complex morphogenesis and patterning of a simple epithelial precursor, the renal vesicle. Renal vesicles are established from a mesenchymal progenitor population in response to inductive signals. Several lines of evidence support the sequential roles of two Wnt family members, Wnt9b and Wnt4, in renal vesicle induction. Using genetic approaches to specifically manipulate the activity of β-catenin within the mesenchymal progenitor pool in mice, the potential role of the canonical Wnt pathway in these inductive events was investigated. Progenitor-cell-specific removal of β-catenin activity completely blocked both the formation of renal vesicles and the expected molecular signature of an earlier inductive response. By contrast, activation of stabilized β-catenin in the same cell population causes ectopic expression of mesenchymal induction markers in vitro and functionally replaces the requirement for Wnt9b and Wnt4 in their inductive roles in vivo. Thus, canonical Wnt signaling is both necessary and sufficient for initiating and maintaining inductive pathways mediated by Wnt9b and Wnt4. However, the failure of induced mesenchyme with high levels of β-catenin activity to form epithelial structures suggests that modulating canonical signaling may be crucial for the cellular transition to the renal vesicle (Park, 2007).
The liver and pancreas are specified from the foregut endoderm through an interaction with the adjacent mesoderm. However, the earlier molecular mechanisms that establish the foregut precursors are largely unknown. This study identified a molecular pathway linking gastrula-stage endoderm patterning to organ specification. In gastrula and early-somite stage Xenopus embryos, Wnt/β-catenin activity must be repressed in the anterior endoderm to maintain foregut identity and to allow liver and pancreas development. By contrast, high β-catenin activity in the posterior endoderm inhibits foregut fate while promoting intestinal development. Experimentally repressing β-catenin activity in the posterior endoderm is sufficient to induce ectopic organ buds that express early liver and pancreas markers. β-catenin acts in part by inhibiting expression of the homeobox gene hhex, which is one of the earliest foregut markers and is essential for liver and pancreas development. Promoter analysis indicates that β-catenin represses hhex transcription indirectly via the homeodomain repressor Vent2. Later in development, β-catenin activity has the opposite effect and enhances liver development. These results illustrate that turning Wnt signaling off and on in the correct temporal sequence is essential for organ formation, a finding that might directly impact efforts to differentiate liver and pancreas tissue from stem cells (McLin, 2007).
The mammalian kidney is organized into a cortex where primary filtration occurs, and a medullary region composed of elongated tubular epithelia where urine is concentrated. The cortico-medullary axis of kidney organization and function is regulated by Wnt7b signaling. The future collecting duct network specifically expresses Wnt7b. In the absence of Wnt7b, cortical epithelial development is normal but the medullary zone fails to form and urine fails to be concentrated normally. The analysis of cell division planes in the collecting duct epithelium of the emerging medullary zone indicates a bias along the longitudinal axis of the epithelium. By contrast, in Wnt7b mutants, cell division planes in this population are biased along the radial axis, suggesting that Wnt7b-mediated regulation of the cell cleavage plane contributes to the establishment of a cortico-medullary axis. The removal of beta-catenin from the underlying Wnt-responsive interstitium phenocopies the medullary deficiency of Wnt7b mutants, suggesting a paracrine role for Wnt7b action through the canonical Wnt pathway. Wnt7b signaling is also essential for the coordinated growth of the loop of Henle, a medullary extension of the nephron that elongates in parallel to the collecting duct epithelium. These findings demonstrate that Wnt7b is a key regulator of the tissue architecture that establishes a functional physiologically active mammalian kidney (Yu, 2009).
Previous studies have highlighted a role for the Notch signalling pathway during pronephrogenesis in the amphibian Xenopus laevis, and in nephron development in the mammalian metanephros, yet a mechanism for this function remains elusive. This study furthers the understanding of how Notch signalling patterns the early X. laevis pronephros anlagen, a function that might be conserved in mammalian nephron segmentation. The results indicate that early phase pronephric Notch signalling patterns the medio-lateral axis of the dorso-anterior pronephros anlagen, permitting the glomus and tubules to develop in isolation. This novel function acts through the Notch effector gene hrt1 by upregulating expression of wnt4. Wnt-4 then patterns the proximal pronephric anlagen to establish the specific compartments that span the medio-lateral axis. Pronephric expression was identified of lunatic fringe and radical fringe that is temporally and spatially appropriate for a role in regulating Notch signalling in the dorso-anterior region of the pronephros anlagen. On the basis of these results, a mechanism is proposed by which the Notch signalling pathway regulates a Wnt-4 function that patterns the proximal pronephric anlagen (Naylor, 2009).
