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IntroductionThe molecular basis underlying osteoblast development ispoorly understood. Collagen I and alkaline phosphatase (Ap)are among the earliest markers expressed by the osteoblastlineage (Aubin, 1998). Runx2, a runt-domain transcriptionfactor, is essential for osteoblast differentiation as Runx2–/–

mice possess a completely cartilaginous skeleton due to thelack of osteoblasts (Ducy et al., 1997; Komori et al., 1997; Ottoet al., 1997). Similarly, mice lacking the osterix (Osx; Sp7 –Mouse Genome Informatics) gene, which encodes a zinc-fingertranscription factor, do not develop bone, indicating anessential role of this factor in osteoblast differentiation(Nakashima et al., 2002). Osx is believed to functiondownstream of Runx2 as Runx2 is expressed normally in Osx–/–

mice but Osx is not expressed in the Runx2–/– animals(Nakashima et al., 2002).

Indian hedgehog (Ihh) signaling is indispensable forosteoblast development in the endochondral skeleton. Indeveloping cartilage, Ihh is primarily expressed byprehypertrophic chondrocytes (chondrocytes immediatelyprior to hypertrophy) as well as in early hypertrophicchondrocytes, and Ihh signals to both immature chondrocytesand the overlying perichondrial cells (St-Jacques et al., 1999;Vortkamp et al., 1996). Ihh–/– mice completely lack osteoblastsin the endochondral skeleton, at least in part because of thefailure to express Runx2 (St-Jacques et al., 1999). Furthermore,Ihh is required for the formation of ectopic bone collar ingrowth plates containing both wild-type and PTH/PTHrPreceptor null chondrocytes (Chung et al., 2001). Geneticmanipulation of Smoothened (Smo), which encodes anobligatory component of the Hh signaling pathway, has

revealed that cells devoid of Smo, hence Hh signaling, fail toundergo osteoblast differentiation (Long et al., 2004).Therefore, direct Ihh input in early osteogenic progenitors isnecessary for osteoblast differentiation. However, it is notknown whether Ihh interacts with other osteogenic signals inthis regulation, or whether Ihh also functions at later stages ofosteoblast development.

Wnt signaling has been implicated in postnatal bone masshomeostasis. Wnt molecules exert their functions by activatingseveral distinct intracellular pathways, including that mediatedby β-catenin, known as the canonical pathway (Huelsken andBirchmeier, 2001; Wodarz and Nusse, 1998). In this pathway,Wnt proteins signal through the Frizzled family of receptorsand the low-density lipoprotein receptor-related proteins (Lrp5and Lrp6, Arrow in Drosophila) as co-receptors, resulting instabilization of β-catenin (Mao et al., 2001b; Pinson et al.,2000; Tamai et al., 2000; Wehrli et al., 2000). The stabilizedβ-catenin transports to and accumulates in the nucleus whereit interacts with transcription regulators including the lymphoidenhancer-binding factor 1 (Lef1) and T-cell factors (Tcf1, Tcf3and Tcf4), leading to transcriptional activation of downstreamtarget genes (Eastman and Grosschedl, 1999). Recently, Lrp5was found to be inactivated in patients with the osteoporosis-pseudoglioma syndrome (Gong et al., 2001). Conversely, anactivating mutation in Lrp5 has been linked in two separatecases to individuals with a high bone density syndrome(Boyden et al., 2002; Little et al., 2002). Furthermore, micedeficient in Lrp5 were viable but postnatally developed a lowbone mass phenotype because of reduced osteoblastproliferation and function (Kato et al., 2002). The endogenousWnt ligand(s) that signal through Lrp5 have not beenidentified. Moreover, the role of the Wnt/β-catenin pathway in

Signals that govern development of the osteoblast lineageare not well understood. Indian hedgehog (Ihh), a memberof the hedgehog (Hh) family of proteins, is essential forosteogenesis in the endochondral skeleton duringembryogenesis. The canonical pathway of Wnt signalinghas been implicated by studies of Lrp5, a co-receptor forWnt proteins, in postnatal bone mass homeostasis. In thepresent study we demonstrate that β-catenin, a centralplayer in the canonical Wnt pathway, is indispensable for

osteoblast differentiation in the mouse embryo. Moreover,we present evidence that Wnt signaling functionsdownstream of Ihh in development of the osteoblastlineage. Finally Wnt7b is identified as a potentialendogenous ligand regulating osteogenesis. These datasupport a model that integrates Hh and Wnt signaling inthe regulation of osteoblast development.

Key words: Hh, Wnt, Osteoblast, Mouse

Summary

Sequential roles of Hedgehog and Wnt signaling in osteoblastdevelopmentHongliang Hu1,*, Matthew J. Hilton1,*, Xiaolin Tu1, Kai Yu2, David M. Ornitz2 and Fanxin Long1,2,†

1Department of Medicine, Washington University Medical School, St. Louis, MO 63110, USA2Department of Molecular Biology and Pharmacology, Washington University Medical School, St Louis, MO 63110, USA*These authors contributed equally to this work†Author for correspondence (e-mail: flong@wustl.edu)

Accepted3 November 2004

Development 132, 49-60Published by The Company of Biologists 2005doi:10.1242/dev.01564

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the development of the osteoblast lineage remains unclear asmice deficient in β-catenin die because of gastrulation defectsby E8.5 before skeletal development occurs (Haegel et al.,1995; Huelsken et al., 2000). Interestingly, several studies havesuggested that activation of canonical Wnt signaling caninduce osteoblast differentiation in cell culture models (Bainet al., 2003; Gong et al., 2001; Rawadi et al., 2003).

Here, we examine the role of β-catenin in osteoblastdevelopment by removing it from early osteogenic tissuesusing the Cre/LoxP-mediated gene inactivation. Wedemonstrate that Wnt signaling is essential for osteoblastdevelopment and acts downstream of Hh. We also identifyWnt7b as a potential ligand controlling osteogenesis in vivo.

Materials and methodsMouse strainsAll mouse strains, including Ihh+/–, β-cateninc/c and Dermo1-Cre areas previously described (Brault et al., 2001; St-Jacques et al., 1999;Yu et al., 2003). The β-cateninc/c strain was acquired through theJackson Laboratory (Bar Harbor, ME).

