armadillo
ß-catenin, development, patterning and morphogenesis In early embryos of the ascidian Ciona savignyi (phylum Urochordata), nuclear accumulation of ß-catenin is most probably the first step of endodermal cell specification. If ß-catenin is mis- and/or over-expressed, presumptive notochord cells and epidermal cells change their fates into endodermal cells, whereas if ß-catenin nuclear localization is downregulated by the overexpression of cadherin, the endoderm differentiation is suppressed, accompanied by the differentiation of extra epidermal cells. Subtractive hybridization screens of mRNAs between ß-catenin overexpressed embryos and cadherin overexpressed embryos were conducted to identify potential ß-catenin target genes that are responsible for endoderm differentiation in Ciona savignyi embryos. A LIM-homeobox gene (Cs-lhx3: Drosophila homolog - Lim3), an otx homolog (Cs-otx) and an NK-2 class gene (Cs-ttf1) were among ß-catenin downstream genes. In situ hybridization signals for early zygotic expression of Cs-lhx3 are evident only in the presumptive endodermal cells as early as the 32-cell stage, those of Cs-otx in the mesoendodermal cells at the 32-cell stage and those of Cs-ttf1 in the endodermal cells at the 64-cell stage. Later, Cs-lhx3 is expressed again in a set of neuronal cells in the tailbud embryo, while Cs-otx is expressed in the anterior nervous system of the embryo. Expression of all three genes is upregulated in ß-catenin overexpressed embryos and downregulated in cadherin overexpressed embryos. Injection of morpholino oligonucleotides against Cs-otx does not affect the embryonic endoderm differentiation, although the formation of the central nervous system is suppressed. Injection of Cs-ttf1 morpholino oligonucleotides also fails to suppress the endoderm differentiation, although injection of its synthetic mRNAs results in ectopic development of endoderm differentiation marker alkaline phosphatase. By contrast, injection of Cs-lhx3 morpholino oligo suppresses the endodermal cell differentiation and this suppression is rescued by injection of Cs-lhx3 mRNA into eggs. In addition, although injection of delE-Ci-cadherin mRNA into eggs results in the suppression of alkaline phosphatase development, injection of delE-Ci-cadherin mRNA with Cs-lhx3 mRNA rescues the alkaline phosphatase development. These results strongly suggest that a LIM-homeobox gene Cs-lhx3 is one of the ß-catenin downstream genes and that its early expression in embryonic endodermal cells is responsible for their differentiation (Satou, 2001).
A key issue for understanding the early development of the chordate body plan is how the endoderm induces notochord formation. In the ascidian Ciona, nuclear accumulation of ß-catenin is the first step in the process of endoderm specification. Nuclear accumulation of ß-catenin directly activates the gene (Cs-FoxD) for a winged helix/forkhead transcription factor and this gene is expressed transiently at the 16- and 32-cell stages in endodermal cells. The function of Cs-FoxD, however, is not associated with differentiation of the endoderm itself but is essential for notochord differentiation or induction. In addition, it is likely that the inductive signal that appears to act downstream of Cs-FoxD does not act over a long range. It has been suggested that FGF or Notch signal transduction pathway mediates ascidian notochord induction. Previous work suggests that Cs-FGF4/6/9 is partially involved in the notochord induction. The present experimental results suggest that the expression and function of Cs-FGF4/6/9 and Cs-FoxD are not interdependent, and that the Notch pathway is involved in B-line notochord induction (B-line cells represent a secondary notochord lineage) downstream of Cs-FoxD (Iwai, 2002).
A zebrafish recessive maternal effect mutant, ichabod, has been identified that results in severe anterior and dorsal defects during early development. The ichabod mutation is almost completely penetrant, but
exhibits variable expressivity. All mutant embryos fail to
form a normal embryonic shield; most fail to form a head
and notochord and have excessive development of ventral
tail fin tissue and blood. Abnormal dorsal patterning
can first be observed at 3.5 hpf by the lack of nuclear
accumulation of beta-catenin in the dorsal yolk syncytial layer,
which also fails to express bozozok/dharma/nieuwkoid and
znr2/ndr1/squint. At the onset of gastrulation, deficiencies
in expression of dorsal markers and expansion of
expression of markers of ventral tissues indicate a dramatic
alteration of dorsoventral identity. Injection of beta-catenin
RNA markedly dorsalizes ichabod embryos and often
completely rescues the phenotype, but no measurable
dorsalization is obtained with RNAs encoding upstream
Wnt pathway components. In contrast, dorsalization is
obtained when RNAs encoding either Bozozok/Dharma/
Nieuwkoid or Znr2/Ndr1/Squint are injected. Moreover,
injection of beta-catenin RNA into ichabod embryos results
in activation of expression of these two genes, which can
also activate one another. RNA injection experiments
strongly suggest that the component affected by the ichabod
mutation acts on a step affecting beta-catenin nuclear
localization that is independent of regulation of beta-catenin
stability. This work demonstrates that a maternal gene
controlling localization of beta-catenin in dorsal nuclei is
necessary for dorsal yolk syncytial layer gene activity and
formation of the organizer in the zebrafish (Kelly, 2000).
The ectoderm of the pre-gastrula Xenopus embryo is at least partially patterned along the dorsal-ventral axis. The early expression of the anti-neural homeodomain gene Dlx3 is localized to the ventral ectoderm by a mechanism that occurs prior to gastrulation and is independent of the Spemann organizer. The repression of Dlx3 is mediated by signaling through beta-catenin, but is probably not dependent on the induction of the Xnr3 or chordin genes by beta-catenin. It is proposed that the establishment of
the dorso-ventral axis in Xenopus, which occurs during the
first cell cycle and requires an enrichment of beta-catenin in
prospective dorsal cells, leads to the repression of Dlx3 in the most dorsal
ectoderm, prior to the formation of the Spemann organizer.
This inhibition could be mediated by repression of BMP-4
expression, but could also be direct. Injection experiments
in which beta-catenin and BMP-4 are co-injected indicate that
the repression of Dlx3 by beta-catenin is at least partially
dependent on inhibition of BMP-4 expression. Since Dlx3 is an inhibitor of neural gene expression, this repression could account for the
propensity of dorsal ectoderm to respond to neural inducers, and the tendency of ventral ectoderm to express epidermal markers. This model is compatible with the observation that ventral ectoderm can be induced to become neural by organizer transplants, or equivalent procedures which would repress expression of Dlx3. This model also predicts that neural induction will take place more readily with dorsal versus ventral ectoderm, which is what has been observed (Beanan, 2000).
In order to identify factors involved in posteriorization of the central nervous system, a functional screen was undertaken in Xenopus animal cap explants that involved coinjecting noggin RNA together with pools of RNA from a chick somite cDNA library. In the course of this screen, a clone was isolated encoding a truncated form of ß-catenin, which induced posterior neural and dorsal mesodermal markers when coinjected with noggin in animal caps. Similar results were obtained with Xwnt-8 and Xwnt-3a, suggesting that these effects are a consequence of activating the canonical Wnt signaling pathway. To investigate whether the activation of posterior neural markers requires mesoderm induction, experiments were performed using a chimeric inducible form of ß-catenin. Activation of this protein during blastula stages results in the induction of both posterior neural and mesodermal markers, while activation during gastrula stages induces only posterior neural markers. This posteriorizing activity occurs by an indirect and noncell-autonomous mechanism requiring FGF signaling (Domingos, 2001).
Although FGF signaling plays an integral role in the migration and patterning of mesoderm at gastrulation, the mechanism and downstream targets of FGF activity have remained elusive. FGFR1 orchestrates the epithelial to mesenchymal transition and morphogenesis of mesoderm at the primitive streak by controlling Snail and E-cadherin expression. Furthermore, FGFR1 functions in mesoderm cell fate specification by positively regulating Brachyury and Tbx6 expression. Finally, evidence is provided that the attenuation of Wnt3a signaling observed in Fgfr1-/- embryos can be rescued by lowering E-cadherin levels.
It is proposed that modulation of cytoplasmic ß-catenin levels, associated with FGF-induced downregulation of E-cadherin, provides a molecular link between FGF and Wnt signaling pathways at the streak (Ciruna, 2001).
Results from the Fgfr1 mutant expression analyses, chimeric studies, and in vitro explant experiments can be assembled into a minimal model for FGFR1 function at gastrulation. This study has defined a specific region of the primitive streak that requires FGFR1 signaling activity; this domain encompasses the paraxial and posterior embryonic mesoderm populations, but excludes the node, axial, and extraembryonic mesoderm. In the context of this domain, it is proposed that FGFR1 signaling orchestrates both the morphogenetic movement and cell fate specification events of gastrulation (Ciruna, 2001).
In addition, it is proposed that FGFR1 signaling indirectly regulates Wnt signal transduction at the primitive streak. In Fgfr1 -/- embryos, although Wnt3a is expressed in the late primitive streak, direct targets of Wnt signaling (i.e., Brachyury and the T-lacZ reporter transgene) are not activated. It is suggested that ectopic E-cadherin expression in Fgfr1 mutants attenuates Wnt3a signaling by sequestering free ß-catenin from its intracellular signaling pool, and demonstrates that forced downregulation of E-cadherin in Fgfr1 -/- explants can rescue endogenous Wnt signaling at the primitive streak. Evidence that cadherins act as regulators of ß-catenin signaling is well documented. E-Cadherin and LEF-1 bind to partially overlapping sites in the central region of ß-catenin; consequently, LEF-1 and E-cadherin form mutually exclusive complexes with ß-catenin and compete for the same intracellular signaling pool. Furthermore, overexpression of cadherins during Drosophila and Xenopus embryogenesis has been shown to phenocopy Wnt/ß-catenin signaling mutants (Ciruna, 2001).
It is well established that Wnt signaling stabilizes cytosolic levels of ß-catenin by inhibiting its GSK3ß-mediated phosphorylation and degradation. At gastrulation, loss of E-cadherin expression downstream of FGFR1 may also facilitate a rapid intracellular transfer of membrane-bound ß-catenin to the cytosolic 'signaling' pool. Since downregulation of E-cadherin alone is not sufficient to induce ectopic activation of T-lacZ and Brachyury expression in WT primitive streak cultures, signaling through the ß-catenin pathway is still dependent on the activity of localized Wnt signals. However, FGF-mediated changes in cadherin levels and ß-catenin localization could still regulate the threshold for and/or speed of Wnt signaling responses at gastrulation. It is therefore proposed that normal downregulation of E-cadherin at the primitive streak not only regulates the EMT and migration of mesoderm progenitor cells at gastrulation, but also permits the rapid and uninhibited accumulation of cytosolic ß-catenin levels in response to localized Wnt signals. This competition for and opposing influences on the intracellular localization and function of ß-catenin thus establishes a molecular link between the FGF and Wnt signaling pathways at gastrulation. Consequently, FGFR1 activity plays an indirect but permissive role in the propagation of Wnt signaling responses at the primitive streak. The fundamental interregulation of cell adhesion, morphogenesis, and cell fate determination, as demonstrated in this analysis of FGFR1 function, serves to underscore the interdependent nature of morphogenesis and patterning at gastrulation and the intricate network of inductive interactions that pattern and shape the developing embryo (Ciruna, 2001).
Since the three main pathways (the Wnt, VegT and BMP pathways) involved in organizer and axis
formation in the Xenopus embryo are now characterized, the challenge is to understand their interactions.
This study makes three comparisons. (1) A systematic comparison was made of the expression of zygotic genes in sibling wild-type, VegT-depleted (VegT-), ß-catenin-depleted (ß-catenin-) and double
depleted (VegT-/ß-catenin-) embryos and early zygotic genes were placed into specific groups. In the first group some organizer genes, including chordin, noggin and cerberus, required the activity of both the Wnt pathway and the VegT pathway to be expressed. A second group including Xnr1, 2, 4 and Xlim1 were initiated by the VegT pathway but their dorsoventral pattern and amount of their expression was regulated by the Wnt pathway. (2) The roles of the Wnt and VegT pathways in producing dorsal signals were compared. Explant co-culture experiments showed that the Wnt pathway does not cause the release of a dorsal signal from the vegetal mass independent from the VegT pathway. (3) The extent to which inhibiting Smad 1 phosphorylation in one
area of VegT-, or ß-catenin- embryos would rescue organizer and axis formation was measured. BMP inhibition with cm-BMP7 mRNA
has no rescuing effects on VegT- embryos, while cm-BMP7 and noggin mRNA causes a complete rescue of the trunk, but not of the
anterior pattern in ß-catenin- embryos. One likely missing component required for normal anterior patterning could be later BMP signaling, which would remain inhibited by the over-expression of cm-BMP7 or noggin mRNAs. Also, early organizer elements still missing in these embryos include the
dorsoventral waves of Xnr1, 2 and 4 expression, the expression of siamois, Xnr3 and the correct level of expression of Xhex and Xlim1. Many studies have implicated all of these in aspects of neural, head, heart and anterior endoderm specification. The challenge is to
work out the hierarchy of the regulatory networks. One simple possibility is that the early high dorsal level of Xnr expression is needed for the high
level of expression of siamois, Xnr3, Xhex and Xlim1. This view is supported by the fact that a dose response of Xnr1, 2
and 4 mRNAs injected into VegT- embryos restores increasing amounts of head formation. However, the specific roles played by individual Xnrs have yet to be examined by loss-of-function analysis (Xanthos, 2002).
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).
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).
Cadherin-mediated cell adhesion is involved in muscle differentiation from early stages of myogenic induction to late stages of myoblast interaction and fusion. ß-Catenin is
a major constituent of cadherin-based adherens junctions
and also serves as a signal transduction molecule that
regulates gene expression during development. This study explores the involvement of ß-catenin in myogenic differentiation. Shortly after
a switch from growth to differentiation medium, ß-catenin
translocates to cell-cell junctions and its levels increase. Elevation of ß-catenin levels, induced either by inhibition of its breakdown, using LiCl, or by its overexpression, suppresses the formation of adherens
junctions, resulting in a sharp decline in myogenin
expression and an arrest of myogenic progression.
Recruitment of ß-catenin to adherens junctions after
transfection with N-cadherin restores myogenin expression
in the transfected cells. These results suggest that increased
cadherin-mediated adhesion and translocation of ß-catenin
to adherens junctions are involved in activating the early
steps of myogenic differentiation (Goichberg, 2001).
