Wnt regulation of chondrocyte differentiation

Vicki Church1, Tsutomu Nohno2, Claudia Linker3, Christophe Marcelle3 and Philippa Francis-West1,*

1 Department of Craniofacial Development, Guy's, King's and St Thomas' School of Dentistry, Floor 28 Guy's Tower, Guy's Hospital, London Bridge, London SE1 9RT, UK
2 Department of Molecular Biology, Kawasaki Medical School, Kurashiki, Japan
3 Developmental Biology Institute, LGPD, Campus de Luminy, case 107, University of Aix-Marseille II, 13288 Marseille Cedex 09, France

* Author for correspondence (e-mail: pfrancis{at}hgmp.mrc.ac.uk)

Accepted 6 September 2002


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The Wnt family of growth factors are important regulators of several developmental processes including skeletogenesis. To further investigate the role of Wnts we analysed their expression in the developing chick limb and performed functional analyses in vivo and in vitro. We found that Wnt5b and Wnt11 are restricted within the prehypertrophic chondrocytes of the cartilage elements, Wnt5a is found in the joints and perichondrium, while Wnt4 is expressed in the developing joints and, in some bones, a subset of the hypertrophic chondrocytes. These Wnts mediate distinct effects on the initiation of chondrogenesis and differentiation of chondrocytes in vitro and in vivo. Wnt4 blocks the initiation of chondrogenesis and accelerates terminal chondrocyte differentiation in vitro. In contrast, Wnt5a and Wnt5b promote early chondrogenesis in vitro while inhibiting terminal differentiation in vivo. As Wnt5b and Wnt11 expression overlaps with and appears after Indian hedgehog (Ihh), we also compared their effects with Ihh to see if they mediate aspects of Ihh signalling. This showed that Ihh and Wnt5b and Wnt11 control chondrogenesis in parallel pathways.

Key words: Wnt, Ihh, Chondrocyte differentiation


    Introduction
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 Introduction
 Materials and Methods
 Results
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 References
 
Endochondral bone forms from a cartilaginous template. The first step involves the recruitment and condensation of mesenchymal cells. Cells in the core of the condensation differentiate into proliferating chondroblasts, while those cells in the periphery form the perichondrium, the fibrous sheath surrounding the cartilage element. Subsequently, cells in the centre of the condensation differentiate into prehypertrophic chondrocytes which specifically express the marker Indian hedgehog [Ihh (Vortkamp et al., 1996Go)]. Later, these cells undergo hypertrophy which is characterised by an enlargement of the cells and the production of a characteristic matrix: type II and type IX collagens are downregulated, whilst alkaline phosphatase (ALP) and type X collagen are induced, the latter being expressed specifically by hypertrophic chondrocytes (reviewed by Volk and Leboy, 1999Go). This is accompanied by differentiation of cells in the perichondrium into osteoblasts which synthesise the subperiosteal bone surrounding the prehypertrophic and hypertrophic zones. The hypertrophic cells are thought to undergo apoptosis which is accompanied by vascular invasion and bone deposition on the mineralised cartilage (reviewed by Gerber and Ferrara, 2000Go).

Differentiation is controlled by signals from both the prehypertrophic chondrocytes and perichondrium. The secreted factor Ihh expressed in the prehypertrophic region negatively regulates chondrocyte differentiation through the induction of PTHrP in the periarticular perichondrium (Vortkamp et al., 1996Go). Thus, overexpression of Ihh in the developing chick limb prevents chondrocyte terminal differentiation (Vortkamp et al., 1996Go). However, experiments in vitro have shown that, in the absence of the perichondrial signal, Ihh promotes terminal differentiation suggesting that Ihh may signal directly to the chondrocytes to promote differentiation (Stott and Chuong, 1997Go; Akiyama et al., 1999Go). This has been supported by the observation that the hedgehog receptor patched-1, which is induced by Ihh signalling, is expressed in the chondrocytes adjacent to Ihh-expressing cells (Vortkamp et al., 1998Go). Ihh can also act locally on the adjacent perichondrium to initiate bone collar formation and can promote the proliferation of early differentiating chondrocytes in a PTHrP-independent manner (St-Jacques et al., 1999Go; Karp et al., 2000Go; Chung et al., 2001Go).