Analyses of mouse embryosEmbryonic tissues were collected in PBS, fixed in 10% formalinovernight at room temperature, then processed and embedded inparaffin prior to sectioning at 6 µm. For detection of mineralization,sections were stained with 1% silver nitrate (von Kossa method) andcounterstained with Methyl Green. In situ hybridization wasperformed as described previously, using 35S-labeled riboprobes(Long et al., 2001) or using digoxigenin-labeled riboprobes (Brent etal., 2003). For BrdU analysis, pregnant females were injected withBrdU at 0.1 mg/g body weight at 2 hours prior to harvest. Embryoswere collected in ice-cold PBS, processed and sectioned as above.BrdU detection was performed using a kit from Zymed Laboratories(South San Francisco, CA) as per instructions. Labeling index wasscored for a least four sections from various planes of section throughthe cartilage. Multiple wild-type and mutant pairs of littermates wereanalyzed and results from a representative pair are reported here. Forimmunohistochemistry, sections were prepared as above and stainedwith a monoclonal antibody against β-catenin (BDBiosciences/Pharmingen, San Diego, CA) following antigen retrievalusing SDS (BD Biosciences/Pharmingen protocol). Signal wasdetected using the Vectorstain Elite ABC kit (Vector Laboratories,Burlingame, CA) and DAB as chromagen (Zymed Laboratories).

Cell lines and osteogenesis assaysThe C3H10T1/2 cells, the Wnt3a-expressing cell line and the controlcell line were obtained from ATCC (Manassas, VA) and maintainedas per instructions. Wnt3a-conditioned medium and the controlconditioned medium were harvested as per instructions. Therecombinant N-terminal fragment of Shh (N-Shh) was purchased fromR&D Systems (Minneapolis, MN) and used at 1 µg/ml. C3H10T1/2cells were cultured to confluence prior to N-Shh or Wnt3a stimulation.AP expression was detected either by substrate staining or a chemicalassay as previously described (Katagiri et al., 1994). In experimentswhere both GFP fluorescence and AP staining were visualized, BCIPand NBT were used as substrates for AP (Roche DiagnosticsCooperation, Indianapolis, IN). To assess the effects of cycloheximideon Hh-induced gene expression, C3H10T1/2 cells were treated withN-Shh for 24 hours with the addition of either DMSO or 10 µg/mlcycloheximide (Sigma, dissolved in DMSO) before harvested for real-time PCR (see below).

Expression constructs and retroviral infectionsRetroviral expression constructs were generated by a two-step

procedure. The various cDNA fragments were first cloned into thevector pCIG which contains the GFP sequence following the internalribosomal entry site (IRES) and the nuclear localization signal (NLS)(Megason and McMahon, 2002). The fragment containing the clonedcDNA along with the IRES-NLS-GFP sequence was subsequentlycloned into the retroviral vector pSFG (Ory et al., 1996). The Smo*cDNA was as previously described (Long et al., 2001). Dkk1 wasdonated by Dr Christopher Niehrs (Glinka et al., 1998). Thedominant-negative form of Tcf4 (dnTcf4) was originally from DrMcCormick (Tetsu and McCormick, 1999). A control virus was alsogenerated by cloning the NLS-GFP sequence into pSFG. Viruses werepackaged as previously described (Ory et al., 1996). Viral titers weredetermined by counting the GFP-positive cells under a fluorescencemicroscope. For infections, cells ~50% confluent were incubated withthe virus-containing medium for 24 hours, and then cultured in regularcomplete medium for an additional 4 days before harvest for eitherAP assay or RNA extraction. For bone nodule formation assays,C3H10T1/2 cells were infected with either the Smo* or the controlvirus for 24 hours and then cultured in regular complete medium inthe presence of ascorbic acid and β-glycerophosphate for 2 weekswith medium changed as needed. The cells were finally fixed in 10%formalin and stained with Alizarin Red. For transient transfections ofWnt genes, full-length sequences of Wnt5a, Wnt7b or Wnt9a (ATCC)were cloned into pCIG and then transfected with Lipofectamine(GibcoBRL, Gaithersburg, MD).

Real-time PCRTotal cellular RNA was isolated using Trizol reagent (GibcoBRL).The cDNA was prepared using reagents from Roche DiagnosticsCooperation. Real-time PCR was performed on GeneAmp5700(Applied Biosystems, Foster City, CA) as per instructions. All primerswere designed using the ABI software Primer Express and sequencesare available upon request. Three independent samples were analyzedfor each condition and results were normalized to GAPDH in eachsample. For analyses of cartilage samples, cartilage elements wereisolated from the hindlimbs of E14.5 embryos and collected in liquidnitrogen. The cartilage was pulverized in liquid nitrogen with a smallpestle fit in 1.5 ml tubes, and then extracted for RNA using the Trizolreagent.

Resultsβ-Catenin is critical for osteoblast developmentTo determine the role of canonical Wnt signaling in osteoblastdevelopment, we generated Dermo1-Cre; β-cateninc/c

conditional knockout (CKO) embryos in which β-catenin wasspecifically removed from the progenitor cells giving rise tothe skeleton. The Dermo1-Cre line directs Cre expression inthe mesenchymal precursors of both chondrocytes andosteoblasts (Yu et al., 2003). The T-cell factor 1 (Tcf1) anddickkopf 1 (Dkk1) have been shown to be transcriptionaltargets of the Wnt canonical signaling pathway (Cong et al.,2003; Roose et al., 1999). At E14.5 both molecules areexpressed in the perichondrium flanking the prehypertrophicand the hypertrophic regions in wild-type embryos (Fig. 1A,B,arrows), indicating active Wnt signaling within this domain. Inthe CKO littermates, however, Tcf1 and Dkk1 are no longerdetectable in the perichondrium (Fig. 1A′,B′, arrows). Theseresults confirm that removal of β-catenin abolishes canonicalWnt signaling in the perichondrial cells of Dermo1-Cre; β-cateninc/c embryos.

At E18.5 the mutant embryos exhibited shortened limbs anda twisted body axis. Whole-mount skeletal preparationsrevealed that the mutant skeleton lacked bone although

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cartilage was present (data not shown). The von Kossa stainingconfirmed that bone was not present in either the endochondralor the intramembranous skeleton. Specifically, in the humerusof E18.5 wild-type embryos, a bone collar surrounding thecartilage characteristically extends from the diaphysis to theperichondrial region flanking the hypertrophic cartilage (Fig.1C,C′, arrow), which also undergoes mineralization (Fig. 1C′,asterisk). In the humerus of E18.5 CKO embryos, no bonecollar was detected on the cartilage surface (Figures 1D,D′,arrow) even though mineralization of hypertrophic cartilageoccurred (Fig. 1D′, asterisk). Similarly, in the skull of wild-type embryos, the parietal bone is well ossified by E18.5 (Fig.