An effector of intercellular adhesion, beta-catenin also functions in Wnt signaling, associating with Lef-1/Tcf DNA-binding proteins to form a functional transcription
factor. This pathway operates in keratinocytes: mice expressing a stabilized beta-catenin controlled by an epidermal promoter undergo
a process resembling de novo hair morphogenesis. The new follicles form sebaceous glands and dermal papilla, normally established only in
embryogenesis. As in embryologically initiated hair germs, transgenic follicles induce Lef-1, but follicles are disoriented and defective in sonic hedgehog
polarization. Additionally, proliferation continues unchecked, resulting in two types of tumors also found in humans. These findings suggest that transient
beta-catenin stabilization may be a key player in the long-sought epidermal signal leading to hair development and implicate aberrant beta-catenin activation
in hair tumors (Gat, 1998).
ß-Catenin is an essential molecule in Wnt/wingless signaling, which controls decisive steps in embryogenesis. To study the role of ß-catenin in skin development, a conditional mutation of the gene was introduced in the epidermis and hair follicles using Cre/loxP technology. When ß-catenin is
mutated during embryogenesis, formation of placodes that generate hair follicles is blocked. ß-Catenin is required genetically downstream of tabby/downless and upstream of bmp and shh in
placode formation. If ß-catenin is deleted after hair follicles have formed, hair is completely lost after the first hair cycle. Further analysis demonstrates that ß-catenin is essential for fate decisions of skin stem cells: in the absence of ß-catenin, stem cells fail to differentiate into follicular keratinocytes, but instead adopt an epidermal fate (Huelsken, 2001).
An important role for ß-catenin during hair follicle related development and tumorigenesis has recently been
established, though little is known of its endogenous expression during the development of these structures. The expression of ß-catenin in relation to markers for proliferation, differentiation and Wnt signaling was examined during the development of three hair
follicle related structures, i.e., whiskers, normal body hair and the preputial gland, and a hair follicle-derived tumor, the epidermal cyst. Nuclear accumulation of ß-catenin, the hallmark of Wnt signaling, is observed in the upper matrix, the dermal papilla, the developing ringwulst
of the whisker (a gland surrounding the whisker follicle) and in the tumor, though nuclear accumulation was never in association with proliferation or terminal differentiation. Co-localization of nuclear ß-catenin
with Tcf-3/4 was found only in the dermal papilla and the developing ringwulst of the whisker, but not in the upper matrix or in the
tumor. These results further elucidate the role of the Wnt signal transduction pathway during hair follicle related development and
tumorigenesis and illustrate the dynamic role of ß-catenin in signal transduction and cell-adhesion (Ridanpaa, 2002).
Wnts have key roles in many developmental processes, including hair follicle growth and differentiation. Stabilization of ß-catenin is essential in the canonical Wnt signaling pathway. Transgenic mice were developed expressing a regulated form of ß-catenin in the skin. Chronic activation of ß-catenin in resting (telogen) hair follicles results in changes consistent with induction of an exaggerated, aberrant growth phase (anagen). Transient activation of ß-catenin produces a normal anagen. These data lend strong support to the notion that a Wnt/ß-catenin signal operating on hair follicle precursor cells serves as a crucial proximal signal for the telogen-anagen transition (Van Mater, 2003).
The role of beta-catenin in chicken skin morphogenesis has been examined. Initially beta-catenin mRNA is expressed at homogeneous
levels in the epithelia over a skin appendage tract field that become transformed into a periodic pattern corresponding to
individual primordia. The importance of periodic patterning is shown in scaleless mutants, in which beta-catenin is
initially expressed normally, but fails to make a punctuated pattern. To test beta-catenin function, a truncated armadillo
fragment was expressed in developing chicken skin from the RCAS retrovirus. This produces a variety of phenotypic
changes during epithelial appendage morphogenesis. In apteric and scale-producing regions, new feather buds with
normal-appearing follicle sheaths, dermal papillae, and barb ridges are induced. In feather tracts, short, wide, and curled
feather buds with abnormal morphology and random orientation form. Epidermal invaginations and placode-like
structures form in the scale epidermis. PCNA staining and the distribution of molecular markers (SHH, NCAM,
Tenascin-C) are characteristic of feather buds. These results suggest that the beta-catenin pathway is involved in modulating
epithelial morphogenesis and that increased beta-catenin pathway activity can increase the activity of skin appendage
phenotypes (Widelitz, 2000).
Regression of the Mullerian duct in the male embryo is
one unequivocal effect of anti-Mullerian hormone, a
glycoprotein secreted by the Sertoli cells of the testis.
AMH is a member of the TGF-beta family, which signals through a receptor complex
formed by two distantly related serine/threonine kinases. This
hormone induces ductal epithelial regression through
a paracrine mechanism originating in periductal
mesenchyme. To probe the mechanisms of action of anti-Mullerian
hormone, the sequence of cellular and molecular events involved in duct regression have been studied. Studies were performed in male rat embryos and in
transgenic mice overexpressing or lacking anti-Mullerian
hormone, both in vivo and in vitro.
Anti-Mullerian hormone causes regression of the cranial
part of the Mullerian duct whereas it continues to grow
caudally. This pattern of regression is
correlated with a cranial to caudal gradient of anti-Mullerian
hormone receptor protein, followed by a wave of
apoptosis spreading along the Mullerian duct as its
progresses caudally. Apoptosis is also induced by AMH in
female Mullerian duct in vitro. Furthermore, apoptotic
indexes are increased in Mullerian epithelium of transgenic
mice of both sexes overexpressing the human anti-Mullerian
hormone gene, exhibiting a positive correlation
with serum hormone concentration. Inversely, apoptosis is
reduced in male anti-Mullerian hormone-deficient mice. Apoptosis is a decisive but not sufficient process; epitheliomesenchymal transformation is
an important event of Mullerian regression.
The most striking result of this study is that anti-Mullerian
hormone action in peri-Mullerian mesenchyme
leads in vivo and in vitro to an accumulation of cytoplasmic
beta-catenin. The co-localization of beta-catenin with lymphoid
enhancer factor 1 in the nucleus of peri-Mullerian
mesenchymal cells, demonstrated in primary culture,
suggests that overexpressed beta-catenin in association with LEF1 may alter transcription of target genes and may lead to changes in mesenchymal gene
expression and cell fate during Mullerian duct regression.
This may well be the first report that beta-catenin, known for its role in Wnt signaling, may mediate anti-Mullerian hormone action (Allard, 2000).
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).
Using Cre/loxP, the ß-catenin gene was conditionally inactivated in cells of structures that exhibit important embryonic organizer functions: the visceral endoderm, the node, the notochord, and the definitive endoderm. Mesoderm formation is not affected in the mutant embryos, but the node is missing, patterning of the head and trunk is affected, and no notochord or somites are formed. Surprisingly, deletion of ß-catenin in the definitive endoderm leads to the formation of multiple hearts all along the anterior-posterior (A/P) axis of the embryo. Ectopic hearts develop in parallel with the normal heart in regions of ectopic Bmp2 expression. Evidence is provided that ablation of ß-catenin in embryonic endoderm changes cell fate from endoderm to precardiac mesoderm, consistent with the existence of bipotential mesendodermal progenitors in mouse embryos (Lickert, 2002).
ß-Catenin regulates important biological processes, including embryonic development and tumorigenesis. The role of ß-catenin in the regulation of the chondrocyte phenotype has been investigated. Expression of ß-catenin is high
in prechondrogenic mesenchymal cells, but significantly decreased in differentiated chondrocytes both in vivo and
in vitro. Accumulation of ß-catenin by the inhibition of glycogen synthase kinase-3ß with LiCl inhibits
chondrogenesis by stabilizing cell-cell adhesion. Conversely, the low level of ß-catenin in differentiated articular
chondrocytes is increased by post-translational stabilization during phenotypic loss caused by a serial monolayer culture or exposure to retinoic acid
or interleukin-1ß. Ectopic expression of ß-catenin or inhibition of ß-catenin degradation with LiCl or proteasome inhibitor causes de-differentiation of
chondrocytes. Transcriptional activation of ß-catenin by its nuclear translocation is sufficient to cause phenotypic loss of differentiated
chondrocytes. The expression pattern of Jun, a known target gene of ß-catenin, is essentially the same as that of ß-catenin both in vivo and in vitro
suggesting that Jun and possibly activator protein 1 is involved in the ß-catenin regulation of the chondrocyte phenotype (Ryu, 2002).
In the small intestine, the progeny of stem cells migrate in precise patterns. Absorptive, enteroendocrine, and
goblet cells migrate toward the villus while Paneth cells occupy the bottom of the crypts. Here it has been shown that ß-catenin and TCF inversely control the expression of the EphB2/EphB3 receptors and their ligand
ephrin-B1 in colorectal cancer and along the crypt-villus axis. Disruption of EphB2 and EphB3 genes reveals that their gene products restrict cell intermingling and allocate cell populations within the intestinal epithelium. In EphB2/EphB3 null mice, the proliferative and differentiated populations intermingle. In adult EphB3-/- mice, Paneth cells do not follow their downward migratory path, but scatter along crypt and villus. It is concluded that in the intestinal epithelium ß-catenin and TCF couple proliferation and differentiation to the sorting of cell populations through the EphB/ephrin-B system (Batlle, 2002).
ß-Catenin is an essential component of the canonical Wnt signaling system that controls decisive steps in development. Two conditional ß-catenin mutant alleles were used to alter ß-catenin signaling in the central nervous system of mice: one allele to ablate ß-catenin and the second allele to express a constitutively active ß-catenin. The tissue mass of the spinal cord and brain is reduced after ablation of ß-catenin, and the neuronal precursor population is not maintained. In contrast, the spinal cord and brain of mice that express activated ß-catenin is much enlarged in mass, and the neuronal precursor population is increased in size. ß-Catenin signals are thus essential for the maintenance of proliferation of neuronal progenitors, controlling the size of the progenitor pool, and impinging on the decision of neuronal progenitors to proliferate or to differentiate (Zechner, 2003).
Gene expression profiling of ß-catenin, Cripto and
Wnt3 mutant mouse embryos has been used to characterize the genetic
networks that regulate early embryonic development. Genes have been defined
whose expression is regulated by ß-catenin during formation of the
anteroposterior axis and the mesoderm; this study identifies Cripto,
which encodes a Nodal co-receptor, as a primary target of ß-catenin
signals both in embryogenesis as well as in colon carcinoma cell lines and
tissues. Groups of genes regulated by Wnt3/ß-catenin
signalling during primitive streak and mesoderm formation have been identified. The data assign a key role to ß-catenin upstream of two distinct gene expression programs during anteroposterior axis and mesoderm formation (Morkel, 2003).
ß-Catenin mutant embryos fail to undergo two crucial developmental
steps: (1) the distal visceral endoderm does not become positioned at the
anterior side at E6.0, and (2) primitive streak and mesoderm formation does
not occur at E6.5. These changes can be interpreted as the sum of the
phenotypes observed in Cripto and Wnt3 mutant mice.
Cripto-/- embryos fail to re-orient the anteroposterior
axis at E6.0, but generate extra-embryonic mesoderm from the proximal epiblast
at E6.5, whereas Wnt3-/- embryos correctly position the
anteroposterior axis at E6.0, but fail to generate mesoderm at E6.5. Using
expression profiling, this study has identified Cripto and other genes whose
expression is absent in ß-catenin mutants at E6.0, when the
anteroposterior axis is normally reoriented in wild-type embryos. Brachyury, Nanog and other genes were identified whose expression depends
on ß-catenin at E6.5, when the primitive streak and mesoderm are formed.
Furthermore, a significant overlap of genes is found whose expression is
deregulated in ß-catenin and Cripto, and in ß-catenin and
Wnt3 mutant embryos at E6.0 and E6.5, respectively. The profiling
data thus support the model of two distinct ß-catenin dependent steps. In the first step,
ß-catenin is essential for the expression of the Nodal co-receptor gene
Cripto in the epiblast, which is required for translocation of the
distal visceral endoderm to the anterior side, and thus the correct
orientation of the anteroposterior axis at E6.0. In the second step,
ß-catenin is required for Wnt3 signalling and thus regulates the
expression of target genes in the proximal/posterior epiblast that are
essential for mesoderm formation (Morkel, 2003).
When ß-catenin signalling is disturbed from mid-gestation onward, lineage commitment is profoundly altered in postnatal mouse epidermis. Whether adult epidermis has the capacity for ß-catenin-induced lineage conversion without prior embryonic priming has been investigated. N-terminally truncated, stabilized ß-catenin was fused to the ligand-binding domain of a mutant estrogen receptor ({Delta}Nß-cateninER). {Delta}Nß-cateninER was expressed in the epidermis of transgenic mice under the control of the keratin 14 promoter and ß-catenin activity was induced in adult epidermis by topical application of 4-hydroxytamoxifen (4OHT). Within 7 days of daily 4OHT treatment resting hair follicles were recruited into the hair growth cycle and epithelial outgrowths formed from existing hair follicles and from interfollicular epidermis. The outgrowths expressed Sonic hedgehog, Patched and markers of hair follicle differentiation, indicative of de novo follicle formation. The interfollicular epidermal differentiation program was largely unaffected but after an initial wave of sebaceous gland duplication, sebocyte differentiation was inhibited. A single application of 4OHT was as effective as repeated doses at inducing new follicles and growth of existing follicles. Treatment of epidermis with 4OHT for 21 days resulted in conversion of hair follicles to benign tumors resembling trichofolliculomas. The tumors were dependent on continuous activation of ß-catenin, and by 28 days after removal of the drug they had largely regressed. It is concluded that interfollicular epidermis and sebaceous glands retain the ability to be reprogrammed in adult life and that continuous ß-catenin signalling is required to maintain hair follicle tumors (Celso, 2004).
