Wnt signaling in development and disease
© Yang; licensee BioMed Central Ltd. 2012
Received: 10 February 2012
Accepted: 20 April 2012
Published: 20 April 2012
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© Yang; licensee BioMed Central Ltd. 2012
Received: 10 February 2012
Accepted: 20 April 2012
Published: 20 April 2012
Cell signaling mediated by morphogens is essential to coordinate growth and patterning, two key processes that govern the formation of a complex multi-cellular organism. During growth and patterning, cells are specified by both quantitative and directional information. While quantitative information regulates cell proliferation and differentiation, directional information is conveyed in the form of cell polarities instructed by local and global cues. Major morphogens like Wnts play critical roles in embryonic development and they are also important in maintaining tissue homeostasis. Abnormal regulation of these signaling events leads to a diverse array of devastating diseases including cancer. Wnts transduce their signals through several distinct pathways and they regulate vertebrate embryonic development by providing both quantitative and directional information. Here, taking the developing skeletal system as an example, we review our work on Wnt signaling pathways in various aspects of development. We focus particularly on our most recent findings that showed that in vertebrates, Wnt5a acts as a global cue to establishing planar cell polarity (PCP). Our work suggests that Wnt morphogens regulate development by integrating quantitative and directional information. Our work also provides important insights in disease like Robinow syndrome, brachydactyly type B1 (BDB1) and spina bifida, which can be caused by human mutations in the Wnt/PCP signaling pathway.
One of the most remarkable biological processes is the formation of a morphologically complex and functional diverse multicellular organism such as a human being from a single fertilized egg within a short period of time. During this tightly regulated process of embryonic morphogenesis, functional tissues and organs are formed and they have to be properly maintained during adult lives. Because cell-cell signaling plays essential and pivotal roles in both embryonic development and adult physiology, understanding the function and the underlying molecular mechanism of key cell signaling pathways in both development and diseases has been a major focus of our lab. Here we primarily focus on Wnt signaling in major developmental events of the skeletal system.
Wnts are evolutionarily conserved major regulatory factors in both development and disease. Wnt signaling is required in most embryonic developmental processes in both invertebrates and vertebrates. Abnormal Wnt signaling causes many types of tumors [1–3]. For instance, ectopic activation of Wnt1 gene expression in the mouse mammary gland leads to tumor formation . Mutations in Wnt signaling components have also been found to cause other human diseases. Weakened Wnt/β-catenin signaling leads to osteoporosis-pseudoglioma syndrome due to reduced bone mass  whereas enhanced Wnt/β-catenin signaling causes thick bone syndrome due to increased bone mass [6, 7]. In addition, Robinow syndrome and Brachydactyly Type B1 that are characterized by shortened skeletal elements are caused by mutations in the Wnt/planar cell polarity pathway components [8–11].
The developing skeleton provides a good system to study major events in embryonic morphogenesis. It is relatively simple in its structure and cellular composition. It only contains three major cell types: chondrocytes that form the cartilage, osteoblasts that secrete bone matrix and osteoclasts that are specialized macrophage cells differentiated from hematopoietic precursor cells [21, 22]. The skeletal system is widely distributed throughout the body, covering, supporting and protecting important organs. Compelling evidence also indicates that the skeletal system plays systemic regulatory roles as well . The skeletal elements exhibit characteristic morphologies and organization that serve as convenient readouts for regulation of morphogenesis. For instance, the entire skeleton can be visualized in great detail in the intact embryo or adult animal after simple staining procedures.
Skeletal development starts from mesenchymal condensation in which mesenchymal progenitor cells are at least bipotential and they are also called osteochondral progenitors. These cells differentiate into either osteoblasts or chondrocytes depending on the mechanism of ossification. During intramembranous ossification that occurs primarily in the skull, osteochondral progenitors differentiate directly into bone forming osteoblasts whereas in endochondral ossification that occurs in most parts of the body, osteochondral progenitors first differentiate into cartilage forming chondrocytes instead. Osteoblast cells then form in the peripheral of the cartilage template and invade the hypertrophic cartilaginous area together with blood vessels to start the formation of trabecular bone. After bone formation, osteoclasts brought in by blood vessels remodels the bone, which is very important in maintaining bone homeostasis in adult lives . The skeletal system is also highly segmented. Individual skeletal elements connect to each other through joints and this feature is required for motilities of vertebrate animals.