1E, arrow). However, no ossification was detected in a similarregion in CKO embryos (Fig. 1E′, arrow).

Consistent with the lack of bone at the histological level,molecular analyses by in situ hybridization demonstrated thatmature osteoblasts failed to develop in Dermo1-Cre; β-cateninc/c embryos. Collagen type I (Col1a1) was expressed ata lower level in early osteoblast progenitors but wassubsequently upregulated with further differentiation. In thehumerus of E18.5 wild-type embryos, robust expression ofColα1(I) was detected in the perichondrium surrounding theprehypertrophic and hypertrophic cartilage (Fig. 1F, arrow), inthe bone collar flanking the marrow cavity (Fig. 1F, ‘M’), as

Fig. 1. β-Catenin is required for bone formation. (A-B′) In situ hybridization using digoxigenin-labeled riboprobes against Tcf1 (A,A′) or Dkk1(B,B′) on sections of E14.5 humeri of wild-type (A,B) or Dermo1Cre; β-cateninc/c (CKO) embryos (A′,B′). Arrows indicate the perichondrium.(C-E′) Von Kossa staining of sections through the humerus (C-D′) or the parietal bone (E,E′) of E18.5 wild-type (C,C′,E) or CKO embryos(D,D′,E′). Boxed areas in C,D are shown at higher magnification in C′,D′, respectively. Arrows indicate bone (stained black, C′ and E) orequivalent regions where bone is missing (D′,E′). Asterisks indicate mineralization in the cartilage (stained black, C′,D′). (F-K′) In situhybridization using digoxigenin-labeled (F-H′) or 35S-labeled riboprobes (I-K′) against Col1a1 (F,F′,I,I′), Bsp (G,G′,J,J′) or OC (H,H′,K,K′) onsections of E18.5 humeri (F-H′) or heads (I-K′) of wild type (F-K) or CKO embryos (F′-K′). Arrows indicate the bone collar (F-H) or thefrontal bone (I-K) in the wild-type animal or the equivalent regions in the mutant embryo (F′-K′). Asterisks indicate the primary spongiosa. (L-O′) Histological analyses of the humerus from wild-type (L-O) versus CKO (L′-O′) embryos. The distal end of the humerus is towards the rightin each panel. Boxed regions are shown at a higher magnification as insets. H, hypertrophic chondrocytes. Red arrow in M indicates a red bloodcell present in a blood vessel invading the cartilage. Magenta arrow in M′ indicates a cell undergoing hypertrophy, whereas the orange arrowsindicate immature chondrocytes. Green arrows in N and O indicate the primary spongiosa and the bone collar, respectively. Blue arrows in O′point to perichondrial cells. B, brain; M, marrow cavity; P, proliferating chondrocytes; H, hypertrophic chondrocytes.

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well as in the primary spongiosa (Fig. 1F, asterisk). In CKOlittermates however, only a low level of Col1a1 expression wasdetected in the perichondrium (Fig. 1F′, arrow), with noCol1a1 expression in the marrow cavity (Fig. 1F′, ‘M’). LikeCol1a1, bone sialoprotein (Bsp; Spp1 – Mouse GenomeInformatics) was normally expressed in the perichondriumsurrounding the prehypertrophic and hypertrophic cartilage(Fig. 1G, arrow), as well as in the primary spongiosa (Fig. 1G,asterisk). In CKO embryos however, Bsp was not detectable ineither the perichondrium or the marrow cavity (Fig. 1G′, arrow,‘M’). Finally, osteocalcin (OC; Bglap1 – Mouse GenomeInformatics) was normally expressed by mature osteoblastspresent either in the bone collar (Fig. 1H, arrow) or in theprimary spongiosa (Fig. 1H, arrowhead), but no OC wasdetectable in CKO embryos (Fig. 1H′, arrow). Similarly in theskull, whereas Col1a1, Bsp and OC are normally all expressedin the frontal bone at E18.5 (Fig. 1I-K, arrows), none of thesemarkers was detected in the equivalent regions of CKOlittermates (Fig. 1I′-K′, arrows). Thus, removal of β-catenin by

Dermo1-Cre disrupted osteoblastdifferentiation.

Histological analyses of the developinglong bones also revealed a significant delayin chondrocyte maturation in CKO embryos.At E14.5, chondrocytes at the diaphysis of anormal humerus became hypertrophic (Fig.1L); this, however, was not the case in theCKO embryo where all chondrocytesremained small (Fig. 1L′). The delay inhypertrophy in the CKO mutant continuedthrough E15.5, when only a small cluster ofchondrocytes at the core of the humerusinitiated hypertrophy (Fig. 1M′, magentaarrow), whereas the vast majority of cellsremained immature (Fig. 1M′, orangearrows). By contrast, in E15.5 wild-typelittermates, blood vessels invaded thehypertrophic cartilage of the humerus (Fig.1M, inset). Extensive hypertrophy dideventually occur in the CKO mutant byE17.5 and this was accompanied by vascularinvasion as evidenced by red blood cells inthe core of the humerus (Fig. 1N′). However,no bone was detected in the mutant humerusat either E17.5 (Fig. 1N′) or E18.5 (Fig. 1O′)even though the primary spongiosa (Fig. 1N,arrows) and a bone collar (Fig. 1O, arrows)were evident in the wild-type littermate.Instead, the humerus cartilage in CKOmutants was surrounded by thin layers ofperichondrial cells at E18.5 (Fig. 1O′,arrows). Thus, β-catenin is required for boththe normal schedule of chondrocytehypertrophy, as well as bone formation.

Molecular basis for osteoblast defectin Dermo1-Cre; β-cateninc/c embryoTo characterize further the arrest ofosteoblast development in Dermo1-Cre; β-cateninc/c (CKO) embryos, additionalmolecular analyses were performed on the

humerus of E14.5 embryos. Consistent with the analyses atE18.5, Col1a1 was normally expressed at a low level in theperichondrium towards the epiphysis (Fig. 2A, arrowhead) butwas strongly upregulated in regions surrounding thehypertrophic cartilage (Fig. 2A, arrow). In CKO embryoshowever, a low level of Col1a1 was maintained throughout theperichondrium without any increase at the diaphysis (Fig. 1A′,arrowhead). Alkaline phosphatase (AP) was expressed in asimilar pattern as Col1a1 in the wild-type embryo (compareFig. 2A with 2B, arrowheads and arrows), except that AP wasalso expressed by early hypertrophic chondrocytes (Fig. 2B,‘H’). In the CKO embryo, only a low level of AP was detectedin the perichondrium (Fig. 2B′, arrowhead). No AP expressionwas evident in the chondrocytes of the E14.5 humerus,consistent with a delay in chondrocyte maturation with theremoval of β-catenin (Fig. 2B′). The failure to upregulateCol1a1 and AP in the perichondrium of the CKO mutant wasnot secondary to a delay in chondrocyte hypertrophy, as bothmarkers remained expressed at low levels even at E18.5 after