Wnt signaling is implicated in many developmental processes, including cell fate changes. Several members of the Wnt family, as well as other molecules involved in Wnt signaling, including Frizzled receptors, LDL-related protein co-receptors, members of the Dishevelled and Dickkopf families, are known to be expressed in the lens during embryonic or postembryonic development. However, the function of Wnt signaling in lens fiber differentiation remains unknown. GSK-3ß kinase has been shown to be inactivated and ß-catenin accumulates during the early stages of lens fiber cell differentiation. In an explant culture system, Wnt conditioned medium (CM) induces the accumulation of ß-crystallin, a marker of fiber cell differentiation, without changing cell shape. In contrast, epithelial cells stimulated with Wnt after priming with FGF elongate, accumulate ß-crystallin, aquaporin-0, p57kip2, and alter their expression of cadherins. Treatment with lithium, which stabilizes ß-catenin, induces the accumulation of ß-crystallin, but explants treated with lithium after FGF priming do not elongate as they do after Wnt application. These results show that Wnts promote the morphological aspects of fiber cell differentiation in a process that requires FGF signaling, but is independent of ß-catenin. Wnt signaling may play an important role in lens epithelial-to-fiber differentiation (Lyu, 2004).
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).
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).
Wnt/ß-catenin signaling pathway is involved in the
maintenance of the progenitor cell population in the skin,
intestine and other tissues, and its aberrant activation caused by
stabilization of ß-catenin contributes to tumorigenesis. In
the mammary gland, constitutive activation of Wnt/ß-catenin
signaling in luminal secretory cells results in precocious
lobuloalveolar differentiation and induces adenocarcinomas, whereas
the impact of this signaling pathway on the function of the second
major mammary epithelial cell lineage, the basal myoepithelial
cells, has not been analyzed. The keratin (K) 5 promoter has been
used to target the expression of stabilized N-terminally truncated
ß-catenin to the basal cell layer of mouse mammary epithelium.
The transgenic mice present an abnormal mammary phenotype:
precocious lateral bud formation, increased proliferation and
premature differentiation of luminal epithelium in pregnancy,
persistent proliferation in lactation and accelerated involution.
Precocious development in pregnancy is accompanied by increased Myc
and cyclin D1 transcript levels, and a shift in p63 variant
expression towards the DeltaNp63 form. The expression of
ECM-degrading proteinases and their inhibitors is altered in
pregnancy and involution. Nulliparous transgenic females develop
mammary hyperplasia that comprise undifferentiated basal
(K5/14-positive, K8- and alpha-smooth muscle-actin-negative) cells.
Multiparous mice, in addition, developed invasive basal-type
carcinomas. Thus, activation of ß-catenin signaling in basal
mammary epithelial cells affects the entire process of mammary
gland development and induces amplification of basal-type cells
that lack lineage markers, presumably, a subpopulation of mammary
progenitors able to give rise to tumors (Teuliere, 2005).
Despite an increasingly sophisticated understanding of transcriptional regulation in pancreas development, relatively little is known about the extrinsic signaling pathways involved in this process. This study shows that the early pancreatic epithelium exhibits a specific enrichment in unphosphorylated ß-catenin protein, a hallmark of activation of the canonical Wnt signaling pathway. To determine if this pathway is functionally required for normal pancreas development, the ß-catenin gene was specifically deleted in these cells. Pancreata developing without ß-catenin are hypoplastic, although their early progenitors appear normal and exhibit no premature differentiation or death. Surprisingly, and in marked contrast to its role in the intestine, loss of ß-catenin does not significantly perturb islet endocrine cell mass or function. The major defect of the ß-catenin-deficient pancreas is an almost complete lack of acinar cells, which normally comprise the majority of the organ. ß-Catenin appears to be cell-autonomously required for the specification of acinar cells, rather than for their survival or maintenance; deletion of ß-catenin specifically in differentiated acinar cells has no effect. Thus, these data are consistent with a crucial role for canonical Wnt signals in acinar lineage specification and differentiation (Murtaugh, 2005).
The mouse embryonic axis is initially formed with a proximal-distal
orientation followed by subsequent conversion to a prospective
anterior-posterior (A-P) polarity with directional migration of visceral
endoderm cells. Importantly, Otx2, a homeobox gene, is essential to this
developmental process. However, the genetic regulatory mechanism governing axis
conversion is poorly understood. Defective axis conversion due to Otx2
deficiency can be shown to be rescued by expression of Dkk1, a Wnt
antagonist, or following removal of one copy of the β-catenin gene.
Misexpression of a canonical Wnt ligand can also inhibit correct A-P axis
rotation. Moreover, asymmetrical distribution of β-catenin localization is
impaired in the Otx2-deficient and Wnt-misexpressing visceral endoderm.
Concurrently, canonical Wnt and Dkk1 function as repulsive and attractive
guidance cues, respectively, in the migration of visceral endoderm cells. It is
proposed that Wnt/β-catenin signaling mediates A-P axis polarization by guiding
cell migration toward the prospective anterior in the pregastrula mouse embryo
(Kimura-Yoshida, 2005).
This study indicates that localization of the dephosphorylated form of
β-catenin is dynamically regulated during A-P axis specification. In the
wild-type visceral endoderm layer, cytoplasmic and nuclear β-catenin expression
are specifically reduced in the prospective anterior side. Notably, in both
Otx2-deficient and Tg(CAG-mWnt8A) embryos, which display failure
of axis rotation, the expression is not downregulated; rather, it is upregulated
throughout the entire visceral endoderm layer. Although further molecular
analysis is necessary in order to elucidate the precise molecular mechanism by
which Dkk1 expression is initially induced in the most proximal portion
of DVE and subsequently downregulated in the prospective posterior side,
Otx2 expression is crucial for Dkk1 expression in the visceral
endoderm. In addition, Dkk1 alone can rescue axis rotation failure
attributable to Otx2 deficiency. These findings suggest that Otx2
specifies A-P axis development primarily via regulation of Wnt/β-catenin
signaling pathways, including Dkk1, in the visceral endoderm
(Kimura-Yoshida, 2005).
Surprisingly, mWnt8A transcripts driven by the CAG promoter are
upregulated primarily in the epiblast, but not in the visceral endoderm, whereas
expression of the dephosphorylated form of β-catenin is not elevated in the
epiblast layer of Tg(CAG-mWnt8A) embryos. This finding suggests the
involvement of unexpected molecular mechanisms via which Wnt signaling can be
transmitted to β-catenin activity mainly in the visceral endoderm, but not in
the epiblast layer (Kimura-Yoshida, 2005).
This genetic evidence affords novel insights into evolutionarily conserved
mechanisms governing primary body axis formation across the metazoans. The
asymmetrical distribution of β-catenin activity along with the A-P axis plays a
pivotal role in the specification of A-P polarity throughout metazoan embryos.
In amphibians, fish, ascidians, sea urchins, and cnidarians, β-catenin is
localized to cell nuclei preferentially at one pole of the cleavage-stage
embryo. In these various organisms, nuclear activity of β-catenin is
required for early axis specification and the subsequent establishment of
critical signaling centers, 'organizers', in the early embryo. The present
investigation suggests that asymmetrical distribution of β-catenin expression
serves as a primary mediator of axis specification in the mammalian embryo
(Kimura-Yoshida, 2005).
During regional patterning of the anterior neural plate, a medially positioned domain of cells is specified to adopt retinal identity. These eye field cells remain coherent as they undergo morphogenetic events distinct from other prospective forebrain domains. Two branches of the Wnt signaling pathway coordinate cell fate determination with cell behavior during eye field formation. Wnt/ß-catenin signaling antagonizes eye specification through the activity of Wnt8b and Fz8a. In contrast, Wnt11 and Fz5 promote eye field development, at least in part, through local antagonism of Wnt/ß-catenin signaling. Additionally, Wnt11 regulates the behavior of eye field cells, promoting their cohesion. Together, these results suggest a model in which Wnt11 and Fz5 signaling promotes early eye development through the coordinated antagonism of signals that suppress retinal identity and promotion of coherence of eye field cells (Cavodeassi, 2005).
These data add to the body of evidence that Wnt/β-catenin signaling regulates the regionalization of the forebrain. Overactivation of Wnt/β-catenin signaling promotes posterior diencephalic fates and suppresses anterior telencephalic and eye field identities. It is further shown that local suppression of Wnt/β-catenin signaling can expand eye field markers caudally into the posterior diencephalon. There are at least three Wnts potentially involved in this process: Wnt1, Wnt10b, and Wnt8b. However, a number of results argue in favor of Wnt8b being the one most likely involved in the regionalization of the forebrain. While wnt8b is expressed in the posterior diencephalon, wnt1 and wnt10b are expressed more posteriorly. Moreover, wnt1/wnt10b double mutants/morphants do not show an obvious patterning defect in the forebrain, and the slight posterior expansion of the eye field found in wnt8b morphants is not significantly enhanced in the wnt8b/wnt10b/wnt1 triple morphants (Cavodeassi, 2005).
The results strengthen the hypothesis that Fz8a is the receptor responsible for transducing the Wnt8b signal. fz8a is expressed in a broad domain within the ANP, consistent with the entire prospective forebrain being susceptible to reception of Wnt8b signals in a graded posterior/high to anterior/low fashion. Still, it is unclear whether Wnts can exert their action at a distance or can act only locally. A scenario is favored in which Wnt8b would be working as a short-range signal, since Wnt8b is required for the formation of diencephalon and midbrain, the main territories where it is expressed, and to establish the posterior boundary of the eye field, which is located no more than a few cell rows away from the anterior boundary of the wnt8b domain. Specification of the eye field more anteriorly requires local suppression of Wnt/β-catenin signaling, but as yet, there is no evidence that Wnts signaling through the β-catenin branch of the pathway significantly encroach throughout the eye field during gastrula stages of normal development (Cavodeassi, 2005).
Similar to the eye field, induction of the telencephalon also requires suppression of Wnt/β-catenin signals. What, then, might specify the difference between eye field and telencephalon? Slight differences in the level of Wnt signaling may be enough to effect the separation of these two territories. Alternatively, additional signals, such as those coming from the margin of the neural plate, may also be required for this patterning process. For instance, early-acting BMP signals promote telencephalic gene expression, but can suppress specification of eye field gene expression (Cavodeassi, 2005).
During formation of the eye, nascent eye field cells must be specified to acquire eye field identity and must undergo a program of morphogenesis quite distinct from that of adjacent forebrain territories. This study shows that a noncanonical Wnt pathway activated by Wnt11 in the eye field helps to coordinate these two events. Wnt11 function may direct the morphogenesis of the eye field by maintaining the coherence of this territory. Simultaneously, noncanonical Wnt activity would consolidate the extent of the territory defined as eye field by keeping it refractory to any residual Wnt/β-catenin signals encroaching from more posterior domains. Thus, through the coordinated antagonism of signals that suppress retinal identity and promotion of cell coherence, Wnt11 and Fz5 signaling would link induction and morphogenesis during the early stages of eye development (Cavodeassi, 2005).
The molecular mechanisms by which liver genes are differentially expressed along a portocentral axis, allowing for metabolic zonation, are poorly understood. Evidence is provided that the Wnt/ß-catenin pathway plays a key role in liver zonation. First, the complementary localization of activated ß-catenin in the perivenous area and the negative regulator Apc in periportal hepatocytes is demonstrated. The immediate consequences are shown of either a liver-inducible Apc disruption or a blockade of Wnt signaling after infection with an adenovirus encoding Dkk1; Wnt/ß-catenin signaling inversely controls the perivenous and periportal genetic programs. Genes involved in the periportal urea cycle and the perivenous glutamine synthesis systems are critical targets of ß-catenin signaling, and perturbations to ammonia metabolism are likely responsible for the death of mice with liver-targeted Apc loss. From these results, it is proposed that Apc is the liver 'zonation-keeper' gene (Benhamouche, 2006).
Wnt signaling orchestrates multiple aspects of central nervous system
development, including cell proliferation and cell fate choices. In this
study, gene transfer was used to activate or inhibit canonical Wnt signaling in
vivo in the developing eye. The expression of Wnt2b or constitutively active (CA) ß-catenin inhibited retinal progenitor gene (RPG) expression and the differentiation of retinal neurons. In addition, Wnt signal activation in the central retina is sufficient to induce the expression of markers of the ciliary body and iris, two tissues derived from the peripheral optic cup (OC). The expression of a dominant-negative (DN) allele of Lef1, or of a Lef1-engrailed fusion protein, leads to the inhibition of expression of peripheral genes and iris hypoplasia, suggesting that canonical Wnt signaling is required for peripheral eye development. It is proposed that canonical Wnt signaling in the developing optic vesicle (OV) and OC plays a crucial role in determining the identity of the ciliary body and iris.
Because wingless (wg) plays a similar role in the induction
of peripheral eye tissues of Drosophila, these findings indicate a
possible conservation of the process that patterns the photoreceptive and
support structures of the eye (Cho, 2006).
These findings provide an additional link between the development of the
vertebrate and invertebrate eye. In Drosophila, photoreceptor cells
are surrounded at the periphery with a non-neural cuticular structure.
wg, the Drosophila homolog of the Wnt genes, is expressed in
the margin of the eye imaginal disc, which is the anlage of peripheral eye
tissues. Activation of wg, or armadillo, the Drosophila ß-catenin, in the eye imaginal disc promotes head cuticle formation at the expense of ommatidia, and has been proposed to act as a morphogen to pattern the peripheral structures (Cho, 2006).
Wnt signaling thus promotes the development of the non-neural, peripheral
support structures in both Drosophila and chicks. The similarity of
wg/Wnt expression and function in eye development provides an
additional line of evidence that strengthens the proposed evolutionary
conservation of the vertebrate and invertebrate eyes. The modern version of
this model originated with the observation of a conserved expression and
activity for the eyeless/Pax6 gene. The fact that wg/Wnt appears to play a role in patterning the central and peripheral eye structures suggests that the visual structure of the last common ancestor of flies and vertebrates had not only a photoreceptive component, but a support structure as well. A conserved unit of neural and non-neural eye tissues has also been suggested by the observation of a
single-celled dinoflagellate that has several of the support
structures of an eye, including pigment, a lens, a cornea and a photoreceptor. The fact that Pax6 plays a role in the development of not only the NR, but also the
supporting tissues, such as the lens, cornea, iris and RPE, might also be seen
as being in keeping with this model (Cho, 2006).