When my lab first got into the field of skeletal biology a decade ago, I decided to start by investigating the regulatory mechanism underlying several fundamentally important processes of skeletal development: the determination of chondrocytes versus osteoblasts when osteochondral progenitor cells are differentiating; The sequential proliferation and hypertrophy of chondrocytes in the long bone cartilage and induction of synovial joint formation. Now we found that it is quite remarkable that all these fundamental processes are controlled by Wnt/β-catenin signaling [17, 24–30]. The functional spectrum of Wnt signaling in skeletal biology is getting broader and Wnt signaling has become a major field in skeletal biology.
Taken together, we have found that the mesenchymal progenitor cells in the condensation are bipotential [28, 30] (Figure 2). During intramembranous ossification, higher Wnt signaling in the condensation leads to inhibition of chondondrocyte differentiation and promotion of osteoblast differentiation. During endochondral ossification, however, Wnt signaling is kept low in the condensation such that only chondrocytes can differentiate. Later, when Wnt signaling is upregulated in the peripheral of the cartilage, osteoblasts will differentiate. Wnt signaling upregulation in the presumptive joint area induces joint formation. Therefore, by manipulating Wnt signaling, mesenchymal progenitor cells can be directed to form only chondrocytes or osteoblasts. These studies, together with others, have indicated that higher canonical Wnt signaling in cartilage may cause cartilage damages such as those observed in osteoarthritis . Moreover, antibody therapies targeting canonical Wnt signaling are quite effective in increasing bone mass and are under clinical trials [33–36].
One of the most important functions of secreted molecules, Wnts included, is to act as morphogens which are critically required in embryonic morphogenesis by coordinating cell proliferation with cell fate determination [37–39]. This function has been the focal attention of developmental biology. However, the entire organism as well as the internal tissues and organs exhibit distinct morphologies and organizations which are essential for their functions. To understand how these are generated during morphogenesis, one has to understand how directional or polarity information is provided globally and locally. If cells in the developing embryo were not polarized and could not sense global or local directional cues, proliferation and differentiation would be the predominant mechanisms driving the development of the embryo. The result would be a three-dimensionally symmetric sphere composed of many different cell types. Therefore, providing directional information is fundamentally important in biology. For example, it is important to understand why the limb preferentially elongates along the proximal-distal axis.
The PCP pathway is best characterized in Drosophila in which a group of core PCP components were identified and they play conserved roles in both invertebrates and vertebrates [13, 42, 43]. These core PCP components include Wnt receptor frizzled, a four transmembrane protein Van Gogh (vang) (the vertebrate homologues are Van Gogh like 1 and 2 (Vangl 1 and 2)), flamingo (the vertebrate homologues are Celsr1, 2 and 3), prickle and disheveled (Figure 1). Wnt ligands have not been found to regulate PCP in Drosophila. However, in vertebrates such the zebrafish, Wnt11 and Wnt5a are both required to regulate PCP during convergent extension [48, 49]. Downstream of the PCP core components, the PCP pathway might act through RhoA to control cytoskeleton remodeling and Jun kinase pathway to regulate gene expression, which still awaits rigorous genetic tests.
To investigate the function and mechanism of PCP, we removed both Vangl1 and Vangl2 as they form the smallest family of core PCP components, so the problem of functional redundancy within a gene family can be managed relatively easily. In addition, unlike Frizzled and Disheveled Vangl1 and 2 are not shared by the canonical Wnt pathway. Therefore, the phenotypic perturbation in Vangl1 and 2 mutants should be solely due to altered PCP. In mammals, PCP has been found to play fundamental roles in development. For instance, loss of PCP in the mouse Vangl2 Looptail (Lp) mutant leads to randomization of the inner ear hair cells and open neural tube [45, 50–55]. Mild condition of incomplete neural tube closure called spina bifida. In humans, spina bifida is the most common neural tube defect and can be caused by mutations in both human VANGL1 and 2 [56, 57]. Since the nature of the Vangl2 Lp mutation was not clear and the Lp homozygous mutant is embryonic lethal at the time, we generated null alleles of Vangl1 and Vangl2 and a floxed allele of Vangl2. Both Vangl1 and 2 regulates embryonic development as Vangl1/2 double mutants are more severe than Vangl2 the single mutant and Vangl1 single mutant has no embryonic defects . The various abnormalities demonstrated by the Vangl1/2 double mutants indicate that PCP is fundamentally important in many developmental processes.
