Studies on the Role of Cux1 in Regulation of the Onset of ... · Studies on the Role of Cux1 in Regulation of the Onset of Joint Formation in the Developing Limb Gail Lizarraga,*

Developmental Biology 243, 44–54 (2002)doi:10.1006/dbio.2001.0559, available online at http://www.idealibrary.com on

Studies on the Role of Cux1 in Regulation of theOnset of Joint Formation in the Developing Limb

Gail Lizarraga,* Alexander Lichtler,† William B. Upholt,*and Robert A. Kosher*,1

*Department of BioStructure and Function, School of Dental Medicine, and†Department of Genetics and Developmental Biology, School of Medicine,University of Connecticut Health Center, Farmington, Connecticut 06030

Joint formation, the onset of which is characterized by the segmentation of continuous skeletal rudiments into two or moreseparate elements, is a fundamental aspect of limb pattern formation, playing a critical role in determining the size, shape,and number of individual skeletal elements. Joint formation is initiated by conversion of differentiated chondrocytes at sitesof presumptive joints into densely packed nonchondrogenic cells of the joint interzone. This conversion is accompanied byloss of Alcian blue-staining cartilage matrix and downregulation of cartilage-specific gene expression. Here, we report thatCux1, which encodes a transcription factor containing a homeodomain and other DNA-binding motifs, is highly expressedat all of the discrete sites of incipient joint formation in the developing limb concomitant with conversion of differentiatedchondrocytes into interzone tissue. Moreover, differentiated limb chondrocytes in micromass cultures infected with a Cux1retroviral expression vector are converted into nonchondrogenic cells which exhibit loss of Alcian blue cartilage matrix anddownregulation of cartilage-specific gene expression as occurs at the onset of normal joint formation. These results suggestthat Cux1 is involved in regulating the onset of joint formation by facilitating conversion of chondrocytes intononchondrogenic cells of the interzone. © 2002 Elsevier Science (USA)

Key Words: joint development; Cux1; cartilage; skeletal development; limb development; pattern formation.

INTRODUCTION

The formation of synovial (diarthrodial) joints plays acritical role in the patterning of the skeletal elements of thedeveloping vertebrate limb. The various skeletal elementsof the limb do not initially develop as discrete individualrudiments, but rather initially form as continuous entitieswhich are subsequently divided into distinct segments as aresult of joint formation. For example, the humerus, radius,and ulna rudiments are initially formed as a continuousuninterrupted Y-shaped unit in which the radius and ulnabranch from the humerus. The separation of this singleY-shaped rudiment into three distinct skeletal elements isdependent on the formation of the elbow joint between thedistal end of the humerus and proximal ends of the radiusand ulna. Similar segmentation processes occur during theformation of the individual carpal/tarsal rudiments and theindividual metacarpal/metatarsal and phalangeal rudi-

1 To whom correspondence should be addressed. Fax: (860) 679-
2910. E-mail: [email protected].
44

ments. Thus, the segmentation of continuous skeletal ru-diments into two or more separate elements is one of thefundamental aspects of limb pattern formation (Shubin andAlberch, 1986). This process plays a critical role in deter-mining the size, shape, and number of individual skeletalelements (Shubin and Alberch, 1986).

The onset of the segmentation process that generatesjoints between the skeletal elements of the limb is charac-terized by the conversion of differentiated chondrocytes atthe site of the presumptive joint into a narrow band ofdensely packed flattened cells called the joint interzone(Mitrovic, 1977, 1978; Archer et al., 1994; Francis-West etal., 1999b). The conversion of differentiated chondrocytesinto joint interzone cells is accompanied by a loss of Alcianblue-staining cartilage matrix at sites of presumptive jointformation and a striking diminution in expression ofcartilage-characteristic type II collagen and other cartilage-specific genes. For example, Alcian blue stainable cartilagematrix and high level expression of type II collagen arepresent throughout the continuous Y-shaped humerus, ra-
dius, and ulna rudiment, including the site at which the
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elbow joint will subsequently form, and Alcian blue stain-ing and type II collagen expression greatly diminish con-comitant with the formation of the interzone of the futureelbow joint (Koyama et al., 1995; see also Fig. 3 in Results).Similarly, digits form as continuous rods of cartilage char-acterized by high level type II collagen expression which aresubsequently divided into separate elements by the forma-tion of joint interzones which exhibit little type II collagenexpression (Craig et al., 1987). Thus, the onset of jointformation is characterized by the conversion of differenti-ated chondrocytes that stain with Alcian blue and expresshigh amounts of type II collagen into the densely packednonchondrogenic flattened cells constituting the interzonewhich exhibit little or no Alcian blue staining or type IIcollagen expression.

