Patterning in the Drosophila Embryo
The power of genetics to analyze Drosophila development has been priceless for dissecting the signaling interactions between the Wnt (wingless) and BMP (decapentaplegic) pathways. In certain tissues, such as during Drosophila leg development, antagonism between these pathways is simply hardwired by mutual repression of each other's ligand-encoding gene, whereby Wnt signaling represses BMP expression and BMP signaling represses Wnt expression (e.g., Theisen et al.,1996).
The patterning of the mesoderm in Drosophila provides a perfect model system for investigating more complex, combinatorial signaling mechanisms between the Wnt and BMP signaling pathways. Here they involve synergy and antagonism in the same tissue and even in some of the same cells depending on the target gene (Fig. 1). Wnt and BMP signaling overlaps in the anterior domain of the segmental units of the Drosophila embryo (parasegments). The homeobox genes bagpipe and even-skipped read this positional information, but interpret it completely differently: while Wnt and BMP synergize to induce even-skipped expression (Carmena et al.,1998), Wnt signaling antagonizes BMP signaling to prevent bagpipe expression in the same domain (Azpiazu et al.,1996).
Figure 1. Combinatorial Wnt and bone morphogenic protein (BMP) signaling regulates homeobox genes in the Drosophila mesoderm. Wnt and BMP signaling overlap in the anterior but not the posterior compartment of the embryonic segmental units in Drosophila embryos. The enhancer of the even-skipped gene (eve) integrates synergy between Wnt and BMP signaling through Smad and Tcf binding sites, which mediate activation of expression in the anterior compartment but repression by Tcf and a transcriptional corepressor (R, i.e., Groucho) in the posterior compartment where there is only BMP, but no Wnt signaling. The enhancer of the bagpipe (bap) gene mediates expression in a complementary pattern; in the absence of Wnt signaling it activates bagpipe expression through its Smad binding site; but it also contains a binding site for the FoxG-related transcription factor, a product of the sloppy paired (slp) gene, which associates with a transcriptional corepressor (R, i.e., Groucho) to repress bagpipe expression in the anterior compartment where Wnt signaling specifically induces sloppy paired (FoxG) expression. β indicates β-catenin/armadillo. Figure modified after Lee and Frasch (2005).
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The Wnt and BMP signaling is differently integrated on the relevant enhancers of these genes; while the even-skipped enhancer has a BMP response element (Smad1/5/8 [Mad] and Smad4 [Medea] binding sites) next to a Wnt response element (Tcf [pangolin] binding sites; Knirr and Frasch,2001) to mediate synergy; the bagpipe enhancer, to integrate antagonism, contains a BMP response element next to the binding site for a FoxG forkhead-family transcriptional repressor (Sloppy paired), which is up-regulated in these cells by Wnt signaling (Lee and Frasch,2005) (Fig. 1).
Combinatorial Wnt (wingless) and BMP (decapentaplegic) signaling regulates development of the Drosophila midgut, and in particular homeotic gene expression in the endoderm (labial) and the associated visceral mesoderm (Ultrabithorax; Fig. 2A). This precise regulation of homeotic gene expression governs morphogenesis and subsequent differentiation of specific cell types in the endoderm, such as in the labial expressing cells into the copper cells (e.g, Hoppler and Bienz,1994). The Wnt ligand is expressed in the visceral mesoderm in a domain (parasegment 8) immediately posterior to the BMP ligand-expressing domain (parasegment 7). Autocrine BMP and low Wnt signaling synergize to maintain Ultrabithorax expression in the BMP expression domain; while just posteriorly, in the Wnt expression domain, high Wnt signaling antagonizes BMP signaling to repress Ultrabithorax expression (reviewed by Bienz,1997). This regulation of Ultrabithorax in the visceral mesoderm layer is mirrored by labial regulation in the endoderm: BMP and low Wnt signaling synergize to induce labial expression in the endoderm next to the BMP expression domain in the visceral mesoderm; while further posterior, high Wnt signaling antagonizes BMP signaling-induced expression of labial adjacent to the Wnt expression domain (Hoppler and Bienz,1995).
Figure 2. Combinatorial Wnt and bone morphogenetic protein (BMP) signaling regulates homeotic genes in the Drosophila midgut. A: Extracellular Wnt and BMP signaling from the visceral mesoderm regulate expression of the homeotic genes labial in the endoderm and Ultrabithorax in the visceral mesoderm itself. At a distance from the Wnt expression domain, relatively low levels of Wnt signaling synergizes with BMP signaling to induce expression of labial and maintain Ultrabithorax expression; while close to the Wnt expression domain, higher levels of Wnt signaling antagonize BMP signaling by inducing expression of Teashirt, which encodes a transcriptional repressor that prevents expression of labial and Ultrabithorax in this domain. B: The Ultrabithorax enhancer integrates the synergistic and antagonistic regulation by Wnt and BMP signaling. A Wnt Response Element (WRE, containing conserved Tcf (pangolin) TCF binding sites) sits next to a BMP Response Element (BRE, containing Smad/MAD binding sites [SMAD]) to mediate synergy between BMP and relatively low levels of Wnt signaling. However, the BRE overlaps with binding sites for the Brinker sequence-specific DNA binding protein (BRK), which recruits Teashirt (TSH) and CtBP and thus forms a transcriptional repression complex on the BRE to antagonize BMP signaling in the domain with high Wnt signaling. β, β-catenin/armadillo. Figure modified after Saller et al. (2002).