Other factors that control chondrocyte differentiation include members of the Wnt and fibroblast growth factor families. The Wnt family of proteins consists of at least 22 cysteine-rich, secreted glycoproteins which participate in a multitude of developmental processes (for reviews, see Cadigan and Nusse, 1997Go; Wodarz and Nusse, 1998Go). Of these, Wnt4, -5a, -5b and -14, together with the secreted Wnt antagonist Frzb, are expressed in the developing skeleton and have been shown to have distinct roles (Hoang et al., 1996Go; Hoang et al., 1998Go; Kawakami et al., 1999Go; Wada et al., 1999Go; Yamaguchi et al., 1999Go; Ladher et al., 2000Go; Hartmann and Tabin, 2000Go; Hartmann and Tabin, 2001Go). Wnt4, which is expressed in the developing joints, has been shown to accelerate hypertrophy whilst Wnt5a, which is expressed in the perichondrium, delays hypertrophy (Kawakami et al., 1999Go; Hartmann and Tabin, 2000Go). Finally, Wnt14, which is expressed in the developing joint interzone, has been implicated in the initial steps of joint development (Hartmann and Tabin, 2001Go). In addition, Wnt1 and -7a have been shown to block chondrogenesis in vitro and/or in vivo (Rudnicki and Brown, 1997Go; Stott et al., 1999Go; Tufan and Tuan, 2001Go). Here we have further examined the role of Wnt signalling during chondrogenesis using the in vitro micromass system and in vivo misexpression studies in the developing chick limb.


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 Materials and Methods
 Results
 Discussion
 References
 
In situ hybridisation to tissue sections
35S-in situ hybridisation was performed on 7 µm wax sections as described by Francis-West et al. (Francis-West et al., 1994Go). Sections were counterstained with toluidine blue O. The riboprobe template for cWnt5b was a PCR product equivalent to amino acids 230-355 in the mouse homologue (a gift from A. McMahon, Harvard University, Cambridge, MA); this was linearised with EcoRI and transcribed with SP6 RNA polymerase. The chick osteocalcin riboprobe template was a PCR product encoding amino acids 12-97; the template was linearised with NotI and transcribed with T7 RNA polymerase. The other riboprobes have been described previously: cWnt4 and cWnt5a (Kawakami et al., 1999Go), cWnt11 (Tanda et al., 1995Go), cPTHrP and cIhh (Vortkamp et al., 1996Go), and chick type X collagen (Kwan et al., 1989Go).

Production of retroviruses
Concentrated retroviral supernatants were prepared by the method of Logan and Francis-West (Logan and Francis-West, 1999Go) and were used at a titre of greater than 108 pfu for the in vitro micromass assays. The RCAS BP(A)-Wnt5b retrovirus encoded mouse Wnt5b. The other retroviral constructs have been described previously: ß-catenin (Kengaku et al., 1998Go), Wnt1 (Rudnicki and Brown, 1997Go), Wnt4 (Hartmann and Tabin, 2000Go), Wnt5a (Kawakami et al., 1999Go), Wnt11 (K. Anakwe, L. Robson, P. Buxton et al., unpublished) and Ihh (Vortkamp et al., 1996Go). For control retroviruses we used RCAS BP(A) expressing enhanced-green fluorescent protein (Clontech) or RCAS BP(A) vector.

Micromass cultures
Micromass cultures were prepared as described by Francis-West et al. (Francis-West et al., 1999aGo). However, the cells were resuspended at 2x107 cells/ml in concentrated viral supernatant before plating in 10 µl aliquots in 24-well dishes (Nunc). After 1 hour, cultures were flooded with 1 ml medium (DMEM supplemented with 10% FBS, 1% chick serum, 50 U/ml penicillin, 50 µg/ml streptomycin) and cultured at 37°C and 5% CO2/air in a humidified atmosphere.

Retroviral transgene expression was verified by RT-PCR. RNA was extracted from cultures and DNase-treated as described by Tufan and Tuan (Tufan and Tuan, 2001Go) and Tufan et al. (Tufan et al., 2002Go). One µg total RNA was reverse transcribed using 1.0 µl M-MLV reverse transciptase (Promega) and 500 ng random hexamers (Promega) according to the manufacturer's protocol. Retroviral transgene expression was determined using primers (25 pmol of each) specific for the transgene (Table 1), 1 µl cDNA, 1.5 mM MgCl2, 0.2 mM each dNTP, 1x Thermo-reaction buffer (Promega) and 1.0 U Taq polymerase (Promega) in a total volume of 50 µl. PCR was performed in a Perkin-Elmer DNA Thermal Cycler using one cycle of 94°C for 5 minutes, followed by 25 (GAPDH), 35 (transgenes after 48, 72 hours) or 40 (transgenes after 24 hours) cycles of one minute for denaturing (94°C), one minute for annealing (TA; Table 1), and one minute for elongation (72°C). Negative control PCR reactions were performed in parallel: these contained all components of the reaction except for cDNA.


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Table 1. Primer sequences and annealing temperatures (TA) used for PCR analysis of retroviral transgene expression

 

To assess the effects of Wnt overexpression on the initiation of chondrogenesis, micromass cultures were cultured for three days after which matrix production was assessed by alcian blue staining (Francis-West et al., 1999aGo). The alcian blue dye was extracted with 4 M GuHCl and quantified by measuring its absorbance at 590 nm. The cultures were then re-stained with alcian blue dye and the number of nodules was counted.