The mammalian kidney is composed of thousands of individual epithelial tubules known as nephrons. Deficits in nephron number are associated with myriad diseases ranging from complete organ failure to congenital hypertension. A balance between differentiation and maintenance of a mesenchymal progenitor cell population determines the final number of nephrons. How this balance is struck is poorly understood. Previous studies have suggested that Wnt9b/β-catenin signaling induces differentiation (mesenchymal-to-epithelial transition) in a subset of the progenitors but needs to be repressed in the remaining progenitors to keep them in the undifferentiated state. This study reports that Wnt9b/β-catenin signaling is active in the progenitors and is required for their renewal/proliferation. Using a combination of approaches, a mechanism has been revealed through which cells receiving the same Wnt9b/β-catenin signal can respond in distinct ways (proliferate versus differentiate) depending on the cellular environment in which the signal is received. Interpretation of the signal is dependent, at least in part, on the activity of the transcription factor Six2. Six2-positive cells that receive the Wnt9b signal are maintained as progenitors whereas cells with reduced levels of Six2 are induced to differentiate by Wnt9b. Using this simple mechanism, the kidney is able to balance progenitor cell expansion and differentiation insuring proper nephron endowment. These findings provide novel insights into the molecular mechanisms that regulate progenitor cell differentiation during normal and pathological conditions (Karner, 2011).
Operational parallels in overall mechanisms of three-dimensional patterning of vertebrate organs are becoming increasingly apparent. Many key mediators, such as FGFs, BMPs, and sonic hedgehog, participate in organization of a number of organs, including the lungs, which exhibit a defined proximodistal (P-D) polarity. Recently, Wnt5a has been shown to underlie the
outgrowth and P-D morphogenesis of the vertebrate limb. Wnt5a is also expressed in the mouse lung and plays an important role in lung distal morphogenesis. Analysis of the mutant phenotype in mice carrying a targeted disruption of the Wnt5a locus shows distinct abnormalities in distal lung morphogenesis as manifested by distinct truncation of the trachea and overexpansion of the distal respiratory airways. In the face of deleted WNT5a activity, both epithelial and mesenchymal cell compartments of the Wnt5a minus lungs exhibit increased cell proliferation. The overall architecture of the mutant lungs is characterized by overexpansion of the distal airways and inhibition of lung
maturation as reflected by persistence of thickened intersaccular interstitium. Absence of WNT5a activity in the mutant
lungs leads to increased expression of Fgf-10, Bmp4, Shh, and its receptor Ptc, raising the possibility that WNT5a, FGF-10,
BMP4, and SHH signaling pathways are functionally interactive (Li, 2002).
Although the Wnt signaling pathway regulates inductive interactions between epithelial and mesenchymal cells, little is known of the role that this pathway plays during lung development. Wnt7b is expressed in the airway epithelium, suggesting a possible role for Wnt-mediated signaling in the regulation of lung development. To test this hypothesis, Wnt7b was mutated in the germline of mice by replacement of the first exon with the lacZ-coding region. Wnt7blacZ–/– mice exhibit perinatal death due to respiratory failure. Defects in early mesenchymal proliferation leading to lung hypoplasia are observed in Wnt7blacZ–/– embryos. In addition, Wnt7blacZ–/– embryos and newborn mice exhibit severe defects in the smooth muscle component of the major pulmonary vessels. These defects lead to rupture of the major vessels and hemorrhage in the lungs after birth. These results demonstrate that Wnt7b signaling is required for proper lung mesenchymal growth and vascular development (Shu, 2002).