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Synergy with low Wnt signaling is mediated by means of direct regulation by Tcf (pangolin) and β-catenin (Armadillo) function (Riese et al.,1997), while high Wnt signaling causes antagonism indirectly by means of up-regulation of Teashirt (Mathies et al.,1994; Waltzer et al.,2001), which encodes a Zinc-finger protein that assembles a transcriptional repressor complex containing the transcriptional corepressor CtBP and the sequence-specific DNA binding factor Brinker (Saller et al.,2002). The relevant enhancer region in Ultrabithorax integrates this intricate regulation perfectly (Fig. 2B). It contains a Wnt response element (Tcf [pangolin] binding site; Riese et al.,1997) next to a BMP response element (Smad1/5/8 [Mad] binding sites; Szuts et al.,1998) to mediate the observed synergy between BMP and low levels of Wnt signaling; but also several Brinker binding sites overlapping with this BMP response element to antagonize the positive regulation by BMP/Smad signaling of the Ultrabithorax enhancer directly by high levels of Wnt signaling (Saller et al.,2002).
Dorsal–Ventral Patterning of the Spinal Cord in Vertebrate Embryos
Dorsal–ventral patterning of the spinal cord in vertebrate embryos involves multiple signaling mechanisms (Helms and Johnson,2003). The dorsal spinal cord is characterized by neural crest production and differentiation of dorsal interneurons, along with expression of genes such as olig3 and Math1 (Gowan et al.,2001; Takebayashi et al.,2002; Zechner et al.,2007). The dorsal spinal cord expresses Wnt (Wnt1, Wnt3a; Hollyday et al.,1995; Galli et al.,2007) and BMP (BMP2,4,7; Basler et al.,1993; Lee et al.,1998) ligands, both of which are involved in conferring generally dorsal-specific character in this region. Expression of BMP ligands is initiated by a contact with the surface ectoderm (Liem et al.,1995), and is responsible for Wnt1 expression (Burstyn-Cohen et al.,2004). Wnt signals enhance the BMP pathway as seen by an increase of phospho-smad1/5/8 and expression of downstream target gene Msx1 (Ille et al.,2007). Overactivation of either pathway causes expansion of dorsal-specific domains (Liem et al.,1995; Timmer et al.,2002; Ille et al.,2007; Zechner et al.,2007; Alvarez-Medina et al.,2008), whereas loss-of-function of either results in a failure to specify dorsal-specific cell fates (Nguyen et al.,2000; Muroyama et al.,2002; Zechner et al.,2007), indicating that both Wnt and BMP signals are required for proper fate-specification at the dorsal neural tube. An important question concerns whether the two pathways are independently responsible for dorsal patterning: i.e., whether the two pathways have different outcomes both of which are required for dorsalization of the neural tube, or whether the dorsalization is induced by one of the signals while the other plays a permissive role. To clarify this issue, the direct effect of each pathway has been studied in this context. Wnt signals promote cell proliferation by up-regulating transcription of cyclin D1 (Burstyn-Cohen et al.,2004), whereas BMP signals are responsible for dorsal patterning (Liem et al.,1995,1997; Chesnutt et al.,2004). BMP signals are also required for cell proliferation; however, this appears to be mediated by transcriptional up-regulation of Wnt1 (Burstyn-Cohen et al.,2004). Hence, BMP signals are mainly responsible for patterning, while the role of Wnt signals is to expand the populations of dorsal neuronal progenitors specified by BMP (Chesnutt et al.,2004), both of which are together required for making the dorsal part of the spinal cord.
It has, however, been noted in Wnt/β-catenin overexpression studies that the cell-proliferating effect of the Wnt/β-catenin pathway is best exerted in the ventral side of the neural tube, where BMP signals are absent (Cheung and Briscoe,2003; Ille et al.,2007). In fact, cell proliferation promoted by activation of the Wnt/β-catenin pathway is counteracted by BMP signals in the dorsal neural tube. Similarly, neuronal differentiation caused by BMP signals is best achieved in the absence of Wnt signals. Hence, there is an underlying mechanism of mutual inhibition between Wnt and BMP pathways behind the scene of their cooperative function. Perhaps the negative feedback is taking place to maintain the balance of cell proliferation and differentiation.