Alternatively, for analysis of terminal differentiation, cultures were grown for seven days and then fixed in 95% MeOH for 10 minutes. For histological detection of alkaline phosphatase activity, the cultures were washed in PBS and stained in Naphthol AS-MX phosphate in 0.4 M Tris-HCl (pH 8.3) and Fast Red Violet LB salt at 37°C in the dark for 10 to 15 minutes. Additionally, ALP activity was determined using the method described by Leboy et al. (Leboy et al., 1989Go). Briefly, each micromass culture was lysed in 500 µl lysis buffer and the ALP activity quantified in a 50 µl aliquot using the substrate p-nitrophenyl phosphate. For type X collagen immunohistochemistry, seven day cultures were fixed with MeOH as above, then incubated with type X collagen antibody [diluted 1:500; a gift from Alvin Kwan, University of Cardiff, UK (Kwan et al., 1986Go)] followed by incubation with Texas red-conjugated anti-mouse IgG antibody (diluted to 20 µg/ml; Vector Labs). Fluorescent antibody staining was visualised on an Axiovert 200 fluorescence microscope (Zeiss) using exactly the same exposure time for all photomicrographs.

To determine the effects of Wnt overexpression on cell proliferation, 24-, 48- or 72-hour-old retrovirus-infected micromass cultures were incubated with 10 µM 5-bromo-2'-deoxy-uridine (BrdU) for 30 minutes at 37°C. The cultures were fixed with ethanol, overlaid with 1% agarose to facilitate removal from the tissue culture surface, and processed into paraffin wax and sectioned at 8 µm. Sections were rehydrated, acidified in 2N HCl for 30 minutes and digested with 0.1% type XXIV protease (Sigma) for 60 seconds. Sections were then incubated overnight with biotinylated anti-BrdU antibody (diluted 1:50; Caltag Labs) which was detected using the Vectastain® Elite ABC kit (Vector Labs) in conjunction with the DAB peroxidase substrate kit (Vector Labs) according to the manufacturer's instructions. Sections were counterstained with toluidine blue O and the number of BrdU-labelled and unlabelled cells was counted.

Retroviral infection of limbs
Chick embryonic (O-line) fibroblasts (CEFs) were transfected with retroviral DNA (encoding Wnt5a, Wnt5b, Wnt11, Ihh or a control construct) as described in Logan and Francis-West (Logan and Francis-West, 1999Go). After seven days when the cells were fully infected, the CEFs were trypsinised from the culture plate, pelleted and allowed to consolidate for one hour (Logan and Francis-West, 1999Go). The cell pellet was grafted into the right wing bud of stage 18/19 embryos and the embryos allowed to develop until day 10, during which time the replication-competent retrovirus spreads throughout the limb (Duprez et al., 1996Go). The embryos were fixed in 4% PFA/PBS, and were processed into paraffin wax and sectioned for analysis by in situ hybridisation as above.


    Results
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 Results
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 References
 
Expression of Wnts during chondrogenesis and joint formation
To determine the possible roles of Wnts during chondrogenesis we first mapped their expression by in situ hybridisation to tissue sections of developing chick limbs. This revealed that Wnt4, -5a, -5b and -11 are expressed in distinct domains (Figs 1,2,3). In summary, Wnt5b and Wnt11 are restricted to within the prehypertrophic chondrocytes of the cartilage elements, Wnt5a to the joints and perichondrium, whilst Wnt4 is expressed in both the developing joints and cartilage (Figs 1,2,3).



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Fig. 1. Light field (A,E,I) and corresponding dark field micrographs comparing the expression of Wnt5b (B,F,J) and Wnt11 (D,H,L) with Ihh (C,G,K) in the skeletal elements of the developing wing at stages 28 (A-D), 34 (E-H) and 37 (I-L). Bars, 500 µm. h, humerus; r, radius; u, ulna. HE, haematoxylin and eosin-stained sections. In H and L, arrowheads point to Wnt11 expression in a subset of the prehypertrophic chondrocytes.

 


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Fig. 2. Light field (A,C,E) and corresponding dark field micrographs showing expression of Wnt4 (B,D) and Wnt5a (F) in the developing joints of the stage 28 wing (A,B), stage 37 wing (C,D) and stage 30 leg (E,F). Bars, 500 µm. h, humerus; j, joint interzone; m, mesenchyme; pc, perichondrium; r, radius; sc, synovial capsule; u, ulna. HE, haematoxylin and eosin-stained sections; TolB, toluidine blue-counterstained section.

 


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Fig. 3. Dark field micrographs comparing the expression of Wnt4 (A), Wnt5b (B) and Wnt11 (C) in the stage 40 tibiotarsus. Bars, 500 µm. pr, perichondrial ring.