Despite the importance of joint formation in the patterningof the skeletal elements of the limbs and its clinical relevanceto degenerative joint diseases, relatively little is known aboutthe genes, signaling molecules, and cellular and molecularmechanisms involved in the regulation of early joint forma-tion. A secreted signaling molecule that plays an importantrole in joint formation is growth and differentiation factor-5(Gdf5), a member of the transforming growth factor-� (TGF-�)superfamily that is closely related to bone morphogeneticproteins (BMPs). Gdf5 is expressed at virtually all of the sitesat which synovial joints will form between the skeletalelements of the limb (Storm and Kingsley, 1996; Wolfman etal., 1997; Merino et al., 1999; Francis-West et al., 1999a), andnull or loss-of-function mutations in Gdf5 result in aberrantjoint formation in mice and humans (Storm and Kingsley,1996; Thomas et al., 1996, 1997; Polinkovsky et al., 1997).The mechanism by which Gdf5 regulates joint formation is,however, unknown, and misexpression of Gdf5 does not causeectopic joint formation in limb skeletal elements (Francis-West et al., 1999a; Merino et al., 1999; Storm and Kings-ley,1999).

Another signaling molecule that has recently been impli-cated in the regulation of the onset of joint formation isWnt14, a member of the Wnt/wingless family of secretedsignaling molecules. Wnt14 is expressed at the sites of forma-tion of the presumptive interphalangeal joints in the develop-ing hind limb of the chick embryo (Hartmann and Tabin,2001). Misexpression of Wnt14 converts differentiating limbcartilage into nonchondrogenic tissue which expresses Gdf5and other molecular markers of early joint formation (Hart-mann and Tabin, 2001). Thus, it has been suggested thatWnt14 may play a key role in initiating joint formation in thedeveloping limb (Hartmann and Tabin, 2001).

In the present study, we provide evidence implicating thehomeobox-containing gene Cux1, a homolog of the Dro-sophila Cut gene (Jack et al., 1991; Blochinger et al., 1993),in the regulation of the onset of joint formation in thedeveloping chick limb. Vertebrate Cux1 homologs of Cut(variously called CDP/Cut, Cux/CDP, Clox, CDP2, andCux1), like the fly gene, encode large transcription factors(about 200 kb) that contain a single homeodomain and twoto three novel DNA-binding motifs about 70 amino acids in

length called cut repeats (Neufeld et al., 1992; VandenHeuvel et al., 1996; Valarche et al., 1993; Andres et al.,1992; Yoon and Chikaraishi, 1996; Tavares et al., 2000).The Drosophila Cut gene is required for the formation ofthe fly wing margin, which plays a crucial role in fly limboutgrowth and patterning (Jack et al., 1991; Blochinger etal., 1993; Ludlow et al., 1996). Recently, Cux1 has also beenimplicated in the formation of the apical ectodermal ridge(AER), a signaling center that directs the outgrowth andpatterning of the vertebrate limb bud (Tavares et al., 2000).

Here, we report that Cux1 is highly expressed at all of thediscrete sites of incipient joint formation in the developinglimb concomitant with the conversion of differentiatedchondrocytes into interzone tissue. We further demonstratethat retroviral misexpression of Cux1 elicits a conversion ofdifferentiated limb chondrocytes into nonchondrogeniccells which exhibit loss of Alcian blue cartilage matrix anddownregulation of cartilage-specific gene expression as oc-curs during the onset of normal joint formation. Thesestudies suggest that Cux1 is involved in regulating theonset of joint formation by facilitating the conversion ofdifferentiated chondrocytes into the nonchondrogenic cellsof the joint interzone.