Although the above studies in chick and mouse embryos suggest major roles for BMP signals in patterning, whereas for Wnt in proliferation, a study using zebrafish embryos clearly showed that Wnt signaling is required for both proliferation and patterning in the dorsal spinal cord (Bonner et al.,2008). In addition, it has been clarified that cell proliferation and patterning are independently regulated events; blocking cell proliferation does not affect dorsal–ventral patterning of the neural tube. This led authors to a further finding that these two events are regulated by different Tcf/Lef family members of the Wnt signaling pathway; Tcf3 mediates the Wnt/β-catenin signaling for proliferation, while Tcf7 (a.k.a. Tcf1) mediates the same signaling for dorsal neural tube patterning. This finding is significant in that Wnt signaling has a direct role in dorsal patterning, as does BMP signaling. In other words, the patterning process in the dorsal spinal cord is likely to involve a direct synergy of BMP and Wnt signaling rather than a secondary effect. These studies also highlight the situation where the same group of cells (dorsal spinal cord) integrate Wnt and BMP signals both synergistically and antagonistically depending on the task; synergistically for patterning and antagonistically for cell proliferation.
From the dorsal neural tube, neural crest cells delaminate, migrate, and differentiate into various cell types including peripheral neurons. While both Wnt and BMP signals support pluripotency of neural crest cells during the proliferation, these two signals are involved differently in the neurogenic differentiation process; while Wnt signals promote sensory neurogenesis, BMP signals suppress it (Kleber et al.,2005). This exemplifies not only that the roles of Wnt and BMP signals change during development, but also that the mode of crosstalk between the two signals changes as well.
Head and trunk induction has been a subject of intensive studies in the field of developmental biology as it is the basis for much of the vertebrate body plan (reviewed by Stern et al.,2006). In Amphibian embryos, Spemann's organizer provides inductive signals for axis formation. Spemann and Mangold have shown this by transplanting the dorsal lip of the blastopore (the organizer tissue) from a donor embryo to the ectopic (prospective ventral) region of a host embryo. This resulted in an embryo with a second dorsal body axis where the ventral side should have been. The secondary axis consisted of host cells except the notochord (which derives from the graft), proving the existence of inductive signals responsible for axis formation in the grafted tissue (Spemann and Mangold,1924). It is now known from Xenopus studies that this secondary axis induction (with head and trunk structures) is recapitulated by inhibition of both BMP and Wnt signals (Glinka et al.,1997,1998), while BMP inhibition alone often only induces a secondary axis without head structures (Suzuki et al.,1994; but see below). Spemann's organizer indeed expresses chordin and noggin, both of which function to inhibit BMP signals, and Dkk1, a Wnt inhibitor (reviewed by Niehrs,2004).
In analogy to Xenopus embryos as mentioned above, failure to inhibit both Wnt and BMP signals in mouse also affects head formation: Head structures are not properly formed in double heterozygous for Dkk1 and noggin (del Barco Barrantes et al.,2003). However, a similar phenotype is also obtained by a null deletion of Dkk1 (Mukhopadhyay et al.,2001), or by double knock-out of noggin and chordin in which Dkk1 expression is not compromised (Bachiller et al.,2000). It is unclear whether in this context the Wnt and BMP pathways have distinct downstream targets, or, whether they function additively or synergistically to work on common targets.
Ventral Mesoderm Patterning in Xenopus Embryos
One of the first indications of instructive synergy between BMP and Wnt signals in vertebrates was shown in specification of the ventral mesoderm in Xenopus embryos (Hoppler and Moon,1998). In Xenopus blastula, the equator region called marginal zone develops into mesoderm, which is further specified along the dorsal–ventral axis: The dorsal side of the marginal zone gives rise to the notochord and somites, whereas the ventral side develops into tissues such as pronephros kidneys and embryonic blood. Expressions of BMP4 and Wnt8 overlap in the ventral marginal zone, and indeed formation of ventral mesoderm requires both BMP and Wnt signals. Using the vent homeobox genes as molecular markers for ventral mesoderm, Hoppler and Moon showed the following: (1) vent gene expression requires both Wnt and BMP signals; loss of either (experimentally achieved with expression of dominant negative Wnt8 or dominant-negative BMP receptor Ia) results in reduced vent gene expression; (2) strong BMP signaling is sufficient to induce vent genes; (3) Expression of Wnt8 requires activation of the BMP pathway (i.e., dominant-negative BMP receptor expression results in a failure of Wnt8 and vent gene expression). These results may be interpreted such as to suggest that BMP signaling functions upstream of Wnt8 expression in a linear regulatory pathway. In other words, the function of BMP4 is to up-regulate Wnt8, which then induces vent expression. However, Wnt8 is not sufficient to induce vent gene expression (coexpression of dominant-negative BMP receptor and Wnt8 failed to induce vent expression). This means that activation of the BMP pathway is required for Wnt8 to exert its function to induce vent genes. It was later found that the Xenopusvent2 promoter region contains BMP response elements where Smad proteins bind (Rastegar et al.,1999; Hata et al.,2000; von Bubnoff et al.,2005), but the mechanisms through which Wnt regulates vent2 expression are still investigated. Very similar mechanisms have also since been discovered in other vertebrates (Ramel et al.,2005). This network of interactions between Wnt and BMP signaling in the ventral mesoderm would predict that strong inhibition of BMP signaling will not only cause an obvious lack of BMP signaling, but additionally, due to reduced Wnt8 expression, also a lack of Wnt signaling leading to complete dorsalization. Indeed, this is exactly what was observed when BMP signaling was completely blocked by injection of an inhibitory Smad (Tsuneizumi et al.,1997).