Recent studies have demonstrated that the LIM homeodomain transcription factor Islet1 (Isl1) marks pluripotent cardiovascular progenitor cells and is required for proliferation, survival, and migration of recently defined second heart field progenitors. Factors that are upstream of Isl1 in cardiovascular progenitors have not yet been defined. This study demonstrates that β-catenin is required for Isl1 expression in cardiac progenitors, directly regulating the Isl1 promoter. Ablation of β-catenin in Isl1-expressing progenitors disrupts multiple aspects of cardiogenesis, resulting in embryonic lethality at E13. β-Catenin is also required upstream of a number of genes required for pharyngeal arch, outflow tract, and/or atrial septal morphogenesis, including Tbx2, Tbx3, Wnt11, Shh, and Pitx2. These findings demonstrate that β-catenin signaling regulates proliferation and survival of cardiac progenitors (Lin, 2007).
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).
In the embryonic kidney, progenitors in the metanephric mesenchyme differentiate into specialized renal epithelia in a defined sequence characterized by the formation of cellular aggregates, conversion into polarized epithelia and segmentation along a proximal-distal axis. This sequence is reiterated throughout renal development to generate nephrons. This study identified global transcriptional programs associated with epithelial differentiation utilizing an organ culture model of rat metanephric mesenchymal differentiation, which recapitulates the hallmarks of epithelialization in vivo in a synchronized rather than reiterative fashion. Activation of multiple putative targets of β-catenin/TCF/Lef-dependent transcription were observed coinciding with epithelial differentiation. It was shown, in cultured explants, that isolated activation of β-catenin signaling in epithelial progenitors induces, in a TCF/Lef-dependent manner, a subset of the transcripts associated with epithelialization, including Pax8, cyclin D1 (Ccnd1) and Emx2. This is associated with anti-apoptotic and proliferative effects in epithelial progenitors, whereas cells with impaired TCF/Lef-dependent transcription are progressively depleted from the epithelial lineage. In vivo, TCF/Lef-responsive genes comprise a conserved transcriptional program in differentiating renal epithelial progenitors and β-catenin-containing transcriptional complexes directly bind to their promoter regions. Thus, β-catenin/TCF/Lef-mediated transcriptional events control a subset of the differentiation-associated transcriptional program and thereby participate in maintenance, expansion and stage progression of the epithelial lineage (Schmidt-Ott, 2007).
The activity of keratinocytes in the hair follicle is regulated by signals from a specialized mesenchymal niche at the base of the follicle, the dermal papilla (DP). In this study, mice expressing cre recombinase in the DP were developed to probe the interaction between follicular keratinocytes and the DP in vivo. Inactivation of theβ-catenin gene within DP of fully developed hair follicles results in dramatically reduced proliferation of the progenitors and their progeny that generate the hair shaft, and, subsequently, premature induction of the destructive phase of the hair cycle. It also prevents regeneration of the cycling follicle from stem cells. Gene expression analysis reveals that β-catenin activity in the DP regulates signaling pathways, including FGF and IGF, that can mediate the DP's inductive effects. This study reveals a signaling loop that employs Wnt/β-catenin signaling in both epithelial progenitor cells and their mesenchymal niche to govern and coordinate the interactions between these compartments to guide hair morphogenesis (Enshell-Seijffers, 2010).
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).
DiGeorge syndrome (DGS) is a common genetic disease characterized by pharyngeal apparatus malformations and defects in cardiovascular, craniofacial and glandular development. TBX1 is the most likely candidate disease-causing gene and is located within a 22q11.2 chromosomal deletion that is associated with most cases of DGS. Thus study shows that canonical Wnt-beta-catenin signaling negatively regulates Tbx1 expression and that mesenchymal inactivation of beta-catenin (Ctnnb1) in mice caused abnormalities within the DGS phenotypic spectrum, including great vessel malformations, hypoplastic pulmonary and aortic arch arteries, cardiac malformations, micrognathia, thymus hypoplasia and mislocalization of the parathyroid gland. In a heterozygous Fgf8 or Tbx1 genetic background, ectopic activation of Wnt-beta-catenin signaling caused an increased incidence and severity of DGS-like phenotypes. Additionally, reducing the gene dosage of Fgf8 rescued pharyngeal arch artery defects caused by loss of Ctnnb1. These findings identify Wnt-beta-catenin signaling as a crucial upstream regulator of a Tbx1-Fgf8 signaling pathway and suggest that factors that affect Wnt-beta-catenin signaling could modify the incidence and severity of DGS (Huh, 2010).
The Wnt/beta-catenin pathway is evolutionary conserved signaling system that regulates cell differentiation and organogenesis. Endothelial specific stabilization of Wnt/beta-catenin signaling alters early vascular development in the embryo. The phenotype resembles that induced by upregulation of Notch signaling, including lack of vascular remodeling, altered elongation of the intersomitic vessels, defects in branching, and loss of venous identity. Both in vivo and in vitro data show that beta-catenin upregulates Dll4 transcription and strongly increases Notch signaling in the endothelium, leading to functional and morphological alterations. The functional consequences of beta-catenin signaling depend on the stage of vascular development and are lost when a gain-of-function mutation is induced at a late stage of development or postnatally. These findings establish a link between Wnt and Notch signaling in vascular development. It is proposed that early and sustained beta-catenin signaling prevents correct endothelial cell differentiation, altering vascular remodeling and arteriovenous specification (Corada, 2010).
By conditional gene ablation in mice, ß-catenin, an essential downstream effector of canonical Wnt signaling, was found to be a key regulator of formation of the apical ectodermal ridge (AER) and of the dorsal-ventral axis of the limbs. By generation of compound mutants, ß-catenin was also shown to act downstream of the BMP receptor IA in AER induction, but upstream or parallel to the BMP receptor in dorsal-ventral patterning. Thus, AER formation and dorsal-ventral patterning of limbs are tightly controlled by an intricate interplay between Wnt/ß-catenin and BMP receptor signaling (Soshnikova, 2003).
The Wnt/ß-catenin and TGFß/BMP-signaling pathways coordinately govern many developmental processes. During limb development, Wnt and BMP signals control the formation of the AER and participate in the establishment of the dorsal-ventral axis. The interactions between the two signaling systems in the limb were, however, not understood, and the epistatic relationship between BMP and Wnt signals remained unclear. This study has analyzed the interactions between
Wnt/ß-catenin and BMP receptor signaling during limb development using
conditional mutagenesis, which allows introduction of loss-of-function and
gain-of-function mutations of ß-catenin, the central and essential mediator of canonical Wnt signaling. In addition, compound mutant mice were generated that carry both a gain-of-function mutation in ß-catenin and loss-of-function mutations in Bmp receptor IA. Analysis of these compound
Brn4Cre;DeltaN-ß-catenin: BmpRIAflox/flox mutant mice clearly demonstrates that ß-catenin acts downstream of the BMP receptor IA in AER induction. ß-Catenin-mediated signals do, however, control Bmp4 expression in the ectoderm, and are thus responsible for the formation of a positive feedback loop. In contrast, the data suggest that ß-catenin acts upstream of or in parallel to the BMP receptor IA during dorsal-ventral patterning. These intricate interactions between the Wnt/ß-catenin and BMP-signaling pathways provide the molecular basis that connects the development of proximal-distal and dorsal-ventral axes in the limb, and might thus ensure a tight spatial-temporal control of signaling responses (Soshnikova, 2003).
The cellular and molecular bases allowing tissue regeneration are not well understood. By performing gain- and loss-of-function experiments of specific members of the Wnt pathway during appendage regeneration, it has been demonstrated that this pathway is not only necessary for regeneration to occur, but it is also able to promote regeneration in axolotl, Xenopus, and zebrafish. Furthermore, it has been shown that changes in the spatiotemporal distribution of β-catenin in the developing chick embryo elicit apical ectodermal ridge and limb regeneration in an organism previously thought not to regenerate. The detailed mechanism by which Wnt overexpression in ectodermal cells adjacent to the amputation entails both AER regeneration in the chick, as well as AEC formation in the axolotl/zebrafish/Xenopus, remains to be elucidated. Perhaps, and more importantly, some of the results discussed in this paper -- specifically (1) the observations that changes in Wnt and BMP activities during limb/fin regeneration-limb development induced alterations in the formation of the AEC-AER that are related to spatiotemporal deregulation of p63, and (2) the accomplishment of AER regeneration and subsequent limb development in an embryo not previously shown to have this capability -- support the notion that variations in the concentration and/or spatiotemporal distribution of molecules involved in tissue generation during embryogenesis may be the raw material upon which evolution has granted some animals the ability to regenerate. Understanding the mechanisms responsible for the deployment and fine-tuning of developmental regulators might constitute the basis for inducing tissue regeneration in adult nonregenerating animals (Kawakami, 2006).
Vertebrate muscle arises sequentially from embryonic, fetal, and adult myoblasts. Although functionally distinct, it is unclear whether these myoblast classes develop from common or different progenitors. Pax3 and Pax7 are expressed by somitic myogenic progenitors and are critical myogenic determinants. To test the developmental origin of embryonic and fetal myogenic cells in the limb, Pax3+ and Pax7+ cells were genetically labeled and ablated. Pax3+Pax7- cells contribute to muscle and endothelium, establish and are required for embryonic myogenesis, and give rise to Pax7+ cells. Subsequently, Pax7+ cells give rise to and are required for fetal myogenesis. Thus, Pax3+ and Pax7+ cells contribute differentially to embryonic and fetal limb myogenesis. To investigate whether embryonic and fetal limb myogenic cells have different genetic requirements beta-catenin, an important regulator of myogenesis, was conditionally inactivated or activated in Pax3- or Pax7-derived cells. β-Catenin is necessary within the somite for dermomyotome and myotome formation and delamination of limb myogenic progenitors. In the limb, beta-catenin is not required for embryonic myoblast specification or myofiber differentiation but is critical for determining fetal progenitor number and myofiber number and type. Together, these studies demonstrate that limb embryonic and fetal myogenic cells develop from distinct, but related progenitors and have different cell-autonomous requirements for beta-catenin (Hutcheson, 2009).
Therefore lineage analysis reveals that Pax3+ somitic cells in the limb contribute to both muscle and endothelial cell lineages. Transplantation studies had established that somitic cells contribute to both limb muscle and endothelial cells, and chick lineage studies showed that even single somitic cells are bipotential, contributing to muscle and endothelium. This study shows that Pax3+ somitic cells are bipotential in the limb. Thus, even though Pax3 is a member of the genetic cascade committing cells to myogenesis, in vivo expression of Pax3 in somitic cells is not sufficient to commit these cells to a myogenic fate (Hutcheson, 2009).
It was also found that Pax7+ cells are a subset of Pax3+ somitic cells and only give rise to myogenic, and not endothelial cells. Ablation studies demonstrate that the Pax3 lineage is required for the emergence of Pax7+ cells, even though Pax3 function is not required for the specification of Pax7+ cells (Pax7+ cells are present in axial muscles of Pax3-/- mice). Subsequently, Pax7 somitic derivatives appear to be restricted, and therefore potentially committed, to the muscle lineage. Whether the expression of Pax7 itself commits Pax3+ cells to myogenesis or simply marks committed cells is unclear. Pax7 regulates Myf5 expression, and myogenesis (with the exception of the primary myotome) requires either Pax3 or Pax7 expression. Recently Pax7 has been shown to be more stable than Pax3, which is subject to monoubiquitination and proteasomal degradation. An attractive hypothesis is that Pax3 expression initially establishes intermediate, bipotential precursors. Given the right extrinsic cues, some of these Pax3+ cells differentiate into muscle (or endothelium). However, Pax3+ cells that do not differentiate subsequently express the more stable Pax7, committing these cells to a myogenic fate via Pax7 activation of Myf5 expression (Hutcheson, 2009).
The transcriptional basis of vertebrate limb initiation, which is a well-studied system for the initiation of organogenesis, remains elusive. Specifically, involvement of the β-catenin pathway in limb initiation, as well as its role in hindlimb-specific transcriptional regulation, are under debate. This study shows that the β-catenin pathway is active in the limb-forming area in mouse embryos. Furthermore, conditional inactivation of β-catenin as well as Islet1, a hindlimb-specific factor, in the lateral plate mesoderm results in a failure to induce hindlimb outgrowth. It was further shown that Islet1 is required for the nuclear accumulation of β-catenin and hence for activation of the β-catenin pathway, and that the β-catenin pathway maintains Islet1 expression. These two factors influence each other and function upstream of active proliferation of hindlimb progenitors in the lateral plate mesoderm and the expression of a common factor, Fgf10. These data demonstrate that Islet1 and β-catenin regulate outgrowth and Fgf10-Fgf8 feedback loop formation during vertebrate hindlimb initiation. This study identifies Islet1 as a hindlimb-specific transcriptional regulator of initiation, and clarifies the controversy regarding the requirement of β-catenin for limb initiation (Kawakami, 2011).
The progressive loss of CNS myelin in patients with multiple sclerosis (MS) has been proposed to result from the combined effects of damage to oligodendrocytes and failure of remyelination. A common feature of demyelinated lesions is the presence of oligodendrocyte precursors (OLPs) blocked at a premyelinating stage. However, the mechanistic basis for inhibition of myelin repair is incompletely understood. To identify novel regulators of OLP differentiation, potentially dysregulated during repair, a genome-wide screen of 1040 transcription factor-encoding genes expressed in remyelinating rodent lesions was performed. Approximately 50 transcription factor-encoding genes show dynamic expression during repair, and expression of the Wnt pathway mediator Tcf4 (aka Tcf7l2) within OLPs is specific to lesioned-but not normal-adult white matter. Beta-catenin signaling is active during oligodendrocyte development and remyelination in vivo. Moreover, similar regulation is observed of Tcf4 in the developing human CNS and lesions of MS. Data mining revealed elevated levels of Wnt pathway mRNA transcripts and proteins within MS lesions, indicating activation of the pathway in this pathological context. Dysregulation of Wnt-beta-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination, based on (1) conditional activation of beta-catenin in the oligodendrocyte lineage in vivo and (2) findings from APC(Min) mice, which lack one functional copy of the endogenous Wnt pathway inhibitor APC. Together, these findings indicate that dysregulated Wnt-beta-catenin signaling inhibits myelination/remyelination in the mammalian CNS. Evidence of Wnt pathway activity in human MS lesions suggests that its dysregulation might contribute to inefficient myelin repair in human neurological disorders (Fancy, 2009).
Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a murine hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, a compound genetic model was generated bearing both Pten deletion and β-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of β-catenin alone results in apoptosis of HSCs. However, together, the Wnt/β-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, β-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. A pharmacological approach was developed to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/β-catenin and PI3K/Akt signaling simultaneously. Activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity (Perry, 2011).
The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of ß-catenin to the proteasome. In fact, sustained activation of ß-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. The mechanisms regulating the balance between ß-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes has been investigated. Specifically, it has been shown that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of ß-catenin, which depends on glycogen synthase kinase (GSK) 3ß activity. Mutation of serine 37 (a target of GSK3ß) to an alanine renders ß-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3ß phosphorylation-defective S37A-ß-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-ß-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant ß-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-ß-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKß is a key regulator of the balance between ß-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes (Liu, 2004).
The identity of distinct cell types in the ventral neural tube is generally believed to be specified by sonic hedgehog (Shh) in a concentration-dependent manner. However, recent studies have questioned whether Shh is the sole signaling molecule determining ventral neuronal cell fates. This study provides evidence that canonical Wnt signaling is involved in the generation of different cell types in the ventral spinal cord. Wnt signaling is active in the mouse ventral spinal cord at the time when ventral cell types are specified. Furthermore, using an approach that stabilizes β-catenin protein in small patches of ventral spinal cord cells at different stages, this study shows that Wnt signaling activates different subsets of target genes depending on the time when Wnt signaling is amplified. Moreover, disruption of Wnt signaling results in the expansion of ventrally located progenitors. Finally, this study shows genetically that Wnt signaling interacts with Hh signaling at least in part through regulating the transcription of Gli3. These results reveal a novel mechanism by which ventral patterning is achieved through a coordination of Wnt and Shh signaling (Yu, 2008).
At least three different mechanisms could account for the switching of
ventral progenitor cell fates in response to Wnt signaling. First, the
switching of cell fate is dependent on Wnt signal strength. In this scenario,
a strong Wnt signal induces dorsal cell types, whereas a weak signal induces
ventral cell types. However, because different cell types were induced by
stabilized β-catenin, Wnt signal strength, although possible, is unlikely
to play a crucial role in cell fate determination. The second possibility is
that cell fate switching is dependent on the duration of the Wnt signal. For
example, the longer that Wnt signaling is maintained in a cell, the more
likely it is that the cell will adopt a dorsal cell fate, similar to a
mechanism that has been proposed for Shh action. In this
scenario, the dorsal-most cell type, d1, is specified because these cells
receive the longest exposure to Wnt signaling (from E8.5 to E10.5), whereas
other cells adopt more-ventral cell fates because they receive shorter
exposure to Wnt signaling. However, extending the length of Wnt signaling does
not appear to alter the expression of dorsal markers. Lastly, it is possible
that different cell fates are specified depending on when Wnt signaling is
active. In this scenario, Wnt signaling is capable of activating different
genes at different time points, based on the changing competence of the cells.
Indeed, it was found that early Wnt signaling (induced with TM at E7.5) activated
the expression of several dorsal markers, Pax7, Gsh1/2 and Msx1/2. By
contrast, Wnt signaling induced with TM at E8.5 could only induce the
expression of Msx1/2, and Wnt signaling induced subsequently did not activate
dorsal markers. The results therefore strongly support the time-dependent
mechanism of Wnt signaling in the ventral spinal cord (Yu, 2008).
Only ~70%-90% of ectopic Msx1/2+ or Pax7+ cells
coexpressed TCF/LEF-lacZ. However, this is likely to be an underestimate because the TCF/LEF-lacZ transgenic reporter was not expressed in all E10.5 progenitors that expressed stabilized β-catenin. The action of Wnt signaling on cell fate changes is likely to be cell-autonomous. However,
non-autonomous effects cannot be completely excluded, particularly in light of the reduction in the Irx3 ventral expression domain, which lies outside of the Olig1-Cre expression domain, in β-catenin mutant embryos. Nevertheless, no upregulation of
phosphorylated Smad1/5/8 was observed in embryos expressing stabilized β-catenin, suggesting that BMP pathways were
not activated in response to stabilized β-catenin (Yu, 2008).
Although the removal of β-catenin using Olig1-Cre affects the
morphology of the floor plate at E10.5, the action of Wnt signaling on cell
type switching appears to be direct, based on the following observations.
First, there was no significant change in the level of Shh protein, the number
of Shh-expressing cells or in the response to Shh at E9.5, when cell fate is
being specified. Even at E10.5, when most of the cells have been specified, no significant changes in the expression of Gli2, Shh, Ptch1 or Gli1 were observed, although the floor plate appeared to be less compact. In fact, deletion of floor plate does not affect the generation of most ventral neurons, except for V3 cells, as has been demonstrated in Gli2 mutants. Lastly, activation of Wnt signaling in small patches of cells using Gli1-CreER reveals that activation of Wnt signaling directly affects cell fate (Yu, 2008).
ß-Catenin is a central component of both the cadherin-catenin
cell adhesion complex and the Wnt signaling
pathway. The role of ß-catenin during
brain morphogenesis has been investigated, by specifically inactivating the ß-catenin gene in the region of Wnt1 expression. To achieve
this, mice with a conditional ('floxed') allele of ß-catenin
with required exons flanked by loxP recombination
sequences were intercrossed with transgenic mice that
expressed Cre recombinase under control of Wnt1
regulatory sequences. ß-catenin gene deletion results in
dramatic brain malformation and failure of craniofacial
development. Absence of part of the midbrain and all of
the cerebellum is reminiscent of the conventional Wnt1
knockout (Wnt1-/-), suggesting that Wnt1 acts through ß-catenin
in controlling midbrain-hindbrain development.
The craniofacial phenotype, not observed in embryos that
lack Wnt1, indicates a role for ß-catenin in the fate of
neural crest cells. Analysis of neural tube explants shows
that ß-catenin is efficiently deleted in migrating neural
crest cell precursors. This, together with an increased
apoptosis in cells migrating to the cranial ganglia and in
areas of prechondrogenic condensations, suggests that
removal of ß-catenin affects neural crest cell survival
and/or differentiation. These results demonstrate the pivotal
role of ß-catenin in morphogenetic processes during brain
and craniofacial development (Brault, 2001).
Neural precursor cells (NPCs) have the ability to self-renew and to give rise to neuronal and glial lineages. The fate decision of NPCs between proliferation and differentiation determines the number of differentiated cells and the size of each region of the brain. However, the signals that regulate the timing of neuronal differentiation remain unclear. Wnt signaling is shown to inhibit the self-renewal capacity of mouse cortical NPCs, and instructively promotes their neuronal differentiation. Overexpression of Wnt7a or of a stabilized form of ß-catenin in mouse cortical NPC cultures induces neuronal differentiation even in the presence of Fgf2, a self-renewal-promoting factor in this system. Moreover, blockade of Wnt signaling leads to inhibition of neuronal differentiation of cortical NPCs in vitro and in the developing mouse neocortex. Furthermore, the ß-catenin/TCF complex appears to directly regulate the promoter of neurogenin 1, a gene implicated in cortical neuronal differentiation. Importantly, stabilized ß-catenin does not induce neuronal differentiation of cortical NPCs at earlier developmental stages, consistent with previous reports indicating self-renewal-promoting functions of Wnts in early NPCs. These findings may reveal broader and stage-specific physiological roles of Wnt signaling during neural development (Hirabayashi, 2004).
The mechanism by which Wnt signaling regulates
neurogenesis was investigated. Since the results implicated the ß-catenin/TCF complex in
neuronal differentiation, a proneural gene was sought that might be under
the control of these transcription factors. One such candidate is the bHLH
transcription factor Ngn1, because this gene is expressed during early
neurogenesis in the neocortex, and its expression, together with that of the Ngn2 gene, is essential for development of the neocortex. A consensus sequence for TCF binding was found located at nucleotide (nt) positions -1167 to -1160 relative to the
transcription start site of the mouse Ngn1 gene. This region within
the promoter has been shown to be responsible for expression of the gene in
the dorsal neocortex during neurogenesis. To
determine whether this TCF binding element is functional, the
activities of the Ngn1 gene promoter (nt -2670 to +74) containing
either an intact or mutated version of this DNA sequence were compared. Cultured NPCs were
transfected with a luciferase reporter construct under the control of the
wild-type or mutant Ngn1 gene promoter. The
transcriptional activity of the mutant promoter was markedly reduced compared with that of the wild-type (Hirabayashi, 2004).
Next, a ChIP assay was used to examine whether endogenous ß-catenin was associated with the Ngn1 gene promoter in cultured NPCs. Lysates of
cultured NPCs were subjected to shearing of genomic chromatin followed by
immunoprecipitation with antibodies to ß-catenin. Polymerase chain
reaction (PCR) analysis with primers targeted to the TCF binding element of the Ngn1 gene promoter revealed the presence of this element in the immunoprecipitates.
The level of Ngn1 mRNA was determined by reverse transcription (RT)-PCR in
cultured NPCs. Expression of S33Y ß-catenin markedly increased the level
of Ngn1 mRNA but not that of the control glyceraldehydes-3-phosphate
dehydrogenase (Gapdh) mRNA, suggesting that transcription of the Ngn1 gene is indeed under the control of the canonical Wnt pathway. Together, these results suggest that the ß-catenin/TCF complex directly regulates transcription of the Ngn1 gene during neuronal differentiation of cortical NPCs (Hirabayashi, 2004).
The results clearly indicate that stabilized ß-catenin instructs
neuronal differentiation of cortical NPCs prepared from mouse E11.5 neocortex and cultured for 3 days. However, it has been
shown that ectopic expression of stabilized ß-catenin by the nestin
enhancer results in the expansion of NPC cell number and suppression of cell cycle exit. This difference might be due to the timing at which stabilized ß-catenin was expressed, since the nestin enhancer is known to become active
at around E8.5. To test this idea, the effects were compared of ß-catenin on NPCs prepared from different stages of mouse neocortex development.
Expression of stabilized ß-catenin increased the population of
TuJ1+ cells in NPCs prepared from E13.5 neocortex, but reduced
somewhat the population of TuJ1+ cells among neuroepithelial cells acutely prepared from E10.5 neocortex. This suggests that the response of NPCs to the canonical Wnt
pathway depends on the stage of neural development (Hirabayashi, 2004).
Recent experiments have suggested that Wnt signaling has the capacity to promote self-renewal in various tissue stem cells including neural stem cells and hematopoietic stem cells. In the central nervous system, cells located in the
midbrain or hippocampus are deleted in mice deficient in Wnt1 or Wnt3a,
respectively. Mice lacking both Wnt1 and Wnt3a also manifest a reduction
in the size of the caudal midbrain, rostral hindbrain, cranial and spinal
ganglia, and dorsal neural tube. Furthermore, ectopic expression of Wnt1 or stabilized ß-catenin was shown to lead to a net increase in the size of the precursor pool in the chick spinal cord, in part through transcriptional regulation of cyclinD, and infection of cortical explants with Wnt7aHA-expressing retrovirus induced expansion of neuronal precursors which accompanied expression of the Egf receptor.
Consistently, transgenic mice expressing stabilized ß-catenin in NPCs
under the control of the nestin enhancer or Brn4 promoter also exhibited
overgrowth of the brain and spinal cord, reflecting an expansion of the
precursor population without alteration of the primary patterning of cell
identities. By contrast, in the present study, activation of the
canonical Wnt pathway reduced the size of the precursor pool and promoted
neuronal differentiation in the developing neocortex. It is speculated that this difference might be attributable to differences in the developmental stage of the NPCs. Indeed, activation of the canonical Wnt pathway promoted neuronal differentiation of NPCs derived from E13.5 embryos, but not those acutely dissected from E10.5 embryos. It is possible that the chromatin region encompassing regulatory elements of genes crucial for neuronal differentiation (such as Ngn1) undergoes a change during development from a closed to an open state (Hirabayashi, 2004).
Nodal activity in the left lateral plate mesoderm (LPM) is required to activate left-sided Nodal signaling in the epithalamic region of the zebrafish forebrain. Epithalamic Nodal signaling subsequently determines the laterality of neuroanatomical asymmetries. Overactivation of Wnt/Axin1/β-catenin signaling during late gastrulation leads to bilateral epithalamic expression of Nodal pathway genes independently of LPM Nodal signaling. This is consistent with a model whereby epithalamic Nodal signaling is normally bilaterally repressed, with Nodal signaling from the LPM unilaterally alleviating repression. It is suggested that Wnt signaling regulates the establishment of the bilateral repression. A second role was identified for the Wnt pathway in the left/right regulation of LPM Nodal pathway gene expression, and finally, it was shown that at later stages Axin1 is required for the elaboration of concordant neuroanatomical asymmetries (Carl, 2007).
Structural and functional asymmetries are common features of the nervous systems of both invertebrates and vertebrates. The best described neuroanatomical asymmetries in vertebrates are found in the diencephalic epithalamus, where both the habenulae and the dorsally adjacent pineal complex are lateralized in many species. The epithalamus is part of a conserved output pathway of the limbic system, connecting telencephalic nuclei to the interpeduncular nucleus (IPN) in the ventral midbrain (Carl, 2007).
During early development in zebrafish, bilaterally located parapineal cells migrate leftward from the pineal complex to form a left-sided nucleus that sends ipsilateral axonal projections to the left habenula. The paired habenular nuclei themselves show various asymmetries, including differences in gene expression, subnuclear regionalization, timing of neuronal differentiation, and neuropil organization. Left-right asymmetries in habenular neuronal organization are converted into a dorsal-ventral asymmetry in the targeting of the habenular axons in the midbrain IPN, with left-sided habenular axons predominantly innervating the dorsal IPN and right-sided axons projecting to the ventral IPN (Carl, 2007).
The parapineal influences the elaboration of habenular asymmetries. For instance, the parapineal modulates gene expression in the left habenula, and ablation of parapineal cells results in the left habenula adopting some right-sided character. In contrast, ablation of left-sided habenula precursors can influence the orientation of parapineal migration. Taken together, these results suggest that there is communication between the various structures in the epithalamus to ensure coordinated and consistent elaboration of lateralized neuroanatomical asymmetries (Carl, 2007).