Members of the Dickkopf (Dkk) family of secreted proteins are potent inhibitors of Wnt/ß-catenin signaling. Dkk1, -2, and -3 are expressed distally in the epithelium, while Kremen1, the needed co-receptor, is expressed throughout the epithelium of the developing lung. Using TOPGAL (a ß-galactosidase gene under the control of a LEF/TCF and ß-catenin inducible promoter) mice to monitor the Wnt pathway, canonical Wnt signaling is shown to be dynamic in the developing lung and is active throughout the epithelium and in the proximal smooth muscle cells (SMC) until E12.5. However, from E13.5 onwards, TOPGAL activity is absent in the SMC and is markedly reduced in the distal epithelium coinciding with the onset of Dkk-1 expression in the distal epithelium. To determine the role of Wnt signaling in early lung development, E11.5 organ cultures were treated with recombinant DKK1. Treated lungs display impaired branching, characterized by failed cleft formation and enlarged terminal buds, and show decreased alpha-smooth muscle actin (alpha-SMA) expression as well as defects in the formation of the pulmonary vasculature. These defects coincide with a pattern of decreased fibronectin (FN) deposition. DKK1-induced morphogenetic defects can be mimicked by inhibition of FN and overcome by addition of exogenous FN, suggesting an involvement of FN in Wnt-regulated morphogenetic processes (De Langhe, 2005).
Patterning of the primitive foregut promotes appropriate organ specification along its anterior-posterior axis. However, the molecular pathways specifying foregut endoderm progenitors are poorly understood. This study shows that Wnt2/2b signaling is required to specify lung endoderm progenitors within the anterior foregut. Embryos lacking Wnt2/2b expression exhibit complete lung agenesis and do not express Nkx2.1, the earliest marker of the lung endoderm. In contrast, other foregut endoderm-derived organs, including the thyroid, liver, and pancreas, are correctly specified. The phenotype observed is recapitulated by an endoderm-restricted deletion of beta-catenin, demonstrating that Wnt2/2b signaling through the canonical Wnt pathway is required to specify lung endoderm progenitors within the foregut. Moreover, activation of canonical Wnt/beta-catenin signaling results in the reprogramming of esophagus and stomach endoderm to a lung endoderm progenitor fate. Together, these data reveal that canonical Wnt2/2b signaling is required for the specification of lung endoderm progenitors in the developing foregut (Goss, 2009).
Wnt signal transduction has emerged as an increasingly complex pathway due to the numerous ligands, receptors, and modulators identified in multiple developmental systems. Wnt signaling has been implicated in the renewal of the intestinal epithelium within adult animals and the progression of cancer in the colon. The Wnt family, however, has not been explored for function during embryonic gut development. Thus, to dissect the role of Wnt signaling in the developing gastrointestinal tract, it is necessary to first obtain a complete picture of the spatiotemporal expression of the Wnt signaling factors with respect to the different tissue layers of the gut. This study offers an in depth in situ gene expression study of Wnt ligands, frizzled receptors, and frizzled related modulators over several days of chicken gut development. These data show some expected locations of Wnt signaling as well as a surprising lack of expression of factors in the hindgut (McBride, 2002).
Of the 25 genes in the Wnt pathway analyzed, 18 were
expressed during the window of time studied. The following
Wnt genes have probes from either the isolated chicken
genes or cross-reacting mouse genes: Wnt1, Wnt2, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7b, Wnt8b, Wnt8c, Wnt10a, Wnt11, Wnt13, and Wnt14. Of those genes, Wnt1, Wnt5a, Wnt5b, Wnt6, Wnt7b, Wnt8b, Wnt10a, Wnt11, and Wnt14
were expressed during gut development. Based on the canonical/noncanonical classification system, several of the Wnt genes expressed in the gut fall into each category of the classification system. Wnt1 and Wnt8b cause ß-catenin nuclear localization in other systems that have been tested. Wnt5a, Wnt5b, Wnt11,
and Wnt14 are in the noncanonical class. Several of the Wnt
genes (Wnt6, Wnt7b, and Wnt10a) expressed fall into a separate grouping because they appear to function in either pathway depending on the cell
type tested. This may reflect the different signaling abilities of Wnt proteins with
different frizzled receptors. In the chicken embryo, seven frizzled genes have been isolated (Fz1 Fz2 Fz4 Fz7 Fz8 Fz9, and Fz10) and of those genes all but Fz9 and Fz10 are expressed in the developing chicken gut. In addition, there are four secreted
frizzled related genes, crescent, sfrp-1, sfrp-2, and frzb-1,
isolated in the chicken that are all expressed to varying degrees in the gut. Based on the role of Wnt signaling in maintaining the adult colonic epithelium, it was expected that most Wnt gene expression would be limited to the endoderm. Instead, most of
the genes exhibit expression within the mesoderm, although
a few important exceptions have specific endodermal expression (McBride, 2002).