Stem Cells and Neural Induction
Embryonic stem (ES) cells undergo self-renewal proliferation while maintaining the potential to differentiate into a variety of cell types. Many studies have been conducted to search for factors that control differentiation of ES cells into desired lineages. Among those are studies to identify factors that cause neural differentiation (Gaulden and Reiter,2008). Fibroblast growth factor (FGF) signaling and inhibition of BMP and Wnt signaling sum up the overall requirement for neural differentiation (Wilson et al.,2001; Kleber and Sommer,2004; Bouhon et al.,2005). BMP and Wnt signals act on ES cells to maintain their pluripotency (Ying et al.,2003; Sato et al.,2004; Nusse,2008). Thus, in the context of maintaining pluripotency and adopting neural cell fate, BMP and Wnt signals have similar effects. A question is raised as to whether these pathways have distinct functions or whether they are redundant. For the maintenance of pluripotency, either BMP or Wnt activation appears to be sufficient: A pharmacological Wnt signaling activator (GSK3β inhibitor) is sufficient to maintain the undifferentiated state of ES cells (Sato et al.,2004). Similarly, up-regulation of Id genes, direct downstream targets of the BMP pathway, is able to maintain self-renewal in the presence of LIF, without a need for serum (Ying et al.,2003). It is, however, unclear whether both of them are responsible for a common target or not.
With regard to neural induction in embryos, BMP inhibition was initially found to be sufficient for inducing the neural cell fate in Xenopus ectoderm; whereas BMP signals promote the epidermal fate (reviewed by Hemmati-Brivanlou and Melton,1997). However, in the chick epiblast, BMP antagonism alone does not induce neural fate (Linker and Stern,2004). This led to a search for additional factors and mechanisms required for chick neural induction (that had presumably been masked in Xenopus assays as they were endogenously supplied). One is FGF signaling, which initiates the neural fate in the medial epiblast (Streit et al.,2000; Wilson et al.,2000), and which was later also found to be required for neural induction in Xenopus (Delaune et al.,2005; Kuroda et al.,2005). FGF3 may function either in favor of inhibiting the BMP pathway or independently of BMP inhibition (Streit et al.,2000; Wilson et al.,2000,2001; Pera et al.,2003). Another group of neural inducing factors identified turned out to encode Wnt inhibitors. Similar to BMP inhibition, Wnt inhibitors promote neural differentiation of epiblast cells that would otherwise adopt the epidermal fate (Wilson et al.,2001). This process requires endogenous FGF signaling, hence a model was proposed where Wnt signals in the lateral epiblast (future epidermal, non-neural ectoderm) inhibits cells to respond to FGF signaling by an unknown mechanism, which in turn allows BMP signals to promote epidermal ectoderm. In this context, the BMP pathway appears to be the key for the cell fate specification as seen in Xenopus: BMP inhibition (experimentally caused by a dominant-negative BMP receptor or by noggin) induces neural fate even in the presence of an FGF inhibitor (Wilson et al.,2000) or Wnt ligands (Wilson et al.,2001). Moreover, BMP sufficiently induces epidermal ectoderm even in the presence of a Wnt inhibitor (Wilson et al.,2001). However, high concentration of the FGF blocker or Wnt ligands inhibits the ability of noggin or dominant-negative BMP receptor to induce neural fate (Wilson et al.,2001). Thus, there remains a possible mechanism of BMP inhibition-independent neural induction, which might relate to regulation of FGF or Wnt signals.