 

Wnt5b is detected by stage 28 throughout the developing perichondrium and in the adjacent mesenchyme (Fig. 1A,B). The perichondrial expression continues until at least stage 37 (Fig. 1E,F,I,J). By stage 30, Wnt5b expression is also found in a subset of prehypertrophic chondrocytes (Fig. 1E,F,I,J and data not shown). In the stage 40 tibiotarsus, Wnt5b is more widely expressed being found throughout the element except in the distal part consisting of the proliferating chondrocytes (Fig. 3B). Initially, Wnt5a transcripts are present around the developing cartilage condensations between stages 24 and 26 (data not shown). As the element differentiates, Wnt5a transcripts become restricted to the developing perichondrium (Fig. 2E,F and data not shown). In addition to the perichondrium, Wnt5a is detected in the joint interzone in the digits between stages 30 to 32 but is downregulated as the joint cavitates (Fig. 2E,F and data not shown).

Wnt4 transcripts are found in the developing joint and in differentiating chondrocytes (Fig. 2A-D; Fig. 3A) (Kawakami et al., 1999Go; Hartmann and Tabin, 2000Go). Initially, at stage 28 in the elbow, Wnt4 expression is clearly seen in the joint interzone and in the mesenchyme surrounding the joints (Fig. 2A,B). Between stages 30 to 40, Wnt4 transcripts become localised to the surface articular chondrocytes and the joint capsule (Fig. 2C,D). At stage 40 in the tibiotarsus, Wnt4 is expressed by hypertrophic chondrocytes in an area undergoing lateral resorption (Fig. 3A).

Wnt11 transcripts are not detectable within the developing limb elements at stage 28 but are present in the developing humerus at stage 30; this expression continues until at least stage 37 (Fig. 1A,D,E,H,I,L and data not shown). Like Wnt5b, Wnt11 is expressed in a subset of prehypertrophic chondrocytes but, in contrast to Wnt5b, its expression is restricted to a subpopulation of prehypertrophic chondrocytes just beneath the perichondrium (compare Fig. 1F,J with 1H,L). At stage 40 in the tibiotarsus, when the element has fully differentiated, expression of Wnt11 within the prehypertrophic chondrocytes is highest adjacent to the perichondrial ring, a structure which marks the edge of the bone front (Fig. 3C).

The expression patterns of Wnt5b and Wnt11 correlate with that of Ihh suggesting that there may be a relationship between Wnt and Ihh signalling in developing cartilage. To investigate this, we compared the timing and distribution of Wnt5b, Wnt11 and Ihh expression. This revealed that Ihh expression is first apparent at stage 26 as previously reported [data not shown (Vortkamp et al., 1996Go; Vortkamp et al., 1998Go)] whilst Wnt5b and Wnt11 transcripts are not detectable within the developing humerus until stage 30 when they are expressed within the Ihh-expressing domain (Fig. 1E-L).

Wnts have differential effects on chondrogenesis
To investigate how Wnts control chondrogenesis, Wnts were overexpressed using the chick replication-competent retrovirus RCAS BP in the in vitro micromass assay system. As the expression patterns of Wnt5b and Wnt11 suggested a potential relationship with Ihh, we compared their effects with that of Ihh.

Mesenchymal cells from stage 23/24 wing buds were mixed with concentrated retrovirus particles and plated in high-density micromass cultures. Expression of the Wnt transgenes at 24, 48 and 72 hours was investigated by RT-PCR analysis using PCR primer combinations specific to the transgene. This showed that there was either a low level (control, Wnt5b, -11) or undetectable levels (Wnt4, -5a) of transgene expression after 24 hours but that all transgenes were expressed within 48 hours of infection (Fig. 4A).



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Fig. 4. Comparison of the effects of Wnts on the initiation of chondrogenic differentiation of stage 23/24 wing mesenchymal cells in micromass cultures after three days. (A) RT-PCR analysis of retroviral transgene expression. (B) Quantification of alcian blue staining (solid bars) and nodule numbers (open bars). (C) Micromass cultures stained for cartilaginous matrix with alcian blue. In B, values shown are the mean+s.d. of 11 cultures from three independent experiments. *P<0.0001; **P<0.0005; ***P<0.005 (Student's t-test) of Wnt/Ihh-infected cultures compared to control cultures.

 

After three days, cultures were fixed and stained with alcian blue which stains sulphated glycosaminoglycans in the matrix, and the number of nodules was counted to determine the effect of Wnts on the initiation of chondrogenesis. As reported previously, Wnt1 inhibited chondrocyte differentiation as judged by the reduction in both alcian blue staining and the number of cartilaginous nodules (Fig. 4B,C; P<0.0001) (Rudnicki and Brown, 1997Go). Likewise, ß-catenin, a downstream mediator of Wnt1 signalling, also dramatically reduced chondrogenic differentiation (P<0.05; data not shown). Wnt4 reduced alcian blue staining and the number of nodules to approximately 70% of control (P<0.0005; Fig. 4), while Wnt11 had no detectable effect in these assays (Fig. 4B,C). Wnt5a, Wnt5b and Ihh enhanced alcian blue staining by 1.5-fold (P<0.005, P<0.0001 and P<0.0001, respectively; Fig. 4B,C); however, while Wnt5a and -5b increased the number of nodules by 1.5-fold (P<0.005 and P<0.0001, respectively; Fig. 4B,C), Ihh had no significant effect on nodule number (Fig. 4B,C).