MATERIALS AND METHODS

Hybridization ProbesA 2393-bp chicken cerebellum EST cDNA clone (GenBank

Accession No. T25685) in pUEX1 encoding a highly conservedalternatively spliced product of the Cux1 gene called CASP (CDP/cut alternatively spliced product) (see Lievens et al., 1997) wasprovided by Dr. William Jefferies. A 993-bp fragment correspondingto the 5� end of this clone (T25685) was prepared by digesting withBamHI and Bgl2, and cloned into the BamHI site of the BluescriptSK� vector. A 993-bp hybridization probe was prepared by digest-ing with BamHI and SpeI. The 60 nucleotides at the 5� end of this993-bp fragment encode a 20-amino-acid sequence unique to the Nterminus of CASP, whereas the remainder of the fragment contains933 bp of coding sequence present in both full-length Cux1transcripts and in CASP (Lievens et al., 1997). Therefore, this probewould hybridize to both full-length Cux1 transcripts containingthe homeobox and the DNA-binding cut repeats and to CASP, thesmall alternatively spliced product of the Cux1 gene that lacksDNA-binding motifs.

A cDNA probe that would specifically hybridize to Cux1 tran-scripts and not to either CASP or Cux2, the only other member ofthe vertebrate Cut family (Quaggin et al., 1996; Tavares et al.,2000), was prepared by RT-PCR from total RNA isolated from stage24/25 (Hamburger and Hamilton, 1951) chick embryos with theRNeasy Mini kit (Qiagen). Specifically, degenerate primers wereused to amplify a 281-bp fragment of chicken Cux1 encompassing122 nucleotides upstream of the homeobox and 159 nucleotides ofthe 5� portion of the homeobox. The degenerate primers derivedfrom published mouse and human Cux1 nucleotide sequences(Neufeld et al., 1992; Vanden Heuvel et al., 1996) were 5� primer,5�-ATGGA(A/G)AA(A/G)AA(A/G)GC(A/C/T/G)TA(C/T)ATG,and 3�-primer, 5�-(C/G)(A/T)(A/CT/G)C(T/G)(A/G)TT(A/G)TG(A/G)AACCA. The amplified 281-bp fragment was cloned into thePCR Topo vector (Invitrogen) and sequenced. The deduced amino

45Regulation of Joint Formation by Cux1

© 2002 Elsevier Science (USA). All rights reserved.

acid sequence encoded by the fragment was identical to thecorresponding region of chicken Cux1 (Taveres et al., 2000). Toensure a highly specific Cux1 hybridization probe, the 281-bpfragment isolated by RT-PCR was digested with BamHI and PvuIIto prepare a 122-bp hybridization probe located upstream of thehomeobox. This 122-bp fragment is specific for Cux1 and shares nohomology with CASP or Cux2.

A 741-bp cDNA probe that would specifically hybridize to CASPwas prepared from the 3� portion of chicken CASP cDNA by digestingthe 2393-bp cDNA clone (T25685) with BamHI and MluNI. This741-bp cDNA fragment is unique to the C-terminal end of CASP andshares no homology with Cux1 (Lievens et al., 1997).

A 328-bp Gdf5-specific hybridization probe corresponding to thenonconserved 5� end of chicken Gdf5 cDNA was prepared bydigesting full-length chicken Gdf5 cDNA in Bluescript SK� (pro-vided by Dr. Frank Luyten) with EcoRI and AvaI. A 1.2-kb cDNAprobe corresponding to the entire coding sequence of chickenWnt14 cDNA was prepared by digesting full-length Wnt14 cDNAin Bluescript SK�/� (provided by Dr. A. M. C. Brown; see Bergsteinet al., 1997) with EcoRI. Probes specific for transcripts encodingchicken type II collagen and the core protein of the cartilageproteoglycan aggrecan were prepared as previously described (Nahet al., 1988; Mallein-Gerin et al., 1988).

The cDNA probes were labeled with [33P]- or [32P]dCTP followingthe random primer oligonucleotide procedure.