BMP was originally found as a factor promoting formation of cartilage and bone, hence its name bone morphogenetic protein (Wozney et al.,1988). It is interesting to note that, another protein similarly isolated based on its chondrogenic activity, turned out to encode an extracellular Wnt inhibitor (Hoang et al.,1996; originally named FrzB for Frizzled-related molecule expressed in Bone, now called sFRP3, for secreted Frizzled related protein 3). Since then, many studies have shown that the Wnt pathway is indeed involved in promoting bone formation (reviewed by Baron et al.,2006; Hartmann,2006,2007). This has been manifested by various mutations that primarily affect the Wnt pathway yet exhibit considerable phenotypes in bones. Although, contrary to the effect of sFRP3, most of studies suggest that activation of the Wnt pathway promotes bone formation. For example, loss-of-function mutation of a Wnt co-receptor LRP5 causes a low bone-mass phenotype (Gong et al.,2001), while its gain-of-function mutation causes hypermineralization of bones (Boyden et al.,2002; Little et al.,2002), known as a human syndrome high bone mass (HBM) trait, although it has recently been suggested that indirect mechanisms could contribute to this phenotype (Yadav et al.,2008). LRP6 single nucleotide polymorphism mutation impairing Wnt/β-catenin signaling results in low bone mass (Mani et al.,2007). Constitutive activation of β-catenin caused by APC (Adenomatous Polyposis Coli) deletion results in high bone deposition (Holmen et al.,2005). Moreover, Axin2 knock-out in mice causes skeletal defects, such as craniosynostosis where the skull fuses and ossificates at younger stages than normal (Yu et al.,2005; Liu et al.,2007). Finally, transgenic expression of stabilized β-catenin in osteoblasts causes high bone mass, accompanied by defects in osteoclast differentiation, as osteoprotegerin being as a target of Wnt/β-catenin pathway, while a loss of β-catenin in osteoblasts results in low bone mass (Glass et al.,2005; Holmen et al.,2005). All of these examples suggest that overactivation of the Wnt/β-catenin pathway promotes abnormal mineral deposits in bones while decreased Wnt/β-catenin signaling attenuates it, indicating that the pathway is responsible for regulating the right degree of bone formation and mineral deposition.
How is the Wnt/β-catenin pathway involved in bone formation? Why do opposite activities of the pathway (inhibition by sFRP3 and activation by β-catenin) both result in promotion of bone formation? What is the effect of Wnt pathway activation on the BMP pathway during bone formation? It appears that the interaction of the BMP and Wnt pathways is particularly complex in bone development, probably because the effect of the interaction differs depending on the developmental stage.
Most bones derive from mesenchymal precursor cells that have the ability to differentiate into osteoblast, adipocyte, or chondrogenic precursors, with an exception of the skull, which is formed by direct differentiation of neural crest-derived mesenchymal cells into bone tissues (reviewed in Baron et al.,2006; Hartmann,2006,2007). In skeletal bone formation, the fate of mesenchymal precursors is directed to osteoblast progenitors by activation of the Wnt/β-catenin pathway; without activation, mesenchymal precursors differentiate into chondrocytes or adipocytes (Day et al.,2005; Hill et al.,2005). Cells fated to become osteoblasts are, at this stage, called osteoprogenitors, in which the Wnt/β-catenin pathway functions to promote its proliferation and maintain the precursor status (i.e., attenuating further differentiation). BMP signals can stimulate those cells to become mature osteoblast (Amedee et al.,1994; Hughes et al.,1995). Hence, BMP and Wnt signals have opposing effects in osteoprogenitors. Once osteoprogenitors become osteoblasts, Wnt and BMP signals function cooperatively; both BMP2 and Wnt/β-catenin pathways promote further differentiation seen by expression of alkaline phosphatase (ALP; Bain et al.,2003; Rawadi et al.,2003) and mineralization (Holmen et al.,2005). Thus, the Wnt/β-catenin pathway is crucial at multiple steps of bone formation, and the interaction of Wnt and BMP signals is either opposing or cooperative depending on the differentiation step.
At the step during differentiation when mesenchymal precursor cells choose specific cell fates, Wnt and BMP signals have different roles. In mesenchymal cell line C3H10T1/2, which has the ability to differentiate into chondrocyte, adipocyte, muscle, or osteoblasts by extrinsic factors, BMP2 and β-catenin function distinctly. Muscle differentiation is promoted by β-catenin and not by BMP2 (Bain et al.,2003). On the other hand, chondrogenic differentiation is promoted strongly by BMP2 while β-catenin has no effect or rather functions inhibitory (Fischer et al.,2002; Bain et al.,2003). It was also seen in vivo that deletion of β-catenin in osteoblasts causes enhanced chondrogenesis and decreased osteogenesis (Day et al.,2005; Hill et al.,2005; Rodda and McMahon,2006). Furthermore, in C3H10T1/2 cells, stabilized β-catenin strongly inhibits adipocyte differentiation while BMP2 does not affect it (Bain et al.,2003). It is interesting to note that, in the same cell line, exogenous BMP2 promotes TOPflash reporter activity (Bain et al.,2003). Hence, it appears that BMP2 functions in two ways; one to enhance the Wnt/β-catenin pathway and another to function independently of it, and these mechanisms are selectively used depending on the differentiation stage.
Despite the complex contribution of the two pathways, attempts have been made to pinpoint the function of each pathway at the final stage of osteoblast differentiation. In the induction of ALP expression, the ability of Wnt signals to up-regulate ALP is not blocked by cycloheximide, suggesting no requirement for new protein synthesis, while BMP2-dependent ALP induction is blocked, suggesting that the Wnt/β-catenin pathway plays a direct role in ALP induction (Rawadi et al.,2003). It was also shown in primary culture of mouse osteoblasts that defects in osteoblast differentiation caused by β-catenin deletion is not rescued by additional recombinant BMP2, although it normally increases osteogenic markers (Hill et al.,2005). In addition, sclerostin, a protein responsible for regulating the proper bone density, functions on Wnt signals, not BMP signals, for bone formation (van Bezooijen et al.,2004,2007), despite its ability to bind BMP ligands (Kusu et al.,2003; Winkler et al.,2003). Furthermore, it was found in multiple myeloma patients that the myeloma cells secrete a soluble Wnt inhibitor, sFRP-2, which suppresses bone formation and causes bone destruction (Oshima et al.,2005). These studies suggest that the mineralization step in differentiated osteoblasts is much dependent on the Wnt pathway, consistent with the human conditions involving molecular lesions in genes encoding Wnt signaling components.