The earliest known indication of brain asymmetry in zebrafish is the expression of Nodal pathway genes within the left epithalamus from about 18 hpf. Epithalamic Nodal signaling influences the laterality of the habenulae and parapineal, but asymmetry per se appears to be established independently of this pathway. As the Nodal pathway is activated unilaterally in the epitahalmus, other mechanisms must act upstream to initiate this asymmetry. Within the lateral plate mesoderm (LPM), Nodal signaling has evolutionarily conserved roles in the development of asymmetries, and in zebrafish, it appears that activation of Nodal pathway genes in the left epithalamus is dependent upon the activity of the Nodal ligand Southpaw (Spw) emanating from the left LPM. Whether this activity of Spw is direct or indirect is unknown. It has been proposed that the role of left-sided LPM Nodal signaling may be indirect, through removal of repression of Nodal pathway gene expression in the left epithalamus (Carl, 2007).
This study addresses the role of the Wnt/Axin1/β-catenin signaling pathway in the regulation of asymmetric Nodal pathway gene expression and in the elaboration of brain asymmetries. The role of this pathway in the development of brain asymmetries has not previously been assessed, but some studies suggest that Wnt signaling can influence visceral asymmetries. For instance, overexpression of Xwnt8 in Xenopus can lead to cardiac left-right reversals as can overactivation of the Wnt/β-catenin pathway in medaka. In chick, Wnt/β-catenin signaling is suggested to be a left determinant of Nodal pathway gene expression in the LPM, as early upregulation of the pathway results in bilateral Nodal gene expression. Furthermore, mice lacking Wnt3a exhibit asymmetry defects that are likely due to a requirement for Wnt3a acting in and around the node during the period when asymmetries first become evident (Carl, 2007).
This study used a variety of approaches to establish roles for Wnt/β-catenin signaling and the Wnt pathway scaffolding protein Axin1 in both the regulation of Nodal pathway activation and in the differentiation of lateralized brain nuclei. masterblind (mbl) embryos carry a mutation in Axin1 that disrupts the binding of GSK3β, reducing the ability of GSK3β to degrade β-catenin and consequently leading to overactivation of Wnt/β-catenin signaling in the anterior neural plate. mbl mutant embryos show bilateral activation of Nodal pathway genes in the epithalamus but not the viscera. This activation can occur independently of the activity of Spw, suggesting that overactivation of Wnt signaling bilaterally removes repression of epithalamic Nodal pathway gene expression. Evidence is provided that this likely reflects a role for Wnt signaling during late gastrulation. Later overactivation of Wnt signaling during somitogenesis stages can disrupt lateralized Nodal pathway gene expression concordantly in the LPM and brain in both zebrafish and medaka. This is consistent with a role for Spw in the ipsilateral removal of repression of epithalamic Nodal pathway gene expression. Finally, Axin1 is shown to be required downstream of Nodal signaling during the elaboration of epithalamic asymmetries. These results provide evidence that the Wnt/Axin1/β-catenin signaling pathway plays several critical roles during the establishment and elaboration of asymmetries in the forming CNS (Carl, 2007).
Stem cell-based replacement therapy has emerged as a potential strategy to alleviate specific features of movement disorder in Parkinson's disease. However, the current strategy to produce dopamine (DA) neurons from embryonic stem cells has many limitations, including the difficulty of generating DA neurons with high yields. Further insights into the mechanisms that control the neurogenesis of DA neurons will reduce or mitigate such limitations. It is well established that the ventral midbrain (vMB) contains the neurogenic niche that produces DA neurons. However, it is unclear how the microenvironment within this niche controls DA neurogenesis. This study shows that β-catenin controls DA neurogenesis by maintaining the integrity of the neurogenic niche and the progression from progenitors to DA neurons. Using conditional gene targeting approaches, this study shows that regional deletion of β-catenin in the vMB by using Shh-Cre disrupts adherent junctions of progenitors and the integrity of radial glia in the vMB, which leads to a severe reduction in DA neurogenesis and perturbs the migration and segregation of DA neurons. By contrast, Th-IRES-Cre removes β-catenin in a subset of neural progenitor cells without perturbing the cellular and structural integrity of the vMB. Interestingly, loss of β-catenin in Th-IRES-Cre;β-Ctnfl/fl mutants negatively regulates neurogenesis by interfering with the progression of committed progenitors to DA neurons. Reduced DA neurogenesis in the mutant embryos can be attributed to the loss of cell polarity, the disruption in radial glia processes and the reduced proliferation in DA progenitors. Taken together, these results provide new insights into the indispensable functions of β-catenin at multiple stages during DA neurogenesis. They also suggest that β-catenin-mediated signaling pathways can be targeted to promote and expand DA neurons in cell-based therapeutic strategies (Tang, 2009).
The Disrupted in Schizophrenia 1 (DISC1) gene is disrupted by a balanced chromosomal translocation (1; 11) (q42; q14.3) in a Scottish family with a high incidence of major depression, schizophrenia, and bipolar disorder. Subsequent studies provided indications that DISC1 plays a role in brain development. This study demonstrates that suppression of DISC1 expression reduces neural progenitor proliferation, leading to premature cell cycle exit and differentiation. Several lines of evidence suggest that DISC1 mediates this function by regulating GSK3β. First, DISC1 inhibits GSK3β activity through direct physical interaction, which reduces β-catenin phosphorylation and stabilizes β-catenin. Importantly, expression of stabilized β-catenin overrides the impairment of progenitor proliferation caused by DISC1 loss of function. Furthermore, GSK3 inhibitors normalize progenitor proliferation and behavioral defects caused by DISC1 loss of function. Together, these results implicate DISC1 in GSK3β/β-catenin signaling pathways and provide a framework for understanding how alterations in this pathway may contribute to the etiology of psychiatric disorders (Mao, 2009).
Autosomal recessive primary microcephaly (MCPH) is a neural developmental disorder in which patients display significantly reduced brain size. Mutations in Abnormal Spindle Microcephaly (ASPM) are the most common cause of MCPH. This study investigated the underlying functions of Aspm in brain development; Aspm expression was found to be critical for proper neurogenesis and neuronal migration. The Wnt signaling pathway is known for its roles in embryogenesis, and genome-wide siRNA screens indicate that ASPM is a positive regulator of Wnt signaling. Knockdown of Aspm results in decreased Wnt-mediated transcription, and expression of stabilized β-catenin can rescue this deficit. Coexpression of stabilized β-catenin can rescue defects observed upon in vivo knockdown of Aspm. These findings provide an impetus to further explore Aspm's role in facilitating Wnt-mediated neurogenesis programs, which may contribute to psychiatric illness etiology when perturbed (Buchman, 2011).
Basal progenitors (also called non-surface dividing or intermediate progenitors) have been proposed to regulate the number of neurons during neocortical development through expanding cells committed to a neuronal fate, although the signals that govern this population have remained largely unknown. This study shows that N-myc mediates the functions of Wnt signaling in promoting neuronal fate commitment and proliferation of neural precursor cells in vitro. Wnt signaling and N-myc also contribute to the production of basal progenitors in vivo. Expression of a stabilized form of beta-catenin, a component of the Wnt signaling pathway, or of N-myc increased the numbers of neocortical basal progenitors, whereas conditional deletion of the N-myc gene reduced these and, as a likely consequence, the number of neocortical neurons. These results reveal that Wnt signaling via N-myc is crucial for the control of neuron number in the developing neocortex (Kuwahara, 2010).
Wnt signaling and its downstream target N-Myc play a key role in the production of basal progenitors. Expression of N-myc or stabilized β-catenin increases, while conditional gene deletion of N-myc decreases the numbers of basal progenitors found in the developing neocortex, as determined by the numbers of Tbr2-positive cells and non-surface dividing cells. The increase in basal progenitors by the Wnt-N-myc axis can be ascribed to either: (1) differentiation of apical progenitors into basal progenitors; or (2) proliferation (and survival) of basal progenitors, or both. The observation that retroviral expression of stabilized β-catenin or N-myc in the neocortex reduced the number of apical progenitors while increasing that of the basal progenitors supports a role for the former mechanism (Kuwahara, 2010).
Members of the Myc family have been reported to be involved in differentiation processes in other cell types, including epithelial, neural crest and hematopoietic stem cells, although previous reports have not directly demonstrated that Myc is involved in fate commitment by a lineage-tracing analysis. In this study, the clonal analysis suggests that N-myc instructs commitment of NPC fate into the neuronal lineage at the expense of the glial lineage and reduces multipotent neurosphere-forming NPCs. This function of N-myc is similar to the reported function of Wnt signaling (Kuwahara, 2010).
It is not known what transcriptional targets of N-myc are involved in instructing neurogenesis. Possible candidates include the proneural gene Ngn1, as deletion of N-myc was observed to cause a decrease in the level of Ngn1 mRNA in the developing neocortex. As Ngn1 is also a direct target of the β-catenin/Tcf transcription complex, it would be interesting to examine the interaction between N-myc and these transcription factors on the Ngn1 promoter. The Myc family has also been shown to function in the regulation of the global chromatin state, in addition to its function as a classical transcription factor; thus it is possible that mechanisms other than direct target gene activation are also involved in N-myc regulation of neurogenesis and proliferation of NPCs (Kuwahara, 2010).
This study also provides evidence that N-myc is directly regulated by the β-catenin/Tcf transcription complex and mediates the functions of Wnt signaling to stimulate neocortical NPC proliferation and differentiation: (1) Wnt3a treatment and stabilized β-catenin expression induced N-myc expression, whereas expression of a dominant-negative form of Tcf3 reduced N-myc expression in NPC cultures; (2) misexpression of stabilized β-catenin in the ventral telencephalon induced ectopic N-myc expression in vivo; (3) N-myc is expressed in the developing neocortex in a pattern similar to that of a Tcf reporter transgene; (4) Tcf3 directly binds to a Tcf-consensus site 1.6 kb upstream of the N-myc gene; (5) Wnt stimulation of proliferation and differentiation in NPC cultures was abrogated by deletion of the N-myc gene. These results provide evidence that N-myc is a key downstream mediator of Wnt-β-catenin signaling in the developing neocortex. It is of note that N-myc is not the only downstream target responsible for the functions of Wnt signaling in the neocortex (Kuwahara, 2010).
The Wnt-β-catenin pathway exerts multiple functions in a context-dependent manner. For instance, persistent expression of stabilized β-catenin in NPCs results in overproliferation of apical progenitors and horizontal/tangential expansion of the cortex in addition to the reduction of Tbr2-positive basal progenitors. However, when the same stabilized β-catenin was expressed by retroviral infection in a small proportion of NPCs located at the VZ, it had the opposite effect: increasing the numbers of basal progenitors and decreasing the number of apical progenitors. This difference does not appear to be due to the differential requirement of N-myc, as N-myc gene deletion rescued both proliferative and differentiating effects of activation of β-catenin. This difference might be rather due to the aberrant brain architecture generated in the β-catenin-δEx3 mice (mutant for β-catenin), to other non-cell autonomous effects of β-catenin or to differences in the levels or timing of active β-catenin expression. Indeed, different levels of active β-catenin expression result in different outcomes in hair follicle stem cells (Kuwahara, 2010).
Although it has previously been postulated that β-catenin exerts its different functions via distinct targets, this study observed that both the proliferating and neurogenic functions of Wnt-β-catenin signaling in the developing neocortex are mediated in common by N-myc. It is noteworthy that c-Myc can also exert distinct functions depending on its expression levels, such as in epithelial stem cells, raising the possibility that the levels of N-myc might determine the cellular output. Importantly, heterozygous mutation of N-MYC (MYCN) in humans causes Feingold syndrome, comprising several defects including microcephaly, supporting the notion that the levels of N-myc in the nervous system are crucial for determining the neuronal number and brain size. It is also possible that N-myc alters its function in a developmental-stage-dependent manner. This possibility is consistent with a previous finding that canonical Wnt signaling promotes proliferation of neocortical neural precursor cells at a relatively early stage (E10.5) but promotes their differentiation at a relatively late stage (E13.5) (Kuwahara, 2010).
Which Wnt ligands are responsible for the activation of N-myc and consequent regulation of basal progenitors in the developing brain? Wnt7a is expressed in NPCs at the VZ and might be important for increase in cells localized in the SVZ. Wnt7b, which is expressed in the deep-layer neurons (neurons at the layer VI), might elicit a feed-forward signal to increase the number of basal progenitors that in turn contribute to the generation of the upper-layer neurons. It is plausible that extracellular signals other than Wnt ligands are also involved in the activation of N-myc and regulation of basal progenitors. N-myc is induced by Shh signaling in cerebellar granule cells, and a recent report shows that Shh protein is localized in the IMZ of the neocortex and contributes to the production of basal progenitors. Growth factors expressed in NPCs such as Fgf2 and epidermal growth factor (Egf) might also participate in the activation of N-myc. Growth factor receptors activate the PI3K (Pik3r1 - Mouse Genome Informatics) pathway, which induces phosphorylation and stabilization of N-myc protein. In addition, Egfr as well as Frs2, an adaptor of Fgfr/Egfr, have been shown to regulate the production of basal progenitors. The RNA-binding protein HuC/D is another candidate that could regulate N-myc function in basal progenitors, as it binds to and stabilizes N-myc mRNA and is localized in the SVZ (Kuwahara, 2010).
As a mechanism of neocortical expansion during animal evolution, the increase of basal progenitors is considered to be a key event, given that basal progenitors increase the number of neurons from a given number of apical progenitors through extra cell division and that the number of basal progenitors dramatically increases during animal evolution. The observation in this study that N-myc deletion decreases Tbr2-positive cells and non-surface dividing cells without marked reduction of Pax6-positive cells supports the notion that Wnt signaling, via N-myc, promotes differentiation from apical progenitors to basal progenitors and promotes indirect neurogenesis. It would be interesting to investigate possible roles of this signaling pathway in the neocortical expansion during animal evolution in future studies (Kuwahara, 2010).