Wnts and tissue polarity In the mammalian cochlea, stereociliary bundles located on mechanosensory hair cells within the sensory epithelium are unidirectionally oriented. Development of this planar polarity is necessary for normal hearing because stereociliary bundles are only sensitive to vibrations in a single plane; however, the mechanisms governing their orientation are unknown. Wnt signaling has been found to regulate the development of unidirectional stereociliary bundle orientation. In vitro application of Wnt7a protein or inhibitors of Wnt signaling, secreted Frizzled-related protein 1 or Wnt inhibitory factor 1, disrupts bundle orientation. Moreover, Wnt7a is expressed in a pattern consistent with a role in the polarization of the developing stereociliary bundles. It is proposed that Wnt signaling across the region of developing outer hair cells gives rise to planar polarity in the mammalian cochlea (Dabdoub, 2003).
The regulation of asymmetric cell division (ACD) during corticogenesis is incompletely understood. This study documents that spindle-size asymmetry (SSA) between the two poles occurs during corticogenesis and parallels ACD. SSA appears at metaphase and is maintained throughout division, and it is necessary for proper neurogenesis. Imaging of spindle behavior and division outcome reveals that neurons preferentially arise from the larger-spindle pole. Mechanistically, SSA magnitude is controlled by Wnt7a and Vangl2, both members of the Wnt/planar cell polarity (PCP)-signaling pathway, and relayed to the cell cortex by P-ERM proteins. In vivo, Vangl2 and P-ERM downregulation promotes early cell-cycle exit and prevents the proper generation of late-born neurons. Thus, SSA is a core component of ACD that is conserved in invertebrates and vertebrates and plays a key role in the tight spatiotemporal control of self-renewal and differentiation during mammalian corticogenesis (Delaunay, 2014).
Planar cell polarity (PCP) signaling plays a critical role in tissue morphogenesis. In mammals, disruption of three of the six 'core PCP' components results in polarity-dependent defects with rotated cochlear hair cell stereocilia and open neural tube. Recent studies have demonstrated a role of Prickle1, a core PCP molecule in Drosophila, in mammalian neuronal development. To examine Prickle1 function along a broader developmental window, three mutant alleles were generated in mice. The complete loss of Prickle1 leads to systemic tissue outgrowth defects, aberrant cell organization and disruption of polarity machinery. Curiously, Prickle1 mutants recapitulate the characteristic features of human Robinow syndrome and phenocopy mouse mutants with Wnt5a or Ror2 gene defects, prompting an exploration of an association of Prickle1 with the Wnt pathway. This study shows that Prickle1 is a proteasomal target of Wnt5a signaling and that Dvl2, a target of Wnt5a signaling, is misregulated in Prickle1 mutants. These studies implicate Prickle1 as a key component of the Wnt-signaling pathway and suggest that Prickle1 mediates some of the WNT5A-associated genetic defects in Robinow syndrome (Liu, 2014).
Wnt signaling is important in organogenesis, and aberrant signaling in mature cells is associated with tumorigenesis. Several members of the Wnt family of signaling molecules are expressed in the developing pituitary gland. Wnt5a is expressed in the neuroectoderm, which induces pituitary gland development and has been proposed to influence pituitary cell specification. Mice deficient in Wnt5a display abnormal morphology in the dorsal part of the developing pituitary. The expression of downstream effectors of the canonical Wnt pathway is not altered, and expression of genes in other signaling pathways such as Shh, Fgf8, Fgf10 and Fgfr2b is intact. Prop1 and Hesx1 are also important for normal shape of the pituitary primordium, but their expression is unaltered in the Wnt5a mutants. Specification of the hormone-producing cell types of the mature anterior pituitary gland occurs appropriately. This study suggests that the primary role of Wnt5a in the developing pituitary gland is in establishment of the shape of the gland (Cha, 2004).
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Biological Overview
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