Ihh has been shown to promote terminal differentiation in vitro (Stott and Chuong, 1997Go; Akiyama et al., 1999Go). As Ihh, Wnt5b and Wnt11 are co-expressed in the prehypertrophic chondrocytes, with Ihh being expressed prior to Wnt5b and Wnt11, this raised the possibility that these Wnts may mediate Ihh activity to promote chondrocyte hypertrophy in vitro. To investigate this, type X collagen expression and alkaline phosphatase (ALP) activity, which are markers of chondrocyte hypertrophy, were analysed after seven days of culture. We also investigated the effect of Wnt4 since this has been shown to accelerate chondrogenesis when overexpressed in chick limbs in vivo (Hartmann and Tabin, 2000Go).

After seven days, Ihh promoted ALP activity (Fig. 5A,B; P<0.001) as shown previously for sonic hedgehog (Shh) in micromass cultures (Stott and Chuong, 1997Go). Wnt4 also increased ALP activity but not to the extent of Ihh cultures (Fig. 5A,B; P<0.005). In contrast, Wnt5a, Wnt5b and Wnt11-infected cultures did not detectably alter the levels of ALP activity (Fig. 5A,B). Similarly, whole-mount staining of the cultures revealed that type X collagen was expressed by all cultures, including the control, but was upregulated in cultures misexpressing Ihh and Wnt4. In contrast, type X collagen levels did not appear to be changed by Wnt5a, -5b or -11 misexpression (Fig. 5C; n=6 from two experiments).



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Fig. 5. Comparison of the effects of Wnts and Ihh on chondrocyte terminal differentiation of stage 23/24 wing mesenchymal cells in micromass cultures after seven days. (A) Quantification of alkaline phosphatase activity. (B) Micromass cultures stained for alkaline phosphatase activity (red staining). (C) Micromass cultures stained for type X collagen (red fluorescence; magnification x10); examples of the stained nodules are outlined. In A, values shown are the mean+s.d. of nine cultures from three independent experiments. *P<0.005; **P<0.001 (Student's t-test) of Wnt/Ihh-infected cultures compared with control cultures.

 

Overexpression of Wnts does not change proliferation in vitro
Overexpression of Wnt5a and Wnt5b in vitro increased the number of nodules and hence initiated the first steps of chondrogenesis (Fig. 4B,C). This may occur by an increase in cell proliferation and/or cell adhesion. Conversely, Wnt4 decreased the number of cartilage nodules. To investigate the possible mechanism, we pulse-labelled Wnt4-, Wnt5a- and Wnt5b-infected micromass cultures at 24, 48 and 72 hours with bromodeoxyuridine (BrdU) to label the proliferating cells. We also analysed the effect of Wnt11 in parallel. This revealed that, following Wnt misexpression, there were no detectable changes in the overall proliferation in any of the Wnt-infected micromass cultures when compared to the control cultures (Fig. 6 and data not shown).



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Fig. 6. Comparison of the effects of Wnts on the proliferation of stage 23/24 wing mesenchymal cells in micromass cultures after 48 and 72 hours. Values shown are the mean+s.d. percentage of BrdU-labelled cells from six cultures from two independent experiments.

 

Ihh and Wnt signalling controls chondrocyte differentiation in parallel pathways in vivo
The in vitro analyses suggested that Ihh promotes terminal differentiation through Wnt5b- and Wnt11-independent pathways. However, it is possible that these Wnts may mediate the other functions of Ihh. To investigate this we misexpressed Ihh in the developing limb bud using the replication-competent retrovirus RCAS BP to determine whether Ihh induces either Wnt5b or -11 expression. To infect limbs, fibroblasts infected with the Ihh retrovirus were grafted into the developing limb bud between stages 18 and 19, 24 hours prior to the condensation of the developing cartilage elements. This resulted in ectopic expression of Ihh around the cartilage elements and in small domains within the cartilage itself (n=7; Fig. 7C,D) (Duprez et al., 1996Go; Vortkamp et al., 1996Go). Embryos were allowed to grow to day 10 at which time they were analysed histologically for changes in skeletal development and by in situ hybridisation analysis on tissue sections using molecular markers which are expressed at specific stages of differentiation.



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Fig. 7. Effect of misexpression of Ihh in vivo. Chick embryonic fibroblasts (CEFs) infected with retrovirus were grafted into the stage 18/19 wing bud. Limbs were fixed after a further 7 days (i.e. at embryonic day 10) and processed for in situ hybridisation. A,C,E and B,D,F are adjacent sections from two different embryos. Light field (A,B) and dark field (C-F) micrographs showing endogenous and misexpressed Ihh (C,D) and the effect of Ihh on the expression of Wnt5b (E) and Wnt11 (F). The inset in A shows a high power view of the chondrocytes. Bars, 500 µm (A,B); 200 µm (inset).