In Situ and Northern Blot Hybridization

In situ hybridization on serially sectioned limb buds that hadbeen fixed in Bouin’s solution was performed by using 33P-labeledcDNA probes (see above) and high stringency hybridization condi-tions as previously described (Coelho et al., 1991). In order tocarefully correlate expression patterns of different genes, in mostcases, adjacent sections (within 20 �m of one another) of the samelimb buds were mounted on separate slides and hybridized withdifferent probes. Total RNA was isolated from stage 26/27, stage29, and stage 30 limb buds (Hamburger and Hamilton, 1951) byusing the RNeasy Mini kit (Qiagen), and Northern blot analysiswas performed at high stringency by using 32P-labeled cDNA aspreviously described (Dealy et al., 1993).

Preparation of a Cux1 Retroviral Expression Vector

A Moloney murine leukemia virus (MoMLV)-derived replica-tion-defective retroviral vector was used for misexpression of a4.6-kb cDNA containing the full coding sequence of human Cux1(provided by Dr. Ellis Neufeld; see Neufeld et al., 1992). Areplication-defective vector was used for Cux1 misexpression,since the large size of the Cux1 cDNA (4.6 kb) required a vectorthat would express large cDNA inserts. In addition, the replication-defective vector could be used to simultaneously express bothCux1 cDNA and a cDNA encoding enhanced green fluorescentprotein (EGFP), a bright derivative of green fluorescent proteinwhich would serve as a marker to monitor expression of the vectorin living cells by fluorescence microscopy. The MoMLV-derivedreplication-defective vector used for Cux1 misexpression is amodified version of a vector called MG-1 (provided by Dr. DavidDorsky) which is a derivative of LXSN (Miller and Rosman, 1989)into which a cytomegalovirus (CMV) promoter and an EGFP cDNAhad been cloned upstream of the simian virus 40 (SV40) promoterand neomycin resistance (neo) gene cassette of the LXSN vector. Inpreparation for construction of the Cux1 retroviral expression

vector, first, the modified MG-1 vector was prepared by excising a1.86-kb fragment containing the EGFP cDNA, SV40 promoter, andmost of the neo gene by digestion with AgeI and RsrII (Fig. 1). Thisresulted in a modified MG-1 vector called GL-1 containing aninternal CMV promoter (Fig. 1). Next, the 4.6-kb cDNA containingthe full coding sequence of human Cux1 was cloned by blunt-endligation into the SalI site of an pIRES2/EGFP vector (Clontech),resulting in a construct containing Cux1 cDNA upstream of aninternal ribosome entry site (IRES) followed by an EGFP cDNA(Fig. 1). The Cux1-IRES-EGFP fragment was excised by digestionwith EcoRV and NotI and cloned by blunt-end ligation into GL-1downstream of the CMV promoter. This resulted in a retroviralconstruct containing the CMV promoter upstream of the Cux1cDNA followed by an IRES and an EGFP cDNA (Fig. 1). Thus, inthis construct, the CMV promoter drives expression of both Cux1and EGFP, and the presence of the IRES between the two cDNAsenables initiation of translation of the EGFP cDNA immediatelyafter translation of the Cux1 cDNA.

Stable retroviral vector-producing cell lines were generated byusing the vesicular stomatitis virus (VSV) pseudotype helper cellline 293 GPG (Ory et al., 1996). For packaging, the Cux1/EGFPretroviral construct, prepared as described above, was transfectedby use of the cationic lipid reagent Lipofectamine (Gibco BRL) intoVSV pseudotype helper cells 293 GPG plated at 50% confluence intetracycline-containing medium. The VSV envelope protein isunder the control of the Tet-Off system, since VSV is toxic to cells.Therefore, following transfection, virus production was initiated bythe addition of medium lacking tetracycline. After 3–4 days ofculture, the retrovirus-containing medium was harvested, and thevirus was concentrated by ultracentrifugation as described byBurns et al. (1993). To generate high-titer producer cell lines,aliquots of the concentrated retrovirus and 4 �g/ml polybrene (tofacilitate virus infection) were used to infect 293 GPG cells platedat clonal densities. Three days after infection, single cells highlyexpressing EGFP were detected by fluorescence microscopy. Thesehighly fluorescent cells were picked from the tissue culture dishand used to establish the retroviral vector-producing cell lines. Theindividual producer cells were initially grown in wells of a 24-welltissue culture plate, and after reaching confluence were passagedonto 100-mm tissue culture dishes. After reaching confluence inthe 100-mm tissue culture dishes, the virus-containing mediumwas harvested, virus was concentrated by ultracentrifugation(Burns et al., 1993), and aliquots were stored at �70°C.