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Fig. 2. Molecular basis of osteoblast defects in Dermo1Cre; β-cateninc/c (CKO) and Ihh–/–

embryos. In situ hybridization using 35S-labeled riboprobes against Col1a1 (A-A′′), AP (B-B′′), Runx2 (C-C′′) or Osx (D-D′′, E-F′) on sections of humeri from wild type (A-F), CKO(A′-F′) or Ihh–/– embryos (A′′-D′′) at E14.5 (A-D′′), E15.5 (E,E′) or E18.5 (F,F′). H,hypertrophic chondrocytes; L, ligament; M, marrow cavity. Asterisks indicate expression inchondrocytes. Arrowheads and arrows indicate various regions of the perichondrium (seetext).

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hypertrophy had occurred (Fig. 1F′; data not shown). Thesedata indicate that in the absence of β-catenin, osteoblastdevelopment was arrested after low levels of Col1a1 and APwere expressed in the early progenitors.

Additional analyses were performed to define the arrest inosteoblast development. At E14.5 in the wild-type embryo,Runx2 was most robustly expressed in the perichondrium andin the hypertrophic chondrocytes (Fig. 2C, arrow and ‘H’,respectively), although it was also expressed at a low level inimmature chondrocytes (Fig. 2C, asterisk). In the CKOembryo, Runx2 expression was maintained in both theperichondrium and immature chondrocytes (Fig. 2C′, arrowand asterisk respectively) although chondrocyte hypertrophyhad not yet occurred. Osx was normally expressed at highlevels in the perichondrium flanking the prehypertrophic and

hypertrophic chondrocytes (Fig. 2D, arrow) in addition to alower level in the prehypertrophic cells (Fig. 2D, asterisk).However, it was not detectable in the CKO embryo (Fig. 2D′,arrow). The lack of Osx expression in the perichondrium wasnot secondary to the delay in chondrocyte maturation, as it wasnot detected there in these embryos at either E15.5 (Fig. 2E′)or E18.5 (Fig. 2F′) after hypertrophy had occurred. Therefore,osteoblast development was arrested after Runx2 but prior toOsx expression in the Dermo1-Cre; β-cateninc/c embryo.

Compared with the Dermo1-Cre; β-cateninc/c mutant, Ihh–/–

embryos exhibited an earlier defect in osteoblast development.Specifically, at E14.5 Col1a1 was not detectable in theperichondrium of long bones of the Ihh–/– mutant (Fig. 2A′′,arrowhead), although Col1a1 was expressed in the ligament(Fig. 2A′′, ‘L’). Similarly, no AP-expressing cells were present

Fig. 3. β-Catenin promoteschondrocyte proliferation andmaturation. (A-C′) In situhybridization using 35S-labeledriboprobes against Col1a1 (A,A′),Ihh (B,B′) or Ptc1 (C,C′) onsections of E15.5 humeri of wildtype (A-C) or Dermo1Cre; β-cateninc/c (CKO) embryos(A′,B′,C′). H, hypertrophicchondrocytes. M, marrow cavity.Arrow in A′ denotes a cluster ofhypertrophic chondrocytes. Arrowsin C and C′ indicate theperichondrium. (D) BrdU labelingindex of chondrocytes in thehumeri of E15.5 wild type (WT) orDermo1Cre; β-cateninc/c (MT)embryos.

Fig. 4. Distribution of β-catenin in the developing cartilage.Immunohistochemistry with an antibody against β-catenin onsections of tibia from E14.5 wild-type (A,A′) versus Ihh–/–

embryos (B,B′), or from E16.5 wild type (C,C′) versus Ihh–/–

embryos (D,D′). (E) In situ hybridization using a 35S-labeledriboprobe against Col10a1 on a section immediately adjacent tothat shown in D. Boxed regions in A-D are shown at a higher

magnification in A′-D′. Redarrows in A′ and C′ indicatesnuclear staining of β-catenin.Yellow arrow in C′ marks anucleus negative for β-cateninstaining. Arrows in C and Dindicate peripheralchondrocytes with nuclearstaining. Arrowheads in A′, B′and D′ indicate staining at thecell junctions. Asterisk in B

denotes the cartilage proper. Asterisks in D mark chondrocyteswith β-catenin staining. P, proliferating chondrocytes; PH,prehypertrophic chondrocytes; EH, early hypertrophicchondrocytes; LH, late hypertrophic chondrocytes; C, columnarchondrocytes.

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in the perichondrium of the Ihh–/– mutant (Fig. 2B′′,arrowhead). Moreover, Runx2 was not expressed in theperichondrium of the Ihh–/– mutant (Fig. 2C′′, arrow) althoughscattered expression was detected in chondrocytes (Fig. 2C′′,asterisk). Similarly, the Ihh–/– mutant did not exhibit anyexpression of Osx in the perichondrium (Fig. 2D′′, arrow),although a low level of expression in chondrocytes wasdetectable (Fig. 2D′′, asterisk). These results demonstrate thatIhh signaling is required for the initiation of the osteogenicprogram.

β-Catenin promotes chondrocyte maturation andproliferationConsistent with observations at the histological level asdescribed earlier, molecular analyses confirmed that β-cateninwas necessary for the proper maturation of chondrocytes. AtE15.5 in the wild-type humerus, strong expression of collagentype X Col10a1, a specific marker for hypertrophicchondrocytes (Linsenmayer et al., 1991), characteristicallydefines two hypertrophic zones (Fig. 3A, ‘H’) separated by anascent marrow cavity (Fig. 3A, ‘M’). In the CKO embryo,however, expression of Col10a1 had just begun in a small

cluster of cells in the center of the humerus (Fig. 3A′,arrow), indicating a marked delay in chondrocytematuration. Consistent with the delay, Ihh expression,which delineates two discreet prehypertrophicdomains in the wild-type humerus at E15.5 (Fig. 3B),remained in a contiguous region of the diaphysis in aCKO littermate (Fig. 3B′). Patched 1 (Ptch1), whichencodes the receptor for Hh proteins, is also atranscriptional target of the Hh pathway (McMahon etal., 2003). In the wild-type embryo, Ptch1 wascharacteristically upregulated in the immatureproliferating chondrocytes (Fig. 3C, ‘P’) adjacent tothe Ihh-expressing domain, at the primary spongiosawithin the marrow cavity (Fig. 3C, ‘M’), as well as inthe perichondrium flanking the Ihh-expressingchondrocytes (Fig. 3C, arrow). Remarkably, in theCKO embryo, although Ptc1h expression in thechondrocytes was similar to the wild-type level,expression in the perichondrium was much reduced(Fig. 3C′, arrow). Thus, the osteoblast defect in theCKO embryo correlates with a suppressed response ofthe perichondrial cells to Ihh signaling.