 

Ihh misexpression resulted in truncation and dysmorphology of the skeletal elements which consisted of disorganised chondrocytes (Fig. 7A,B) (Vortkamp et al., 1996Go). Chondrocyte differentiation was delayed prior to prehypertrophy as indicated by the lack of endogenous Ihh and type X collagen expression (Fig. 7C,D and data not shown) (Vortkamp et al., 1996Go); in addition, PTHrP was upregulated in the periarticular region (data not shown) (Vortkamp et al., 1996Go). These phenotypes were not associated with ectopic induction of Wnt5b or -11; in fact their expression was reduced or absent (Fig. 7E,F). This suggests that these Wnts are not downstream targets of Ihh signalling at least in its ability to prevent terminal differentiation.

To further determine the function and mechanism of action of Wnt5a, -5b and -11, these Wnts were misexpressed in vivo in the developing chick limb. Again, as with Ihh, retroviral infection resulted in patches of ectopic expression in and around the skeletal elements (Fig. 8C,D and data not shown). Wnt5a misexpression resulted in truncated limbs and occasionally fused joints (n=5; Fig. 8A and data not shown) (Kawakami et al., 1999Go; Hartmann and Tabin, 2000Go). Histological and molecular analysis showed that these truncations may result from delayed differentiation of the hypertrophic zone which was either absent or smaller (Fig. 8A,I). Formation of the hypertrophic zone is the main mechanism for elongation of the skeletal element and thus its loss would significantly decrease the length of the skeletal elements. In addition, the prehypertrophic zone was smaller as determined by Ihh expression (FIg. 8G). However, the rounded zone of proliferating cells appeared normal (Fig. 8A). Hence, Wnt5a appeared to block or delay differentiation during or before the prehypertrophic step. Wnt5a was also observed to induce ectopic Wnt5b expression within the cartilage and upregulate the expression of Wnt5b in the perichondrium (Fig. 8E). Although not as pronounced an effect as for Wnt5a, misexpression of Wnt5b likewise resulted in shortened limbs (data not shown). Again, this appeared to be due to a delay in terminal chondrocyte differentiation as suggested by the smaller domain and/or lower levels of type X collagen expression (n=11; data not shown).



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Fig. 8. Effect of misexpression of Wnt5a (A,C,E,G,I) or Wnt11 (B,D,F,H,J) in vivo. Chick embryonic fibroblasts (CEFs) infected with retroviruses expressing either Wnt5a or Wnt11 were grafted into the stage 18/19 wing bud. Limbs were fixed after a further 7 days (i.e. at embryonic day 10) and processed for in situ hybridisation. Comparison of light field (A,B) and dark field (C-J) micrographs showing the endogenous and misexpressed Wnt5a (C) or Wnt11 (D). The effects on chondrogenic differentiation were assessed by analysis of Wnt5b (E,F), Ihh (G,H), and type X collagen (I,J), which are expressed at different stages of chondrocyte differentiation. The insets in A and B show high power views of the chondrocytes from the diaphyseal region in the infected elements. In the Wnt5a-infected elements, the majority of cells are rounded with few prehypertrophic chondrocytes in contrast to the control where the chondrocytes are hypertrophic. In the Wnt11-infected element, the chondrocytes are undergoing hypertrophic differentiation. Bars, 500 µm (A,B); 200 µm (insets).

 

Similarly, Wnt11-infected limbs were slightly truncated and the joints were fused in two out of four cases (data not shown). However, in contrast to Wnt5a, overexpression of Wnt11 (n=4; Fig. 8D) did not appear to delay chondrocyte differentiation as determined by the expression of Ihh, Wnt5b and type X collagen (Fig. 8F,H,J). The proximity of Wnt11 expression to the perichondrial ring suggested a possible link between Wnt11 function and formation of the bone collar. However, there was no apparent change in expression of the bone marker osteocalcin in Wnt11-infected limbs compared to control limbs (data not shown).


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 References
 
The Wnt family of signalling molecules are involved in a multitude of developmental processes including chondrogenesis. Deregulation of the Wnt signalling pathway has also been implicated in post-natal skeletal disorders such as rheumatoid arthritis (Sen et al., 2000Go; Sen et al., 2001Go). To date, Wnt4, -5a, -5b and -14, which are expressed in the developing chick cartilage elements, have been demonstrated to control chondrogenesis by overexpression studies in vivo (Kawakami et al., 1999Go; Hartmann and Tabin, 2000Go; Hartmann and Tabin, 2001Go). Thus, Wnt4 and -5a have been proposed to accelerate and delay chondrocyte differentiation respectively (Hartmann and Tabin, 2000Go). Moreover, Wnt1, -7a and -14 have been shown to block chondrogenesis in vitro (Rudnicki and Brown, 1997Go; Stott et al., 1999Go; Hartmann and Tabin, 2001Go; Tufan and Tuan, 2001Go). We have further investigated the roles of Wnts during chondrogenesis and their mechanism of action. This has revealed additional roles of Wnts and new insights into the mechanisms of Wnt action during chondrocyte differentiation together with their relationship with the Ihh signalling pathway.