The retinal pigment epithelium (RPE) consists of a monolayer of cuboidal,
pigmented cells that is located between the retina and the choroid. The RPE is
vital for growth and function of the vertebrate eye and improper development
results in congenital defects, such as microphthalmia or anophthalmia, or a
change of cell fate into neural retina called transdifferentiation. The
transcription factors microphthalmia-associated transcription factor (Mitf)
and orthodenticle homolog 2 (Otx2) are crucial for RPE development and
function; however, very little is known about their regulation. By using
a Wnt-responsive reporter, this study shows that the Wnt/β-catenin pathway is activated in the differentiating mouse RPE. Cre-mediated, RPE-specific
disruption of β-catenin after the onset of RPE specification causes
severe defects, resulting in microphthalmia with coloboma, disturbed
lamination, and mislocalization of adherens junction proteins. Upon
β-catenin deletion, the RPE transforms into a multilayered tissue in
which the expression of Mitf and Otx2 is downregulated, while retina-specific
gene expression is induced, which results in the transdifferentiation of RPE
into retina. Chromatin immunoprecipitation (ChIP) and luciferase assays indicate that β-catenin binds near to and activates potential TCF/LEF sites in the Mitf and Otx2 enhancers. It is concluded that Wnt/β-catenin signaling is required for differentiation of the RPE by directly regulating the expression of Mitf and Otx2. This study is the first to show that an extracellular signaling pathway directly regulates the expression of RPE-specific genes such as Mitf and Otx2, and elucidates a new role for the Wnt/β-catenin pathway in organ formation and development (Westenskow, 2009).
The RPE originates from the optic neuroepithelium of the ventral forebrain, which undergoes morphogenetic movements leading to formation of the optic cup. The resulting inner layer of the optic cup develops into the neural retina and the outer layer differentiates into RPE. Both retina and RPE are specified early, prior to optic cup formation. Subsequent to RPE specification, a period of differentiation and maturation follows, resulting in dramatic morphological, structural and functional changes. Interestingly, the RPE fate is reversible for several days following the initial activation of differentiation, as evidenced by a propensity to downregulate RPE-specific genes, to hyperproliferate and to differentiate into retina, a process considered to be transdifferentiation. Thus, it is crucial that mechanisms exist to maintain RPE differentiation in the optic cup (Westenskow, 2009).
RPE specification and differentiation are regulated by two key regulatory transcription factors, Mitf and Otx2. Disruption of either gene, similar to genetic ablation of the RPE, results in microphthalmia and coloboma during murine eye development. Mitf isoforms and Otx2 transactivate essential genes for terminal pigment differentiation in the RPE and neural crest (e.g. tyrosinase-related protein 1; Tyrp1) and for RPE-specific functions. Initiation and maintenance of Mitf and Otx2 expression is controlled by interaction with surrounding extraocular tissues, including the extraocular mesenchyme. A few candidate regulators have been identified; however, the exact mechanisms controlling the expression of Mitf and Otx2 are not known. The Wnt/β-catenin pathway is an excellent candidate because it is active in the developing RPE; activation results in cytoplasmic stabilization of β-catenin, which then translocates into the nucleus and associates with TCF/LEF transcription factors. Interestingly, Wnt/β-catenin signaling promotes differentiation of neural crest-derived pigmented cells by direct transactivation of the Mitf-M promoter. Although melanocytes and RPE cells originate from different tissues, some aspects of the mechanisms regulating pigment cell differentiation in different lineages could be similar. This report is the first to show a direct role for an extracellular signaling pathway in controlling development of the mammalian RPE. Although it cannot be ruled out that cell adhesion defects can independently interfere with RPE differentiation, the results strongly suggest that β-catenin, via TCF/LEF activation, is essential for maintaining cell fate in the developing RPE by the direct regulation of Mitf and Otx2 expression. Thus, this mechanism of Mitf regulation appears to be evolutionary conserved between the RPE and neural crest-derived melanocytes. It remains to be determined what the source(s) of the actual ligand(s) is and how Wnt/β-catenin signaling integrates with other putative regulatory pathways to control RPE development (Westenskow, 2009).
Beta-catenin and the synapse Cadherins and catenins are thought to promote adhesion between pre and postsynaptic elements in the brain. A role is shown for ß-catenin in localizing the reserved pool of vesicles at presynaptic sites. Deletion of ß-catenin in hippocampal pyramidal neurons in vivo results in a reduction in the number of reserved pool vesicles per synapse and an impaired response to prolonged repetitive stimulation. This corresponds to a dispersion of vesicles along the axon in cultured neurons. Interestingly, these effects are not due to ß-catenin's involvement in cadherin-mediated adhesion or wnt signaling. Instead, ß-catenin modulates vesicle localization via its PDZ binding domain to recruit PDZ proteins such as Veli to cadherin at synapses. This study defines a specific role for cadherins and catenins in synapse organization beyond their roles in mediating cell adhesion (Bamji, 2003).
Beta-catenin and neural plasticity Activity-induced changes in adhesion molecules may coordinate presynaptic and
postsynaptic plasticity. ß-catenin, which mediates interactions between cadherins and the actin cytoskeleton, moves from dendritic shafts into spines upon depolarization, increasing its association with cadherins. ß-catenin's redistribution is mimicked or prevented by a tyrosine kinase or phosphatase inhibitor, respectively. Point mutations of ß-catenin's tyrosine 654 alters the shaft/spine distribution: Y654F-ß-catenin-GFP (phosphorylation-prevented) is concentrated in spines, whereas Y654E-ß-catenin-GFP (phosphorylation-mimic) accumulates in dendritic shafts. In Y654F-expressing neurons, the PSD-95 or associated synapsin-I clusters are larger than those observed in either wild-type-beta-catenin or also Y654E-expressing neurons. Y654F-expressing neurons exhibit a higher minifrequency. Thus, neural activity induces beta-catenin's redistribution into spines, where it interacts with cadherin to influence synaptic size and strength (Murase, 2002).
Beta-catenin and apoptosis Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Specific and ordered changes in the actin cytoskeleton take place during apoptosis. In this context, one of the first hallmarks in cell death has been isolated for study: the severing of contacts among neighboring cells. A mechanism has been demonstrated that may contribute to the disassembly of cytoskeletal organization at the level of cell-cell adhesion. Beta-catenin, a known regulator of cell-cell adhesion, is proteolytically processed in different cell types after the induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated beta-catenin into a form that is similar in size to that observed in cells undergoing apoptosis. beta-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting beta-catenin product is unable to bind alpha-catenin, which is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at the level of cell-cell contacts could be dismantled during apoptosis. These observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton that occur during cell death via cleavage of different regulators of the microfilament system (Brancolini, 1997).
Growth factor deprivation of endothelial cells induces apoptosis, which is characterized by membrane blebbing, cell rounding,
and subsequent loss of cell-matrix and cell-cell contacts. In this study, it is shown that initiation of endothelial apoptosis
correlates with cleavage and disassembly of intracellular and extracellular components of adherens junctions. beta-Catenin and
plakoglobin, which form intracellular links between vascular endothelial cadherin (VE-cadherin) and actin-binding
alpha-catenin in adherens junctions, are cleaved in apoptotic cells. In vitro incubations of cell lysates and immunoprecipitates
with recombinant caspases indicate that CPP32 and Mch2 are involved, possibly by initiating proteolytic processing. Cleaved
beta-catenin from lysates of apoptotic cells does not bind to endogenous alpha-catenin, whereas plakoglobin retains its binding
capacity. The extracellular portion of the adherens junctions is also altered during apoptosis because VE-cadherin, which
mediates endothelial cell-cell interactions, dramatically decreases on the surface of cells. An extracellular fragment of
VE-cadherin can be detected in the conditioned medium, and this "shedding" of VE-cadherin can be blocked by an inhibitor of
metalloproteinases. Thus, cleavage of beta-catenin and plakoglobin and shedding of VE-cadherin may act in concert to disrupt
structural and signaling properties of adherens junctions and may actively interrupt extracellular signals required for endothelial
cell survival (Herren, 1998).
Plakoglobin is a vertebrate cytoplasmic protein and a homolog of beta-catenin and Armadillo in Drosophila, with similar
adhesive and signaling functions. These proteins interact with cadherins to mediate cell-cell adhesion and associate with
transcription factors to induce changes in the expression of genes involved in cell fate determination and proliferation. Unlike the
relatively well characterized role of beta-catenin in cell proliferation via activation of c-MYC and cyclin D1 gene
expression, the signaling function of plakoglobin in regulation of cell growth is undefined. High levels of plakoglobin expression in
plakoglobin-deficient human SCC9 cells leads to uncontrolled growth and foci formation. Concurrent with the change in growth characteristics is observed a
pronounced inhibition of apoptosis. This correlates with an induction of expression of BCL-2, a prototypic member of apoptosis-regulating proteins. The BCL-2
expression coincides with decreased proteolytic processing and activation of caspase-3, an executor of programmed cell death. These data suggest that the growth
regulatory function of plakoglobin is independent of its role in mediating cell-cell adhesion. These observations clearly implicate plakoglobin in pathways regulating cell
growth and provide initial evidence of its role as a pivotal molecular link between pathways regulating cell adherence and cell death (Hakimelahi, 2000).
Frizzled (fz) functions as a 7-transmembrane receptor
in the Frizzled-Dishevelled signal transduction
cascade. It is involved in architectural control of development
in species as divergent as Drosophila and
vertebrates. Regulation of multicellular architecture
requires control of cell alignment, but also involves an
equilibrium among cell proliferation, differentiation,
and apoptosis. Recently, modulation of the Frizzled-Dishevelled
(Dvl) cascade has been related to apoptosis.
However, the role of ß-catenin (a second messenger
in the Frizzled-Dishevelled cascade) in programmed
cell death is a matter of debate. To elucidate the role of
this cascade in apoptosis, the effect of over-expression
of fz1, fz2, dvl1, and ß-catenin was investigated. The signal
transduction pathway and the involvement of ß-catenin
were further investigated by using different inhibitors.
These experiments were performed in different
cell types: COS7, 293, and PC12. Overexpression of fz1, fz2, and dvl1 induce apoptosis in COS7 and 293 cells.
ß-Catenin appears to be the mediator for this process
since ß-catenin overexpression as well as lithium and
valproate induce apoptosis. In contrast, lithium treatment does not result in apoptosis in PC12 cells. It is concluded that different components of the Frizzled-Dishevelled cascade can induce apoptosis, but that this effect is dependent on the cell type (van Gijn, 2001).
A mechanism by which ß-catenin induces apoptosis has recently
been proposed. In the nucleus, ß-catenin interacts with HMG-box transcription factors such as lymphoid enhancer factor/T-cell factor (LEF/Tcf) to regulate gene
expression. A role for the ß-catenin-Tcf signaling
in apoptosis has been described: butyrate, sulindac, and trichostatin A upregulate Tcf activity,
which is reflected by an increase in ß-catenin-Tcf formation
in the SW620 cell line, and induces apoptosis in these cells. Moreover, ß-catenin has been found to activate c-myc and cyclin D1 gene expression, genes
involved in cell proliferation. Intriguingly, c-myc can also act as a potent inducer of apoptosis. Myc-induced apoptosis is dependent upon the level at which myc is expressed and is presumably induced by modulation of target genes. Another possibility could be that the increase in ß-catenin-Tcf complex influences the NF-kappaB signaling, which is implicated in cell survival
and which has been shown to be decreased by GSK-3ß inhibition. However, ß-catenin transfection in 3T3 fibroblasts induces apoptosis independent of its transactivation function with LEF-1, suggesting that LEF is not involved in the apoptosis induced
by ß-catenin overexpression. Further work will be needed to discover what the mechanism is by which ß-catenin influences apoptosis (van Gijn, 2001 and references therein).
Beta-catenin and cancer Beta-catenin has functions as both an adhesion and a signaling molecule. Disruption of these functions
through mutations of the beta-catenin gene (CTNNB1) may be important in the development of
colorectal tumors. The entire coding sequence of beta-catenin was examined by reverse
transcriptase-PCR (RT-PCR) and direct sequencing of 23 human colorectal cancer cell lines from 21
patients. In two cell lines, there is apparent instability of the beta-catenin mRNA. Five different
mutations (26%) were found in the remaining 21 cell lines (from 19 patients). A three-base deletion
(codon 45) was identified in the cell line HCT 116, whereas cell lines SW 48, HCA 46, CACO 2, and
Colo 201 each contained single-base missense mutations (codons 33, 183, 245, and 287, respectively).
All 23 cell lines have full-length beta-catenin protein that is detectable by Western blotting and that
coprecipitate with E-cadherin. In three of the cell lines with CTNNB1 mutations, complexes of
beta-catenin with alpha-catenin and APC are detectable. In SW48 and HCA 46, however, complexes of beta-catenin protein with alpha-catenin and APC, respectively were not detected. These results
show that selection of CTNNB1 mutations occurs in up to 26% of colorectal cancers from which cell
lines are derived. In these cases, mutation selection is probably for altered beta-catenin function, which
may significantly alter intracellular signaling and intercellular adhesion and may serve as a complement
to APC mutations in the early stages of tumorigenesis (Ilyas, 1997).
Hepatocellular carcinoma (HCC) is the major primary malignant tumor in the human liver, but the molecular changes leading to liver cell transformation remain largely unknown. The Wnt-beta-catenin pathway is activated in colon cancers and some melanoma cell lines, but has not yet been investigated in HCC. The status of the beta-catenin gene was examined in different transgenic mouse lines of HCC obtained with either the oncogenes c-myc or H-ras. Fifty percent of the hepatic tumors in these transgenic mice have activating somatic mutations within the beta-catenin gene similar to those found in colon cancers and melanomas. These alterations in the beta-catenin gene (point mutations or deletions) lead to a disregulation of the signaling function of beta-catenin and thus to carcinogenesis. Human HCCs have similar mutations in eight of 31 (26%) human liver tumors tested and in HepG2 and HuH6 hepatoma cells. The mutations lead to the accumulation of beta-catenin in the nucleus. Frequently, alterations in the beta-catenin gene are selected for during liver tumorigenesis; this suggests that disregulation of the Wnt-beta-catenin pathway is a major event in the development of HCC in humans and mice (Coste, 1998).