It has also been proposed in chondrogenesis that Wnt/β-catenin signaling plays a more instructive role than BMP signaling. Chondrogenic differentiation is characterized by Sox9-mediated transcriptional up-regulation of specific collagens (Lefebvre et al.,1997; Zhou et al.,1998; Bi et al.,1999; Akiyama et al.,2002). β-Catenin physically interacts with Sox9 and causes ubiquitination-mediated degradation (Akiyama et al.,2004; Jin et al.,2006). In this context, BMP2 blocks β-catenin–Sox9 interaction through activation of p38 MAPK. This suggests a mechanism where BMP signaling indirectly promote chondrogenesis by blocking Wnt/β-catenin signaling, which negatively works for chondrogenesis.
Tooth development is a particularly rewarding area for studying the functional interaction of signaling pathways. Tooth formation involves tissue interactions between epithelium and underlying mesenchyme, mainly mediated by BMP, hedgehog (shh), and FGF signals (Tucker and Sharpe,1999). Involvement of Wnt/β-catenin signaling, however, has also been suggested. For example, Lef1−/− mice lack both incisor and molar teeth along with lack of whiskers and hairs (van Genderen et al.,1994). Axin2 mutant also displays tooth agenesis (Lammi et al.,2004). Overactivation of β-catenin promotes enlarged and ectopic tooth formation, which is accompanied by expansion of BMP4, Msx1/2, and Lef1 expression domains (Jarvinen et al.,2006; Liu et al.,2008). In contrast, overexpression of Dkk1 blocks teeth formation, which is accompanied by down-regulation of BMP and Msx1/2 expression domains (Liu et al.,2008). In the Dkk1-overexpressed tissues, the ability of BMP4 to induce Msx1/2 is not affected, suggesting that Wnt/β-catenin signals are required upstream of BMP4 function (Liu et al.,2008). Another mouse mutant that exhibits a significant tooth phenotype is a knock-out of Wise (also called Ectodin/USAG-1/SOSTDC1; Kassai et al.,2005; Murashima-Suginami et al.,2007; Ohazama et al.,2008; Munne et al.,2009), a BMP inhibitor and also implicated as a Wnt modulator (Itasaki et al.,2003; Laurikkala et al.,2003; Yanagita et al.,2004). Targeted deletion of Wise/Ectodin shows supernumerary teeth, which is explained by an increase of either BMP or Wnt/β-catenin activity, based on the study of Liu et al (2008). A deletion mutant of LRP4, a negative Wnt signal regulator and also known as Megf7 (Johnson et al.,2005), shows the same phenotype as that of Wise/Ectodin mutant mice (Ohazama et al.,2008), suggesting that Wise/Ectodin may function on LRP4 in this context.
A role for Wise/Ectodin and LRP4 in crosstalk between Wnt and BMP signaling is also suggested in limb development (see below). Limb formation is a classic model for the study of morphogenesis. The process of limb development consists of induction and growth of limb buds, pattern formation along the three axes, and tissue differentiation (Tickle,2006), which are regulated by multiple signaling cascades (Kengaku et al.,1998; Kawakami et al.,2001). In inducing the apical ectodermal ridge (marked by fgf8 expression), BMP signaling is required in the initial step and β-catenin functions subsequently to that; while at the later stage in dorsal–ventral patterning, β-catenin acts upstream of, or in parallel with, BMP signaling (Soshnikova et al.,2003). The role of Wnt and BMP signals further changes during the process of digit separation. Digits are formed in shape by programmed cell death of mesenchymal tissues at interdigital regions and the anterior and posterior margins of the limb buds. BMP ligands are expressed in the right place at the right time to induce cell death (Ganan et al.,1996; Yokouchi et al.,1996; Zou and Niswander,1996). Indeed, when noggin is overexpressed by transgenesis in mouse, interdigital tissue is not completely regressed and thus extra-digits are formed, resulting in soft tissue syndactily (Guha et al.,2002; Plikus et al.,2004). However, despite the apparent reduction of BMP signals, the expression of downstream target genes, Msx1 and Msx2, which are expressed in the interdigital regions and believed to be responsible for the cell death effect (Marazzi et al.,1997), are not affected in noggin-overexpressed limbs (Guha et al.,2002). Strikingly, Dkk1 is expressed in interdigits and its deletion mutant mice exhibit a soft tissue syndactily phenotype similar to the one seen in noggin transgenic mice (Mukhopadhyay et al.,2001). Because BMP2 induces Dkk1 expression as an immediate-early response, it is suggested that the apoptosis caused by BMP signals in normal limb development is mediated by Dkk1, rather than by expression of the direct BMP target Msx1 (Mukhopadhyay et al.,2001; Guha et al.,2002). Deletion of a Wnt signal inhibitor LRP4 (also known as Megf7) also shows a similar phenotype of syndactily (Johnson et al.,2005). LRP4/Megf7 shows a strong homology to LRP5/6 at the extracellular domain while the intracellular domain shows little homology (for example, lacking “PPPSP” motifs that are required for signal transduction: Davidson et al.,2005; Zeng et al.,2005), hence predicted to work as a Wnt signal inhibitor by sequestering ligands.