The earliest defect we can find in the Vangl1/2 double mutant is in the establishment of left-right (L-R) asymmetry . One of the molecular markers that allows one to detect left-right asymmetry in early embryos before any distinct morphological sign of left-right asymmetry shows up is unilateral expression of Nodal in the left lateral plate mesoderm of the early E8.5 mouse embryo [58, 59]. In the Vangl1/2 double mutant, Nodal expression is randomized indicating that L-R asymmetry is randomized and Vangl1/2 mediated PCP acts upstream of Nodal expression to control L-R asymmetry.
The unilateral Nodal expression has to be triggered by an earlier event that breaks the bilateral symmetry. During vertebrate morphogenesis, establishment of L-R asymmetry follows the determination of dorsal-ventral (D-V) and anterior-posterior (A-P) body axes during gastrulation. In mice, bilateral symmetry is broken by a leftward fluid flow across a pit-like, teardrop-shaped node generated by posteriorly localized motile cilia of the node cells [60–63]. Both genetic analysis and mathematical modeling have shown that such leftward nodal flow is both necessary and sufficient to trigger unilateral Nodal expression. Abnormal cilium localization will result in turbulent nodal flow, which disrupts left-right patterning. Therefore, failure to transmit the A-P positional information in the node cells is likely to cause random cilium positioning and randomized L-R patterning. Thus, a mechanism that enables the node cells to interpret A-P positional information to position the nodal cilia sits at the top of the regulatory hierarchy of L-R asymmetry.
We then asked what establishes PCP in the developing embryo and whether PCP also regulates other important directional morphogenetic events such as limb elongation. Because the limb of the Vangl double mutant embryo is much shorter and distal digits were lost, we hypothesized that PCP controls directional limb elongation. Because Wnt5a is expressed in a graded manner in the early developing limb and primitive streak adjacent to the node and Wnt5a -/- mutant embryos exhibits much shortened limb [25, 64], we also hypothesized Wnt5a is a cue that establishes PCP in the embryo. Indeed, Wnt5a genetically interacts with Vangl2 in neural tube closure and hair cell orientation in inner ear .
We then asked whether Wnt5a directs P-D limb elongation of the developing limb by regulating PCP. In the wild type limb, cartilage formation extends distally. But in the Wnt5a -/- distal limb, cartilage failed to extend distally, forming ball-like structure [17, 64]. We found previously that loss of distal digits in the Wnt5a -/- embryo may be caused by upregulation of the anticondrogenic canonical Wnt signaling activity . The ball-like structure of the forming digit cartilage led us to hypothesize that Wnt5a may also control cartilage elongation along the PD axis in limb development by regulating PCP.
To test whether the early differentiated chondrocytes are indeed polarized and show PCP, we examined Vangl2 protein localization and found that Vangl2 was asymmetrically localized along the P-D axis only in the Sox9-positive chondrocytes, not in the Sox9-negative interdigital mesenchymal cells in the limb . These results demonstrate for the first time with a definitive molecular marker that chondrocytes are indeed polarized by PCP. Importantly, in the Wnt5a -/- mutant limb, such polarized Vangl2 localization disappeared indicating that Wnt5a controls chondocyte polarity through PCP .