Chondrocyte proliferation was also impaired in theDermo1-Cre; β-cateninc/c mutant. The BrdU labelingexperiments showed that at E15.5 in CKO embryos theproliferation rate of chondrocytes was reduced by~40% compared with that of wild-type littermates(Fig. 3D). The defects in chondrocyte maturation andproliferation described here are similar to thatobserved in the Col2a1-Cre; β-cateninc/c mutant(Akiyama et al., 2004).

Canonical Wnt signaling is disrupted in Ihh–/–

embryosWe next examined the potential relationship betweenIhh and canonical Wnt signaling in osteoblastdevelopment. Immunohistochemistry was performedon sections of long bones to determine the distributionof β-catenin. At E14.5, β-catenin was normally

detected in both prehypertrophic and early hypertrophicchondrocytes (Fig. 4A, ‘PH’ and ‘EH’ respectively) as well asin the perichondrium (Fig. 4A, box). Immature andproliferating chondrocytes, on the other hand, were largelynegative for the β-catenin signal (Fig. 4A, ‘P’). At highermagnification, β-catenin clearly accumulated in the nucleus ofthe perichondrial cells flanking the prehypertrophicchondrocytes (Fig. 4A′, arrows). Some staining was alsoobserved at the boundaries of the cells (Fig. 4A′, arrowheads),probably reflecting β-catenin in association with the adherensjunction. Remarkably, in the Ihh–/– mutant, no nuclear β-catenin was detectable in perichondrial cells although lowlevels of β-catenin were detected at the cell boundaries (Fig.4B′, arrowheads). In addition, in Ihh–/– embryos, no signal wasdetected in chondrocytes (Fig. 4B, asterisk), consistent with theabsence of hypertrophic chondrocytes at this stage.

To discern whether the lack of nuclear β-catenin in theperichondrial cells was due to the delay in chondrocytehypertrophy in Ihh–/– embryos, we examined β-cateninlocalization at E16.5 when hypertrophy has occurred in themutant animals. In E16.5 wild-type embryos, β-catenincontinued to be present in the prehypertrophic and early

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Fig. 5. Canonical Wnt signaling impaired in the Ihh–/– embryo. In situhybridization using digoxigenin-labeled riboprobes against Tcf1 (A,A′), Dkk1(B,B′), Lef1 (C,C′), Tcf3 (D,D′) or Tcf4 (E,E′) on sections of E14.5 tibia of wildtype (A-E) versus Ihh–/– embryos (A′-E′). Arrows indicate the perichondrium.P, proliferating chondrocytes; PH, prehypertrophic chondrocytes; H,hypertrophic chondrocytes; C, chondrocytes.

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hypertrophic chondrocytes, but was not found in latehypertrophic chondrocytes (Fig. 4C, ‘PH’, ‘EH’ and ‘LH’respectively). In addition β-catenin also accumulated in the‘columnar chondrocytes’ (chondrocytes organized in columnsin the growth plate, Fig. 4C, ‘C’), as well as in chondrocytesat the periphery abutting the perichondrium (Fig. 4C, arrow).Importantly, the perichondrial cells flanking theprehypertrophic chondrocytes accumulated a high level of β-catenin in the nucleus (Fig. 4C, box; Fig. 4C′, red arrows). Thehigh level of nuclear accumulation appeared to be restricted toa small population of cells in the perichondrium, as theneighboring perichondrial cells closer either to the epiphysisor to the diaphysis did not exhibit such a significant level of β-catenin in the nucleus (Fig. 4C′, yellow arrow). In E16.5 Ihh–/–

embryos, no significant nuclear staining for β-catenin wasdetected in the perichondrium throughout the length of the longbone, although signals at the cell boundaries remained (Fig.4D, box; Fig. 4D′, arrowheads). In the cartilage proper,scattered expression of β-catenin was observed (Fig. 4D,asterisks), probably reflecting a dysregulated process ofchondrocyte maturation in the Ihh–/– mutant. Finally,chondrocytes adjacent to the perichondrium maintained a wild-type level of β-catenin (Fig. 4D, arrows). In situ hybridizationfor Col10a1 confirmed that a majority of chondrocytes becamehypertrophic at E16.5 in the Ihh–/– embryo (Fig. 4E).Therefore, β-catenin normally accumulates in the nucleus ofperichondrial cells within the osteogenic region; however, thisaccumulation is disrupted in the absence of Hh signaling.

We next examined canonical Wnt signaling in the long bonesof wild type versus Ihh–/– embryos by assaying the expressionof target genes Tcf1 and Dkk1. As described earlier, in E14.5wild-type embryos, both Tcf1 and Dkk1 were expressed in theperichondrium flanking the prehypertrophic and the

hypertrophic regions (Fig. 5A,B, arrows). Tcf1 was alsoexpressed at a lower level in prehypertrophic and proliferatingchondrocytes (Fig. 5A, ‘PH’ and ‘P’ respectively). In the Ihh–/–

littermate, however, no expression was detected in theperichondrium for either Tcf1 or Dkk1 (Fig. 5A′,B′, arrows),although expression of Tcf1 in the chondrocytes wasmaintained (Fig. 5A′,‘C’). Other members of the Lef/Tcffamily were not detectable in the perichondrium of the E14.5wild-type tibia (Fig. 5C-E, arrows), but were expressed inchondrocytes in distinct patterns. In particular, Lef1 expressionwas largely restricted to proliferating chondrocytes (Fig.5C,‘P’); Tcf3 was detected throughout the cartilage proper(Fig. 5D); Tcf4 was expressed both in the proliferatingchondrocytes and at a higher level in hypertrophicchondrocytes (Fig. 5E, ‘P’ and ‘H’ respectively). In the Ihh–/–

littermate, whereas Tcf3 and Tcf4 remained expressed in thechondrocytes (Fig. 5D′,E′, ‘C’), Lef1 was no longer detectable(Fig. 5C′,‘C’). Thus, Tcf1 is the primary member of theLef/Tcf family expressed in the perichondrium. Moreover, thecanonical Wnt signaling pathway is impaired in the Ihh–/–

mutant.