Reciprocal epithelial–mesenchymal interactions mediated by multiple signaling pathways are a fundamental aspect of vertebrate kidney development. In metanephros formation it involves two groups of tissues: Epithelial ureteric buds branch out of the Wolffian duct by signals derived from the metanephrogenic mesenchymal cells, which surround the ureteric buds and regulate further branching of the ureteric bud. In turn, at the tips of the branches, the ureteric epithelial cells induce mesenchymal condensation. The mesenchymal cells differentiate into different types of cells to eventually form nephrons, while ureteric bud gives rise to the collecting ducts and ureter. These processes require multiple signaling pathways including BMP and Wnt/β-catenin pathways (Schedl and Hastie,2000; Perantoni,2003; Carroll et al.,2005). BMP7 is enriched at the tip of the ureteric bud epithelium (Caruana et al.,2006), while BMP receptors are in both the branching epithelium and mesenchyme cells (Martinez et al.,2001). Mice with targeted deletion of BMP7 show severe dysgenesis of kidneys with little or no glomeruli, due to the failure of mesenchymal condensation at the initial stage (Dudley et al.,1995; Luo et al.,1995). Wnt4 and Wnt6 are expressed in ureteric buds and include tubulogenesis (Stark et al.,1994; Itaranta et al.,2002). Loss of β-catenin in the ureteric bud cell lineage causes defects in branches of ureteric epithelium, resulting in dysplasia or aplasia of kidneys (Bridgewater et al.,2008). Thus both Wnt and BMP pathways are required for kidney morphogenesis.
Because a very high dose of BMP7 signaling inhibits branching morphogenesis of ureteric buds (Piscione et al.,2001), an attempt was made to make a model animal of renal dysplasia by introducing a transgene expressing constitutively activated ALK3 (BMP receptor Ia, the receptor for BMP2,4,7) in mouse. Rosenblum and colleagues then found that mice with high BMP signaling show elevated activity of the Wnt/β-catenin pathway revealed by Tcf-reporter transgene expression (Hu et al.,2003; Hu and Rosenblum,2005). This led the authors to the discovery of a Smad1/Tcf4/β-catenin complex, which drives expression of c-myc in excess amounts (Hu and Rosenblum,2005). Hence, both Wnt and BMP signals function synergistically in c-myc expression in the kidney.
In vitro analyses showed that BMP7 functions in a dose-dependent manner in kidney explants and in cell lines; low doses of BMP7 stimulate cell proliferation and tubular formation in a Smad1-independent manner, while high doses inhibit proliferation and induce apoptosis by means of activation of Smad1 (Piscione et al.,2001). The endogenous level of BMP7 plays beneficial roles for the recovery of renal cells from damages such as ischemia, injuries, and renal failure (Gould et al.,2002; Mitu et al.,2007). Both BMP7 and the BMP inhibitor Wise (also called USAG-1, SOSTDC1, and Ectodin) are abundantly expressed in adult kidneys (Yanagita et al.,2004), which may function to maintain the level of BMP7 signals beneficial for the kidney. After kidney damage, BMP7 plays a critical role in tissue repair (Wang et al.,2003; Zeisberg et al.,2003); while the BMP-antagonist Wise/USAG-1 prevents recovery; indeed, a mouse deletion mutant of Wise/USAG-1 shows a better than normal recovery from nephrotoxin-induced kidney damages, with prolonged survival of renal cells and preserved renal function (Yanagita,2006; Yanagita et al.,2006). In addition, it was found in renal cell carcinoma that Wise/USAG-1 is down-regulated in 20 of 20 cases, although the mechanism is not known (Blish et al.,2008). Because Wise/USAG-1 has also been found to function as a Wnt signal inhibitor in different tissues (Itasaki et al.,2003) it is intriguing that other Wnt inhibitors such as sFRP1 and sFRP2 are also down-regulated in renal cell carcinomas (Gumz et al.,2007; Kawamoto et al.,2008).