Mutations in the adenomatous polyposis coli (APC) tumor-suppressor gene occur in most human colon cancers. Loss of functional APC protein results in the
accumulation of beta-catenin. Mutant forms of beta-catenin have been discovered in colon cancers that retain wild-type APC genes, and also in melanomas,
medulloblastomas, prostate cancer and gastric and hepatocellular carcinomas. The accumulation of beta-catenin activates genes that are responsive to transcription
factors of the TCF/LEF family, with which beta-catenin interacts. Beta-catenin activates transcription from the cyclin D1 promoter, and
sequences within the promoter that are related to consensus TCF/LEF-binding sites are necessary for activation. The oncoprotein p21ras further activates
transcription of the cyclin D1 gene, through sites within the promoter that bind the transcriptional regulators Ets or CREB. Cells expressing mutant beta-catenin
produce high levels of cyclin D1 messenger RNA and protein constitutively. Furthermore, expression of a dominant-negative form of TCF in colon-cancer cells
strongly inhibits expression of cyclin D1 without affecting expression of cyclin D2, cyclin E, or cyclin-dependent kinases 2, 4 or 6. This dominant-negative TCF
causes cells to arrest in the G1 phase of the cell cycle; this phenotype can be rescued by expression of cyclin D1 under the cytomegalovirus promoter. Abnormal
levels of beta-catenin may therefore contribute to neoplastic transformation by causing accumulation of cyclin D1 (Tetsu, 1999).
beta-Catenin plays a dual role in the cell: it links the cytoplasmic side of cadherin-mediated cell-cell contacts to the actin cytoskeleton and it acts in signaling that involves transactivation in complex with transcription factors of the lymphoid enhancing factor (LEF-1) family. Elevated beta-catenin
levels in colorectal cancer caused by mutations in beta-catenin or by the adenomatous polyposis coli molecule, which regulates beta-catenin degradation,
result in the binding of beta-catenin to LEF-1 and increased transcriptional activation of mostly unknown target genes. The cyclin D1
gene is a direct target for transactivation by the beta-catenin/LEF-1 pathway through a LEF-1 binding site in the cyclin D1 promoter. Three inhibitors of
beta-catenin activation, wild-type adenomatous polyposis coli, axin, and the cytoplasmic tail of cadherin, suppress cyclin D1 promoter activity in colon
cancer cells. Cyclin D1 protein levels are augmented by beta-catenin overexpression and reduced in cells overexpressing the cadherin cytoplasmic domain.
Increased beta-catenin levels may thus promote neoplastic conversion by triggering cyclin D1 gene expression and, consequently, uncontrolled progression
into the cell cycle (Shtutman, 1999).
beta-Catenin is an important regulator of cell-cell adhesion and embryonic development that associates with and regulates the function of the LEF/TCF family of transcription factors. Mutations of beta-catenin and the tumor suppressor gene, adenomatous polyposis coli, occur in human cancers, but it is not known if, and by what mechanism, increased beta-catenin causes cellular transformation. The demonstration of an interaction between beta-catenin and the product of the tumor suppressor gene,
adenomatous polyposis coli (APC), suggests that beta-catenin is involved in oncogenesis. Tumor cell lines with a loss of one copy of
APC, and harboring mutations in the other allele, have high levels of cytoplasmic (signaling) beta-catenin, which are markedly reduced when functional APC is
reintroduced. Importantly, mutant forms of APC found in human cancers are unable to reduce beta-catenin levels in tumor cells. The
importance of elevated beta-catenin in human cancer was further substantiated when mutations in the beta-catenin gene were described in colon cancer and melanoma cell
lines. At least one of these mutations results in a more stable form of the protein.
This study demonstrates that modest overexpression of beta-catenin in a normal epithelial cell results in cellular transformation. These cells form colonies in soft agar, survive in suspension, and continue to proliferate at high cell density and following gamma-irradiation. Endogenous cytoplasmic beta-catenin levels and signaling activity were found to oscillate during the cell cycle. Taken together, these data demonstrate that beta-catenin functions as an oncogene by promoting the G1 to S phase transition and protecting cells from suspension-induced apoptosis (anoikis) (Orford, 1999).
Although total beta-catenin protein levels do not vary appreciably during the cell cycle,
cytoplasmic beta-catenin levels increase significantly from G1 to S phase. The increase begins in late G1 and continues through S phase. Densimetric scanning reveals a 23-fold increase in cytoplasmic levels from early G1/G0 to S phase.
The c-myc promoter is known to be regulated by the APC/ß-catenin signaling pathway. The upregulation of c-myc by beta-catenin may constitute one
mechanistic link between beta-catenin and tumor formation. c-myc is a potent oncogene that regulates cell cycle progression. However, c-myc overexpression cannot
induce cellular transformation on its own. In fact, when overexpressed alone, c-myc markedly increases the susceptibility of cells to apoptosis. To transform cells, c-myc requires an accompanying survival signal to prevent cells from
undergoing apoptosis. Advancement through the G1 phase of the cell cycle can result in either progression into S phase or apoptosis, depending on the presence or
absence of certain survival signals, for example, IGF-1. In addition to stimulating c-myc, beta-catenin may transduce the requisite antiapoptotic
signal that would permit cell cycle progression. The increase of cytoplasmic beta-catenin protein before S phase during the cell cycle may serve this purpose in normal
cells. Additionally, beta-catenin would protect against anoikis if overexpressed in epithelial cells (Orford, 1999 and references).
WISP-1 (Wnt-1 induced secreted protein 1) is a member of the CCN family of growth factors. This study identifies WISP-1 as a
beta-catenin-regulated gene that can contribute to tumorigenesis. The promoter of WISP-1 was cloned and shown to be
activated by both Wnt-1 and beta-catenin expression. TCF/LEF sites play a minor role, whereas the CREB site played an
important role in this transcriptional activation. WISP-1 demonstrates oncogenic activities; overexpression of WISP-1 in normal rat
kidney fibroblast cells (NRK-49F) induces morphological transformation, accelerates cell growth, and enhances saturation density. Although these cells did not
acquire anchorage-independent growth in soft agar, they readily form tumors in nude mice, suggesting that appropriate cellular attachment is important for signaling
oncogenic events downstream of WISP-1. Taken together, these data suggest that beta-catenin is able to regulate downstream events through multiple factors,
in addition to the TCF/LEF family members. The heterogeneity of genetic elements regulated by Wnt-1 and beta-catenin also suggests the functional diversity
of the Wnt-signaling pathway (Xu, 2000).
Beta-catenin can function as an oncogene when it is translocated to the nucleus, binds to T cell factor or lymphoid enhancer factor family members, and transactivates its target genes. Cyclin D1 is one of the targets of beta-catenin in breast cancer cells. Transactivation of beta-catenin correlates significantly with cyclin D1 expression both in eight breast cell lines in vitro and in 123 patient samples. Beta-catenin activity significantly correlates with poor prognosis of the patients and is a strong and independent prognostic factor in breast cancer. These studies, therefore, indicated that beta-catenin can be involved in breast cancer formation and/or progression and may serve as a target for breast cancer therapy (Lin, 2000).
ß-catenin and plakoglobin (gamma-catenin) are homologous molecules
involved in cell adhesion, linking cadherin receptors to the cytoskeleton. ß-catenin is also a key component of the Wnt pathway by
being a coactivator of LEF/TCF transcription factors. To identify novel
target genes induced by ß-catenin and/or plakoglobin, DNA microarray
analysis was carried out with RNA from cells overexpressing either
protein. This analysis revealed that Nr-CAM is the gene most
extensively induced by both catenins. Overexpression of either ß-catenin or plakoglobin induced Nr-CAM in a variety of cell types and the LEF/TCF binding sites in the Nr-CAM promoter were required for
its activation by catenins. Retroviral transduction of Nr-CAM into
NIH3T3 cells stimulates cell growth, enhances motility, induces transformation, and produces rapidly growing tumors in nude mice. Nr-CAM and LEF-1 expression is elevated in human colon cancer tissue
and cell lines and in human malignant melanoma cell lines but not in
melanocytes or normal colon tissue. Dominant negative LEF-1 decreases
Nr-CAM expression and antibodies to Nr-CAM inhibit the motility of
B16 melanoma cells. The results indicate that induction of Nr-CAM
transcription by ß-catenin or plakoglobin plays a role in melanoma
and colon cancer tumorigenesis, probably by promoting cell growth and motility (Conacci-Sorrell, 2002).
The transactivation of TCF target genes induced by Wnt pathway mutations constitutes the primary transforming event in colorectal cancer (CRC). Disruption of ß-catenin/TCF-4 activity in CRC cells induces a rapid G1 arrest and blocks a genetic program that is physiologically active in the proliferative compartment of colon crypts. Coincidently, an intestinal differentiation program is induced. The TCF-4 target gene c-MYC plays a central role in this switch by direct repression of the p21CIP1/WAF1 promoter. Following disruption of ß-catenin/TCF-4 activity, the decreased expression of c-MYC releases p21CIP1/WAF1 transcription, which in turn mediates G1 arrest and differentiation. Thus, the ß-catenin/TCF-4 complex constitutes the master switch that controls proliferation versus differentiation in healthy and malignant intestinal epithelial cells (van de Wetering, 2002).
c-MYC plays a central role in the proliferative capacity of many cancers, including CRC. tHE data imply that c-MYC blocks the expression of the cell cycle inhibitor p21CIP1/WAF1. The region responsible for p21CIP1/WAF1 regulation has been mapped to a 200 bp fragment of the proximal promoter.
The presence of MIZ-1 and c-MYC on this promoter suggests that c-MYC-mediated repression of p21CIP1/WAF1 occurs by a mechanism resembling c-MYC control of p15INK4b, i.e., through preventing promoter activation by the transcription factor MIZ-1. Decreased expression of c-MYC would allow MIZ-1 to activate p21CIP1/WAF1 transcription. The complementarity in the expression of c-MYC and p21CIP1/WAF1 in the intestine supports this mechanism (van de Wetering, 2002).
The APC tumor suppressor gene is mutated in most colon cancers. A major role of APC is the downregulation of the beta-catenin/T-cell factor (Tcf)/lymphoid enhancer factor (LEF) signalling pathway; however, there are also suggestions that it plays a role in the organization of the cytoskeleton, and in cell adhesion and migration. Stable expression of wild-type APC has been achieved in SW480 colon cancer cells, which normally express a truncated form of APC. The ectopically expressed APC is functional, and results in the translocation of beta-catenin from the nucleus and cytoplasm to the cell periphery, and reduces beta-catenin/Tcf/LEF transcriptional signalling. E-cadherin is also translocated to the cell membrane, where it forms functional adherens junctions. Total cellular levels of E-cadherin are increased in the SW480APC cells and the altered charge distribution in the presence of full-length APC suggests that APC is involved in post-translational regulation of E-cadherin localization. Changes in the location of adherens junction proteins are associated with tighter cell-cell adhesion in SW480APC cells, with consequent changes in cell morphology, the actin cytoskeleton and cell migration in a wound assay. SW480APC cells have a reduced proliferation rate, a reduced ability to form colonies in soft agar and do not grow tumors in a xenograft mouse tumor model. By regulating the intracellular transport of junctional proteins, it is proposed that APC plays a role in cell adhesion in addition to its known role in beta-catenin transcriptional signalling (Faux, 2003).
Smad4 is a central mediator for TGFß signals, which play important functions in many biological processes. To study the role of Smad4 in mammary gland development and neoplasia, this gene was disrupted in mammary epithelium using a Cre-loxP approach. Smad4 is expressed in the mammary gland throughout development; however, its inactivation did not cause abnormal development of the gland during the first three pregnancies. Instead, lack of Smad4 gradually induced cell proliferation, alveolar hyperplasia and transdifferentiation of mammary epithelial cells into squamous epithelial cells. Consequently, all mutant mice developed squamous cell carcinoma and/or mammary abscesses between 5 and 16 months of age. Absence of Smad4 resulted in ß-catenin accumulation at onset and throughout the process of transdifferentiation, implicating ß-catenin, a key component of the Wnt signaling pathway, in the development of squamous metaplasia in Smad4-null mammary glands. TGFß1 treatment degrades ß-catenin and induces epithelial-mesenchymal transformation in cultured mammary epithelial cells. However, such actions are blocked in the absence of Smad4. These findings indicate that TGFß/Smad4 signals play a role in cell fate maintenance during mammary gland development and neoplasia (Li, 2003).
ß-catenin signaling is heavily involved in organogenesis. This study
investigated how pancreas differentiation, growth and homeostasis are affected
following inactivation of an endogenous inhibitor of ß-catenin,
adenomatous polyposis coli (Apc). In adult mice, Apc-deficient pancreata are
enlarged, solely as a result of hyperplasia of acinar cells, which accumulate
ß-catenin, with the sparing of islets. Expression of a target of
ß-catenin, the proto-oncogene c-myc (Myc), is
increased in acinar cells lacking Apc, suggesting that c-myc
expression is essential for hyperplasia. In support of this hypothesis, it was
found that conditional inactivation of c-myc in pancreata lacking Apc
completely reverse the acinar hyperplasia. Apc loss in organs such as the
liver, colon and kidney, as well as experimental misexpression of c-myc in pancreatic acinar cells, lead to tumor formation with high penetrance. Surprisingly, pancreas tumors failed to develop following conditional pancreas Apc inactivation. In Apc-deficient acini of aged mice, these studies revealed a cessation of their exaggerated proliferation and a reduced expression of c-myc, in spite of the persistent accumulation of ß-catenin. In conclusion, this work shows that ß-catenin
modulation of c-myc is an essential regulator of acinar growth
control, and unveils an unprecedented example of Apc requirement in the pancreas that is both temporally restricted and cell-specific. This provides new insights into the mechanisms of tumor pathogenesis and tumor suppression in the pancreas (Strom, 2007).
Tumor progression is a multistep process in which proproliferation mutations must be accompanied by suppression of senescence. In melanoma, proproliferative signals are provided by activating mutations in NRAS and BRAF, whereas senescence is bypassed by inactivation of the p16Ink4a gene. Melanomas also frequently exhibit constitutive activation of the Wnt/β-catenin pathway that is presumed to induce proliferation, as it does in carcinomas. Contrary to expectations, stabilized β-catenin reduces the number of melanoblasts in vivo and immortalizes primary skin melanocytes by silencing the p16Ink4a promoter. Significantly, in a novel mouse model for melanoma, stabilized β-catenin bypasses the requirement for p16Ink4a mutations and, together with an activated N-Ras oncogene, leads to melanoma with high penetrance and short latency. The results reveal that synergy between the Wnt and mitogen-activated protein (MAP) kinase pathways may represent an important mechanism underpinning the genesis of melanoma, a highly aggressive and increasingly common disease (Delmas, 2007).
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