Hh-induced osteogenesis requires Wnt signalingTo gain insight in the functional relationship between Hh andWnt signaling, we investigated osteogenesis induced by Hhand Wnt in C3H10T1/2 cells. The pluripotent mouseembryonic mesenchymal cell line C3H10T1/2 gives rise tomesodermal cell types, including osteoblasts, chondrocytesand adipocytes in response to external stimuli (Taylor andJones, 1979). Consistent with previous reports (Kinto et al.,1997; Nakamura et al., 1997; Spinella-Jaegle et al., 2001;Yuasa et al., 2002), recombinant Hh protein (N-Shh) inducedalkaline phosphatase (AP) expression in these cells as early as

Fig. 6. Hh signaling induces osteogenesis in C3H10T1/2cells. (A) Induction of AP by N-Shh. Results are shown fortriplicate samples from a representative experiment. Thisapplies to all AP assays that follow. (B,B′) Cell-autonomous induction of AP by the constitutively activeSmo*. (B) A fluorescence picture showing cells expressingGFP in the nucleus. (B′) A bright-field picture of the samefield showing cells stained for AP activity. Numbersindicate cells expressing both GFP and AP. Asteriskindicates a cell expressing GFP but not AP. (C,C′) Bonenodule formation induced by the constitutively activeSmo*. Retroviruses encoding Smo* induced bone nodules(arrows) stained positive by Alizarin Red (C′), whereas a

control virus did not (C). (D-G) Expression ofCol1a1 (D), Bsp (E), Runx2 (F) or Osx (G) inHh-treated (red bars) versus control cells (greybars) by real-time PCR. The mRNA levels werenormalized to the level of glyceraldehyde-3-phosphate dehydrogenase (GAPD) mRNA.Results are shown for triplicate samples from arepresentative experiment. This also applies toother real-time PCR experiments that follow.(H,I) Effects of cycloheximide on Hh inductionof Runx2 (H) and Osx (I) expression. Runx2 andOsx mRNA levels were determined by real-timePCR and normalized to Gapd for cellsstimulated for 24 hours with N-Shh in thepresence of either DMSO (C) or 10 µg/mlcyclohexamine (CHX).

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day 2 of Hh stimulation (Fig. 6A). AP expression reached veryhigh levels by day 4 and continued to rise at day 6 (Fig. 6A).To examine whether Hh signaling induced osteogenesis inC3H10T1/2 cells in a cell-autonomous manner, a constitutivelyactive allele of Smo (Smo*) that cell-autonomously activatesHh signaling (Long et al., 2001; Xie et al., 1998) was expressedin the cells via a retroviral vector. The green fluorescent protein(GFP) with a nuclear localization signal (NLS) was co-expressed with Smo* via an internal ribosome entry site (IRES)to monitor expression. The cells were infected at a low titer ofvirus to ensure a low percentage of infected cells and the cellswere eventually stained for AP activity. This experimentdemonstrated that AP expression was activated exclusively incells expressing nuclear GFP hence Smo* (Fig. 6, compare Bwith B′, where the same number represents the same cell).Cells that were in direct contact with the GFP-positive cells,but were themselves negative, were never found to express AP.Interestingly, all GFP-positive cells did not express AP (Fig. 6,compare B with B′, asterisks), indicating heterogeneity of theculture such that not all cells responded to Hh to undergoosteoblast differentiation. Furthermore, with ascorbic acid andβ-glycerophosphate, constitutive Hh signaling by virally

expressed Smo* in C3H10T1/2 cells wassufficient to induce bone nodules stained byAlizarin Red (Fig. 6C′, arrows). Thus, consistentwith the previous finding that Hh signaling isdirectly required for osteoblast development invivo (Long et al., 2004), Hh signaling cell-autonomously stimulates osteogenesis inC3H10T1/2 cells.

To gain insight to the osteogenic processinduced by Hh signaling, expression ofosteoblast markers were examined by real-timePCR following stimulation with N-Shh. By day3 of Hh treatment, the mRNA levels for Col1a1and Bsp were both induced significantly morethan controls (Fig. 6D,E). Interestingly, Runx2was only modestly induced by Hh during 3 daysof treatment (Fig. 6F), whereas expression ofOsx was increased approximately threefold overthe control at all time points (Fig. 6G).Moreover, stimulation of Osx by Hh did notrequire de novo protein synthesis, ascycloheximide was unable to inhibit itsinduction (Fig. 6I), although it did repress theinduction of Runx2 (Fig. 6H). Thus, Hh-inducedosteogenesis in C3H10T1/2 cells correlates witha modest increase of Runx2 and a markedinduction of Osx expression. These resultsdemonstrate dual roles for Hh signaling inosteoblast development, which were not possibleto discern in the Ihh–/– mutant owing to the earlyblock in osteogenesis.

We next investigated osteogenesis induced byWnt signaling in C3H10T1/2 cells. Wnt3a-conditioned medium induced AP as early as day2 of treatment, and AP expression reached amaximum after 4 days of stimulation (Fig. 7A).Like AP, other osteoblast markers, includingCol1a1, osteopontin (OP; Spp1 – MouseGenome Informatics) and OC were all

significantly induced within 48 hours of Wnt3a stimulation(Fig. 7D-F, respectively). However, neither Runx2 nor Osx wasinduced during this time (Fig. 6B,C, respectively). Thus, inC3H10T1/2 cells Wnt3a can induce osteogenesis withoutchanges in either Runx2 or Osx. These results therefore reveala second role for Wnt signaling that is downstream of Runx2and Osx, and is in addition to its requirement for Osxexpression as described earlier.

We next determined whether Wnt signaling was required forHh-induced osteogenesis. To this end, we generatedretroviruses that express either Dkk1, a secreted antagonist thatprevents Wnt ligands from binding to the LRP5/6 co-receptor(Mao et al., 2001a), or a dominant-negative form of Tcf4(dnTcf4) that prevents transcriptional activation by canonicalWnt signaling (Tetsu and McCormick, 1999). Consistent withthe results from the staining experiment described earlier (Fig.6B and 6B′), virally expressed Smo* induced AP expressionin C3H10T1/2 cells (Fig. 7G, red bar). Importantly, wheneither the Dkk1 or the dnTcf4 virus was co-infected at similartiters with the Smo* virus, AP expression was reduced by~50% (Fig. 7G). Similar inhibition was detected at the mRNAlevel by real-time PCR for both AP and Bsp (Fig. 7H,I,

Development 132 (1) Research article

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respectively). Thus, Hh-induced osteogenesis is at least in partmediated by canonical Wnt signaling.