Involvement of deregulated activation of β-catenin in carcinogenesis is evident (Giles et al.,2003; Kikuchi,2003; Logan and Nusse,2004). While a wide variety of cancers show elevated β-catenin–dependent transcription, the causal relationship has been most clearly demonstrated in colorectal cancers. Approximately 80% of cases of colorectal cancer show mutations in APC, a protein required to degrade free β-catenin. In addition, 10% of cases show mutations in β-catenin itself, in the residues that normally get phosphorylated for degradation. Thus, 90% of colorectal cancers are associated with molecular lesions that cause overactivation of the Wnt/β-catenin pathway in the gut epithelium (Giles et al.,2003). It is noteworthy that down-regulation of the BMP pathway can also be a cause of intestinal cancer. Loss-of-function mutations of BMP receptor Ia (Howe et al.,2001; Zhou et al.,2001; He et al.,2004) or smad4 (Howe et al.,1998; Hohenstein et al.,2003) causes polyposis in colon. Overexpression of noggin by transgenesis in mouse also causes polyposis (Haramis et al.,2004; Batts et al.,2006). Furthermore, lacking one allele of smad4 increases the chance of developing malignancy in APC-deficient mice (Takaku et al.,1998). Thus BMP signals may antagonize β-catenin–dependent cell proliferation. In the normal gut, β-catenin dependent transcription is active at the bottom of crypts, where stem cells continue to proliferate (Sancho et al.,2003). In contrast, BMP4 is expressed in the intravillus mesenchyme and activates the BMP pathway in the overlying villi epithelium expressing BMPR1a (Haramis et al.,2004; Batts et al.,2006), while crypts express several BMP antagonists (Kosinski et al.,2007). Thus a balance is maintained between production of new cells in the crypt and differentiation of cells at the lumen/villi side: in the stem cell niche environment where the Wnt/β-catenin pathway is active at the bottom of the crypt, new epithelial cells are produced, which then move toward the lumen side where they cease proliferation and differentiate in response to BMP signals to renew the villi epithelium and function to absorb nutrients. This balance can be broken either when Wnt/β-catenin signaling is overactivated and cells continue to proliferate, or when BMP signal dependent differentiation is attenuated (Brabletz et al.,2009). It has been reported that colon cancer cells with stabilized β-catenin express significantly high levels of BMP4 (Kim et al.,2002), presumably reflecting a negative-feedback mechanism, which colon epithelial cells may have. It is also noted that colorectal cancer cell lines are resistant to BMP's tumor suppressing function (Nishanian et al.,2004).
Summary of Functional Interactions Between Wnt and BMP Signaling
Above examples of crosstalk between Wnt and BMP pathways reveal that the mode of interaction might be categorized into at least four groups (Fig. 3). The first is that these pathways have distinct roles at the same time, both of which contribute to a common goal or achievement (Fig. 3A). As seen in the dorsal neural tube, for example, Wnt and BMP signals are responsible for proliferation and patterning, respectively, both of which are required for formation of the dorsal neural tube. The second is that Wnt and BMP pathways seem to work on a common target, and two signals show additive or synergistic effects (Fig. 3B). This was clearly demonstrated in c-myc expression in the kidney. It is possible that they might play redundant roles in such contexts. The third is that Wnt and BMP signals function sequentially and have different roles in the course of developmental stages (Fig. 3C). As seen in osteogenesis and in the gut epithelium, Wnt signals promote proliferation and maintain undifferentiated status, while BMP signals cause differentiation. In those cases, while causing differentiation, BMP signaling blocks the effect of Wnt signals. This would prevent cells from receiving two signals that have opposing functions (maintaining undifferentiated status vs. causing differentiation), thus perhaps helping a smooth transition of differentiation processes. It is interesting to note that, once cells are differentiated, Wnt signals have different roles from the one at earlier stages, and cooperate with BMP signals during osteogenesis. The fourth case is that BMP signals induce expression of Wnt ligands, as seen in the dorsal neural tube and in the ventral mesoderm in Xenopus embryos (Fig. 3D). Once coexpressed, the two signals show further complex crosstalk. Up-regulation of Wnt ligand expression by BMP signaling suggests importance of having both signals in these contexts.
Figure 3. Functional interactions between Wnt and bone morphogenetic protein (BMP) signaling. The four fundamental modes of functional interactions between Wnt and BMP signaling observed in a variety of tissues, illustrated on a highly generalized cell differentiation pathway. A: Wnt and BMP signaling independently regulate different targets in the same cells at the same stage, which are separately required and subsequently contribute toward a common biological goal. B: Wnt and BMP signaling integrate the regulation of a common target in the same cells, which leads to a biological outcome. C: Wnt and BMP signaling independently regulate distinct aspects of a cellular differentiation pathway at different stages of this differentiation pathway. D: Complex cross-regulation between Wnt and BMP signaling (in any of the above ways) additionally relies on regulation of expression of signaling components of one pathway by the other pathway. This generalized representation should, however, not distract from the tissue-, cell-, stage-, and sometimes gene-specific manners of interaction, which represent a fundamental aspect of integrated Wnt and BMP signaling.
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