As Wnt5a genetically interacts with Vangl2 and Wnt5a has been found to bind Ror2, a receptor tyrosine kinase [16, 65], we further hypothesized that Wnt5a transduces its signal through a novel receptor complex containing Vangl2 and Ror2. Mutations in both WNT5A and ROR2 are found to cause Robinow syndrome characterized by shortened limb dwarfism [8–11]. To test this hypothesis further, we generated Ror2 and Vangl2 double mutant embryos and found that they phenocopied the Wnt5a -/- embryo in the limb, craniofacial processes and the tail. The limb phenotypes of the Wnt5a mutants and the Ror2/Vangl2 double mutants are almost identical . In addition, we found in the developing limb bud, Vangl2 antibodies can pull down Ror2. Furthermore, Vangl2 and Ror2 association is significantly enhanced by Wnt5a .
Then we asked how Wnt5a gradient leads to establishment of PCP. In another word, how the cells in the limbs sense the Wnt5a morphogen gradient and interpret the directional information . We found that Wnt5a and many other Wnt ligands, when coexpressed with Ror2, enhanced Vangl2 phosphorylation. Expression of Wnt5a or Ror2 alone also enhanced Vangl2 phorsphorylation to a lesser extent. The smear gel mobility shift pattern suggests that there are hyperphosphorylated and hypophosphrylated Vangl2, indicating that Vangl2 can be phosphorylated on multiple sites. Vangl2 phosphorylation is also detected in vivo in mouse tissues including the limb and brain . Phosphorylation is abolished by calf intestinal phosphatase (CIP) treatment. In addition, Wnt5a -induced Vangl2 phosphorylation is much reduced in Ror2 -/- MEF cells, indicating that Wnt5a induces Vangl2 phosphorylation through Ror2 .
To further understand how Vangl2 phosphorylation is regulated by Wnt5a/Ror2 signaling, we took a great effort to map the phosphorylation sites of Vangl2 . We found that phosphorylation of Vangl2 occurs on conserved Serine (Ser) and threonine (Thr) residues that are organized in two different clusters in the protein. We also demonstrated that phosphorylation within both clusters occurs in a progressive manner such that founder residues are phosphorylated before others . The progressive nature of Vangl2 phosphoylation suggests that different levels of Vangl2 phosphorylation is regulated by distinct Wnt5a dosages and this may be an underlying mechanism whereby responding cells interpret Wnt5a dosages. Because in the developing limb bud, there is a Wnt5a expression gradient from distal to proximal sides, we tested this in vivo first in the limb bud. We dissected the limb bud into distal parts with higher Wnt5a expression, the middle parts with medium Wnt5a expression and proximal parts with lower Wnt5a expression. Indeed, we found that there is a progressive reduction of Vangl2 phosphoryation distal to proximal limb bud . Importantly, in the Ror2 -/- and Wnt5a -/- distal limb bud, hyperphosphorylated forms of Vangl2 were replaced by hypophosphorylated forms that are observed in proximal parts of limb bud. To further test that different levels of Vangl2 phosphorylation are induced by distinct dosages of Wnt5a, cells expressing Vangl2 and Ror2 were cocultured with various numbers of Wnt5a-expressing cells. Indeed, increasing Wnt5a dosages led to progressively more extensive Vangl2 phosphorylation . These in vivo and in vitro studies demonstrate that regulating levels of Vangl2 phosphorylation is a way to sense the Wnt5a morphogen gradient.
Apart from its critical role in controlling PCP, our lab also showed that Wnt5a can promote chondrocyte differentiation by inhibiting the canonical Wnt signaling activity . Therefore, Wnt5a controls both cell proliferation and polarity by coordinating canonical Wnt signaling with Wnt/PCP signaling. It is possible that these two Wnt pathways are mutually inhibitory to each other . In the case of skeletal development, it will be interesting to test whether altered bone morphology due to disrupted PCP signaling also led to abnormal bone mass caused by alteration of canonical Wnt signaling.
This review is organized largely based on my young investigator award lecture at the SCBA biannual meeting in Guang Zhou, 2011.
This review is based on Dr Yingzi Yang's Young Investigator Award Lecture at the SCBA biennial meeting in Guangzhou, China, in 2011. Dr Yang would like to thank all current and previous members of the Yang lab for the hard work that led to this award. This work was supported by the Division of Intramural Research of the National Institutes of Health.
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