Wnt7b is a potential endogenous signal controllingosteogenesisAs an initial step to identify endogenous Wnt ligands thatmediate Hh-induced osteogenesis, we examined the expressionof Wnt genes in relation to Hh signaling. We first surveyed all19 murine Wnt molecules by real-time PCR in C3H10T1/2cells following Hh stimulation. These experiments revealedthat Wnt5a, Wnt7b and Wnt9a were consistently induced overcontrol levels following 24 or 48 hours of Hh treatment (Fig.8A,B, respectively). Moreover, induction of Wnt7b and Wnt9adid not require de novo protein synthesis, as cycloheximide didnot inhibit it although it did repress Wnt5a induction. Themechanism for the induction of Wnt7b and Wnt9a expressionby cycloheximide is not understood but a similar stimulatoryeffect has been previously reported (Mullor et al., 2001).

We next examined expression of Wnt5a, Wnt7b and Wnt9ain the developing long bones of both wild-type and Ihh–/–

embryos. At E14.5, Wnt5a was normally expressed byprehypertrophic chondrocytes (Fig. 8F, asterisk) as well asperichondrial cells flanking the prehypertrophic andhypertrophic chondrocytes (Fig. 8F, arrow). This expressionpattern was largely maintained in the Ihh–/– embryo, althoughchondrocyte hypertrophy was delayed at this stage (Fig. 8F′).Wnt9a was normally expressed in the outer layers of theperichondrium surrounding either the shaft or the joint regionof the cartilage element (Fig. 8H, red arrow and orange arrow,respectively); the expression was much reduced in the Ihh–/–

embryo (Fig. 8H′). Finally, in the wild-type embryo, Wnt7bwas strictly expressed by perichondrial cells flanking theprehypertrophic chondrocytes (Fig. 8G, arrow), but theexpression was abolished in the Ihh–/– mutant (Fig. 8G′, arrow).Wnt7b downregulation was confirmed by real-time PCR withRNA from limb cartilage elements isolated from E14.5 Ihh–/–

versus wild-type embryos (Fig. 8I). Thus, Wnt7b is specificallyexpressed by potential osteogenic cells in the developing longbone and is regulated by Hh signaling.

We next determined whether Wnt5a, Wnt7b or Wnt9a could

Fig. 8. Wnt7b as a potential endogenous signal regulating osteogenesis. (A,B) Induction of Wnt genes in C3H10T1/2 cells stimulated with N-Shh for 24 hours (A) or 48 hours (B), as determined by real-time PCR. Results are presented as fold changes over unstimulated cells. Wntproteins not detectable in either stimulated or unstimulated cells are not included in the graphs. *P�0.05. (C-E) Effects of cycloheximide onHh induction of Wnt5a (C), Wnt7b (D) or Wnt9a (E) expression. mRNA levels were determined by real-time PCR and normalized to Gapd forcells stimulated for 24 hours with N-Shh in the presence of either DMSO (C) or 10 µg/ml cyclohexamine (CHX). (F-H′) In situ hybridizationusing 35S-labeled riboprobes against Wnt5a (F,F′), Wnt7b (G,G′) or Wnt9a (H,H′) on sections of E14.5 humeri (F,F′,H,H′) or ulna (G,G′) fromwild type (F-H) or Ihh–/– (F′-H′) embryos. Red arrows indicate the perichondrium. Yellow arrows indicate the joint regions. PH,prehypertrophic chondrocytes; H, hypertrophic chondrocytes. (I) Expression of Wnt7b in cartilage isolated from the hindlimbs of E14.5 wild-type or Ihh–/– embryos, assayed by real-time PCR. The average is presented for three pairs of wild type versus Ihh–/– littermates. The levels ofmRNA were normalized to Gapd and presented in arbitrary units with the level in the wild-type embryo set at 1. (J) AP assay for C3H10T1/2cells transfected with either the empty vector pCIG or constructs expressing Wnt5a, Wnt7b or Wnt9a. Average is shown for triplicate samplesfrom a representative experiment.

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induce osteoblast differentiation. AP activity was assayedfollowing transient transfection of constructs expressing one ofthe Wnt ligands in C3H10T1/2 cells. Wnt7b, but neither Wnt5anor Wnt9a, increased AP expression approximately fourfoldover the control (Fig. 8J). Thus, Wnt7b can induce pluripotentmesenchymal cells to undergo osteoblast differentiation.

Sequential roles of Hh and Wnt signaling inosteoblast developmentOverall, these data suggest a model in which Hh and Wntsignals control osteoblast development in a sequential manner(Fig. 9). On the one hand, Hh acts on very early progenitors toinitiate the osteogenic program by activating expression ofRunx2, low levels of Col1a1 and AP (Fig. 9, ‘1’). This phaseof signaling appears to require only a weak Hh response, asindicated by low levels of Ptch1 expression. On the other hand,Hh induces expression of Wnt ligands that signal through β-catenin, which is in turn required for Osx expression andfurther osteoblast differentiation (Fig. 9, ‘2’). Whereas Wnt/β-catenin signaling is not sufficient to induce Osx, Hh candirectly stimulate Osx expression (Fig. 9, ‘3’), and this phaseof signaling correlates with a strong Hh response, as reflectedby robust Ptch1 expression. Finally, Wnt signaling can promoteosteoblast differentiation without inducing Osx expression(Fig. 9, ‘4’). In summary, Hh and Wnt signals orchestrate theprogression of osteoblast development.

DiscussionIn this study, we provide genetic evidence that β-catenin, a keycomponent of the canonical Wnt pathway is required forosteoblast development. A comparative analysis with the Ihh–/–

mutant places Wnt signaling downstream of Ihh signalingduring early phases of osteogenesis. Results from C3H10T1/2cells demonstrate that Wnt signaling at least in part mediatesosteogenesis induced by Hh proteins. Finally, Wnt7b isidentified as a potential endogenous Wnt signal important forosteoblast development.

Despite its role in the adherens junctions, removal of β-catenin did not cause any noticeable changes in themorphology and organization of cells. Moreover,immunostaining with an antibody against α-catenin, which

normally interacts with β-catenin, did not show any changes inthe distribution in either chondrocytes or the perichondrial cellsof the Dermo1Cre; βcatc/c mutant (data not shown). It is likelythat plakoglobin, which is closely related to β-cateninsubstitutes for β-catenin to maintain the integrity of adherensjunctions, as previously reported in the β-catenin–/– embryo(Huelsken et al., 2000). Similarly, deletion of β-catenin fromthe apical ectodermal ridge (AER) of the limb bud did notchange the distribution of E-cadherin in the ectoderm (Barrowet al., 2003). Thus, the osteoblast defect observed in the presentstudy is most likely due to disruption of canonical Wntsignaling. In keeping with this notion, β-catenin normallyaccumulates at high levels in the nucleus of potentialosteogenic cells in the developing long bone, and expressionof the Wnt target genes Tcf1 and Dkk1 was abolished in theDermo1Cre; βcatc/c mutant.