To examine how Wnt signalling affects the initiation of chondrogenesis we used a micromass assay system which revealed that Wnt5a and -5b, but not Wnt4 and -11, can promote the first steps of chondrogenesis. Thus, there was an increase in nodule number in Wnt5a- and Wnt5b-infected micromass cultures. In contrast, Wnt4 decreased the number of nodules. These effects are consistent with the expression patterns of Wnt4 and -5a during skeletal development. Wnt5a is initially expressed in the mesenchyme around the developing condensations where it may act to promote chondrogenesis by recruitment of the mesenchymal cells into the chondrogenic lineage. Later, Wnt5a is expressed in the perichondrium which contributes to appositional growth. The ability of Wnt5a to recruit cells into the chondrogenic lineage is further supported by the widening of the diaphysis observed in Wnt5a-infected limbs. In contrast, Wnt4 expression is associated with the developing joint (Kawakami et al., 1999Go; Hartmann and Tabin, 2000Go). The joint develops within a precartilaginous condensation and requires the de-differentiation of these early prechondrocytes (reviewed by Francis-West et al., 1999bGo). Therefore, one function of Wnt4 may be to act together with Wnt14, which is also expressed in the developing joint and inhibits chondrogenesis in vitro and in vivo, to control joint formation (Hartmann and Tabin, 2001Go).

Initiation of chondrogenesis, and hence the formation of nodules in vitro, is associated with an increase in cell proliferation and/or adhesion which can be modulated by Wnt signalling. For example, Wnt1 and -7a increase cell adhesion by maintaining the expression of cell adhesion molecules such as NCAM, N-cadherin and ß1-integrin during chondrogenesis (Stott et al., 1999Go; Tufan and Tuan, 2001Go). This is associated with the arrest of chondrocyte differentiation at the early cell aggregation step (Rudnicki and Brown, 1997Go; Stott et al., 1999Go). As other studies have shown that Wnt5a does not appear to change cell adhesion, as assessed by the expression of N-cadherin, a factor crucial for the condensation step, we determined whether Wnt overexpression affected cell proliferation (Oberlender and Tuan, 1994Go; Tufan and Tuan, 2001Go). However, although Wnt5a controls the proliferation of undifferentiated limb mesenchymal cells in vivo, BrdU-labelling studies showed that the overexpression of Wnt5a did not significantly affect the levels of cell proliferation in micromass culture (Yamaguchi et al., 1999Go). Similarly, Wnt4 and -5b did not change cell proliferation. Although it is still possible that Wnts specifically change proliferation in the forming aggregates/nodules, which form a subpopulation of the culture, these data do suggest that other mechanisms may be involved. For example, these Wnts could affect the expression of other cell adhesion molecules which have not yet been analysed. It is also possible that Wnts affect cell sorting which occurs during chondrogenesis in micromass culture (Cottrill et al., 1987Go).

The changes in nodule number following overexpression of Wnt4, -5a and -5b were paralleled by changes in the levels of matrix, as assessed by staining for sulphated glycosaminoglycans with alcian blue, suggesting that Wnt4, -5a and -5b signalling does not significantly change matrix production/degradation. However, this analysis does not rule out effects of Wnt signalling on other matrix molecules. For example, Wnt4 is expressed in regions of hyaluronan synthesis and thus it is possible that Wnt4 regulates hyaluronan production. High levels of hyaluronan prevent chondrogenesis by inhibiting the early steps of cell aggregation, and hyaluronan is also a key component of joint cavitation (Toole, 1981Go; Dowthwaite et al., 1998Go).

Wnt4, which accelerates terminal differentiation in vivo, also promoted terminal differentiation in vitro (Hartmann and Tabin, 2000Go). Therefore, Wnt4 is likely to modulate the rate of chondrocyte differentiation. It has been previously proposed, as for other signalling factors such as GDF-5, that Wnt4 signals from the joint to control the development of the adjacent skeletal elements (Francis-West et al., 1999aGo; Hartmann and Tabin, 2000Go). However, in addition to its expression in the developing joints we also found Wnt4 transcripts in a subpopulation of hypertrophic cells at stage 40 in some but not all developing bones. Thus, it is possible that Wnt4 may directly promote differentiation rather than acting through a relay of signals or controlling cell cycle progression from the developing joint.

The expression of Wnt5b and Wnt11 overlapped with Ihh and its receptor patched-1 (Vortkamp et al., 1998Go). As Ihh is expressed prior to both Wnt5b and Wnt11, this raised the possibility that these Wnts may mediate the effects of Ihh. However, the in vitro and in vivo studies suggested that these Wnts are components of distinct pathways. First, unlike Ihh, Wnt5b and -11 did not promote terminal differentiation in vitro. Second, overexpression of Wnt5b and -11 did not block differentiation prior to the prehypertrophic step. Finally, Ihh overexpression did not induce the ectopic expression of either Wnt5b or -11.