Although bone was largely absent throughout theDermo1Cre; βcatc/c embryo, some bone formation wasobserved in the scapula of the mutant. Interestingly, expressionof Tcf1 and Dkk1 was detected in the scapula of the mutantembryo at E15.5 (data not shown). This most probably reflectsincomplete removal of β-catenin in the scapula by Dermo1-Cre, resulting in residual Wnt signaling in this tissue.Consistent with the crucial role of β-catenin in osteogenesis, aprevious study showed that removal of β-catenin from thecranial neural crest cells by Wnt1-Cre resulted in agenesis ofmost craniofacial bones (Brault et al., 2001).

Whereas Wnt/β-catenin is required for osteogenesisthroughout the skeleton, Ihh is required only for theendochondral bones. Recently, Smo, which encodes anobligatory component of the Hh pathway, was removed withWnt1-Cre to eliminate Hh response in cranial neural crest cells(CNCC) that normally give rise to many of the craniofacialbones via intramembranous ossification (Jeong et al., 2004).This resulted in the absence of most CNCC-derived headbones. However, owing to the early growth and patterningdefects associated with an increase in apoptosis and a decreasein proliferation, it is difficult to conclude whether osteogenesisper se was impaired by the removal of Hh responsiveness.Thus, it remains to be determined whether Hh or other signalsact upstream of Wnt/β-catenin signaling in the head skeleton.

Interestingly, Hh response in the perichondrium iscompromised in the Dermo1Cre; βcatc/c embryo. Ptch1 wasexpressed at a much-reduced level in the perichondrial cells inthe absence of β-catenin. It is possible that Wnt/β-cateninsignaling directly regulates the amplitude of Hh response inosteogenic cells. Alternatively, strong Hh response may reflecta certain stage of osteoblast development and the loss of astrong response could simply reflect a developmental arrest inthe absence of β-catenin. The present study does not discernthese possibilities. Regardless of the mechanism, Osxexpression requires β-catenin and correlates with robust Hhsignaling.

The transcriptional effectors of canonical Wnt signaling inthe osteoblast lineage remain to be determined. In situhybridization experiments identified Tcf1 as the predominantmember of the Lef/Tcf family expressed in the perichondriumof embryonic long bones. Tcf1–/– mice, however, developed anormal skeleton (Verbeek et al., 1995). It is not known whetherother family members were upregulated and thus compensatedfor the loss of Tcf1 in the Tcf1–/– mouse. It is also possible that

Development 132 (1) Research article

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Fig. 9. A model that integrates Hh and Wnt signaling in osteoblastdevelopment. Red arrows represent events supported by geneticevidence. Blue arrows indicate events revealed by studies inC3H10T1/2 cells. See text for details.

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other members, although expressed at a much lower level,either alone or in combination are responsible for normalosteogenesis in the Tcf1–/– mutant. Tcf1 is unique in the familyin the sense that it is itself a target of the canonical Wntpathway. Its high level of expression in perichondrial cellscould simply reflect robust Wnt/β-catenin signaling. Moreover,Tcf1 encodes a number of isoforms including bothtranscriptional activators and repressors (Van de Wetering etal., 1996). Thus, it is conceivable that deletion of both formsmay maintain the overall osteogenic program without causingany obvious phenotype. Further insight on this subject willrequire analyses of various compound mutants among theLef/Tcf family members.

Osteogenesis induced by Smo* in C3H10T1/2 cells was notcompletely inhibited by either Dkk1 or dnTcf4. The partialinhibition was not due to lower titers of Dkk1 or dnTcf4 viruses,as deliberately higher titers for these viruses did not change thedegree of inhibition (data not shown). This result could indicatethat other pathways in addition to canonical Wnt signalingcontribute to Hh-induced osteogenesis. Of note, other groupsreported that Hh-induced osteogenesis in C3H10T1/2 cellsrequired BMP signaling (Spinella-Jaegle et al., 2001; Yuasa etal., 2002).

The interaction between Hh and Wnt signaling is probablycomplex. Our studies demonstrate that nuclear localization ofβ-catenin as well as expression of target genes for the Wntcanonical pathway were abolished in the perichondrium inIhh–/– embryos. This could, among other possibilities, be dueto the downregulation of Wnt expression in the absence of Hhsignaling. Indeed, expression of Wnt9a and Wnt7b was eitherreduced or abolished in the perichondrium in Ihh–/– embryos.In addition, both genes were induced by Hh signaling inC3H10T1/2 cells. Interestingly, a previous study showed thatthe Gli molecules, transcriptional effectors of the Hh pathway,induced expression of several Wnt genes in frog embryos(Mullor et al., 2001). Alternatively, the Hh and Wnt signalingpathways could intersect intracellularly via common regulatorssuch as Suppressor of fused [Su(fu)] (Meng et al., 2001) andGSK3 (Jia et al., 2002; Price and Kalderon, 2002).

The expression studies as well as in vitro osteogenesisassays indicate that Wnt7b may function as an osteogenicsignal in vivo. Interestingly, a recent study reported that Wnt7bwas induced during osteogenesis in primary cultures of bonemarrow stromal cells (Zhang et al., 2004). Wnt7b–/– mice havebeen generated by two different targeting strategies. Whereasone group reported placental defects (Parr et al., 2001) thatresulted in death by E11.5, the other concluded that defects inlung development caused perinatal death (Shu et al., 2002).The latter case probably represents a hypomorph of Wnt7b. Itremains to be determined whether Wnt7b is required forosteogenesis in vivo.

We thank Drs E. Schipani, C. Niehr, H. Clevers, R. Shivdasani, B.de Crombrugghe, A. McMahon and J. Mao for providing reagents.We also thank Dr Suli Cheng for technical advice on retrovirusproduction. The work was supported in part by a start-up fund fromthe Department of Medicine, Washington University Medical Schooland a BIRCWH award from Washington University to F.L.; NIHgrants DK065789 (F.L.) and HD39952 (D.M.O.); and a NIH traininggrant 5T32AR07033 (M.J.H. and K.Y.).

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