The expression of Wnt11 in a subset of prehypertophic chondrocytes just underlying the presumptive bone collar suggested that Wnt11 has a role in bone collar formation. Furthermore, in the tibiotarsus at stage 40, Wnt11 expression clearly demarcates the perichondrial ring, which is the edge of the developing cortical bone. Wnt5b is also expressed in this region. However, these Wnts are unlikely to be involved in the Ihh-regulation of bone collar formation as the hedgehog receptor, patched-1, is expressed in the perichondrium and periosteum suggesting that Ihh signals directly to the cells involved in bone collar formation and not through a relay in the prehypertrophic zone (St-Jacques et al., 1999Go). Consistent with this, overexpression of Wnt11 (or Wnt5a and -5b) did not induce development of the bone collar, although this does not rule out other roles for these Wnts such as controlling vascularisation. The joint fusions observed in Wnt11-misexpressed limbs may result from defects in the musculature, since Wnt11 misexpression in vivo affects myogenic differentiation (K. Anakwe, L. Robson, P. Buxton et al., unpublished).

Whilst these overexpression studies suggested that Wnt5b is not part of the Ihh signalling pathway, they did suggest that it has a role in controlling the rate of differentiation. In Wnt5a- and Wnt5b-infected limbs, chondrocyte differentiation was delayed as shown by either the absence or smaller domain of type X collagen expression. Consistent with its expression in prehypertrophic chondrocytes, the function of Wnt5b may be to delay terminal differentiation, and thus act to balance Ihh signalling, which can promote terminal differentiation. These studies also showed that Wnt5b may autoregulate its own expression or be controlled by Wnt5a in the adjacent perichondrium. Strikingly, ectopic Wnt5b expression was even found in the developing muscles following Wnt5a misexpression.

Bothe Wnt5a and -5b led to different effects on terminal chondrocyte differentiation in vivo and in vitro: differentiation was delayed or blocked in vivo whilst it was not affected in vitro (see also Kawakami et al., 1999Go; Hartmann and Tabin, 2000Go). As demonstrated by others and also shown here, hedgehog signalling has different effects in vivo and in vitro, blocking and promoting differentiation respectively (Vortkamp et al., 1996Go; Stott and Chuong, 1997Go). The ability of hedgehog to promote differentiation in vitro, but not in vivo, has been attributed to the absence of perichondrial signals in early micromass cultures (Stott and Chuong, 1997Go). Thus, like Ihh, the ability of Wnt5a/5b to block or delay terminal differentiation in vivo may be mediated through the perichondrium. Alternatively, there may be other factors expressed in vivo, but not in vitro, which modify the effect of Wnt5a/5b signalling.

The Wnts have been simply divided into two families based on their ability to transform the C57MG mammary epithelial cell line and their effect when injected into Xenopus embryos: the Wnt1 class which includes Wnt1, -3a, -7a and -8, and the Wnt5a class which contains Wnt4, -5a and -11. However, recent data has suggested that this classification is biased and has raised the complexity of subdivision of the Wnt family which may be dependent on the receptor profile. For example, Wnt5a can induce a secondary axis in Xenopus when co-injected with the Wnt receptor Frizzled5, whilst on its own Wnt5a cannot (He et al., 1997Go). Furthermore, the Wnt1 class does not always signal through the canonical Wnt pathway (Kengaku et al., 1998Go). Indeed, in our study, members of the Wnt5a class exhibited distinct effects of chondrogenesis which may be related to different signalling pathways. Analyses in zebrafish and Xenopus embryos have shown that Wnt5a signals through the phosphatidyl-inositol/Ca2+ or protein kinase C (PKC) pathways whereas Wnt11 is suggested to signal via JNK (Slusarski et al., 1997Go; Djiane et al., 2000Go; Kuhl et al., 2000Go; Tada and Smith, 2000Go). PKC is required for chondrogenesis of mesenchymal stem cells and, therefore, Wnt5a may initiate chondrogenesis by PKC activation (Chang et al., 1998Go; Yang et al., 1998Go). In contrast, ß-catenin has been implicated in the Wnt4 signalling pathway (Hartmann and Tabin, 2000Go). Finally, it is clear that the Wnt family of growth factors are fundamental players in the control of skeletal development and degenerate abnormalities. The challenge will be to unravel the diverse effects of Wnt signalling and how they interact with other signalling pathways. Recent advances have already emphasised the increasing complexity of these networks showing that Wnt3a interacts with the BMP pathway to promote chondrogenesis in the C3H10T1/2 cell line (Fischer et al., 2002Go).


    Acknowledgments
 
We thank A. Brown, C. Healy, A. Kwan, A. McMahon and C. Tabin for reagents and the Arthritis Research Campaign for funding.


    References
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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