Titers were determined essentially as described by Fekete andCepko (1993), by adding limiting dilutions of concentrated retrovi-rus stock and polybrene to subconfluent cultures of chick embryofibroblasts (CEF) and counting EGFP-positive colonies to obtain thenumber of cfu/ml. Retroviral titers ranged from 2 to 8 � 107.

To confirm that the retrovirus efficiently expressed Cux1 andEGFP, subconfluent CEF cultures in 10 ml of medium wereinfected with 20 �l of concentrated retrovirus stock and polybrene.After 72 h, the extent of EGFP expression was examined byfluorescence microscopy and the expression of Cux1 was examinedby Western blotting by using an affinity-purified goat polyclonalantibody against human Cux1 (CDP; Santa Cruz). Virtually all ofthe CEFs expressed EGFP detectable by fluorescence microscopy.In addition, Western blotting with the anti-Cux1 antibody ofprotein lysates from CEFs infected with the Cux1 retrovirusrevealed a major band of about 200 kDa, which is the size expected forhuman Cux1 protein (Neufeld et al., 1996). In contrast, no immuno-reactive Cux1 protein was detected in Western blots of protein lysates

46 Lizarraga et al.


from uninfected CEFs or from CEFs infected with control retrovirusexpressing EGFP but lacking the Cux1 cDNA insert.

Micromass Cultures: Preparation, RetroviralInfection, and Analysis

Micromass cultures were prepared from the uniform populationof mesenchymal chondrogenic precursor cells of the distal tip ofstage 25 (Hamburger and Hamilton, 1951) embryonic chick wingbuds as previously described (Gay and Kosher, 1984). For retroviralinfection, 1–2 �l of concentrated retrovirus stock (2–8 � 107cfu/ml;see above) and 0.1 �l of polybrene (to facilitate virus infection) wereadded per 10 �l of a cell suspension containing 2 � 107 cells/ml,after which 10-�l drops of the cell suspension (2 � 105 cells) weredispensed onto the surface of 35-mm tissue culture dishes (Ahrenset al., 1977; Gay and Kosher, 1984). The retrovirus-containingmicromass cultures were then incubated for 3 h at 37°C in ahumidified CO2 incubator to allow for cell attachment, after whichthe cultures were supplied with 2 ml of medium [F12 supple-mented with 10% fetal bovine serum (FBS) and antibiotics]. Thus,in these experiments, micromass cultures were exposed to concen-trated retroviral vectors for 3 h during the initial plating andattachment of the cells.

In subsequent experiments, standard micromass cultures wereestablished in the wells of 24-well tissue culture dishes, andcultured for 24 h in 1 ml of standard F12 medium containing 10%FBS. At 24 h of culture, the medium was removed and replacedwith 200 �l of medium containing 20 �l of concentrated retrovirusstock and 2 �l of polybrene. After 5 h incubation at 37°C, thevirus-containing medium was removed, and replaced with 1 ml offresh F12 medium containing 10% FBS. Thus, in these experi-

ments, micromass cultures were exposed to the retroviral vectorsfor 5 h at the beginning of the second day of culture.

The morphology of the micromass cultures was monitoreddaily by phase microscopy, and the expression of the EGFPreporter gene of the retroviral constructs was monitored byfluorescence microscopy. The accumulation of cartilage matrixwas monitored histochemically by staining with Alcian blue, pH1.0, as previously described (Gay and Kosher, 1984). Total RNAwas prepared from cultures by using the RNeasy Mini kit(Qiagen), and steady-state levels of transcripts for type II colla-gen, the core protein of the cartilage proteoglycan aggrecan,Gdf5, and Wnt14 were determined by dot blot hybridizationusing the specific cDNA probes described above and the high-stringency conditions previously described (Kosher et al.,1986a,b; Kulyk et al., 1991). Levels of hybridizable RNA se-quences were determined by densitometry using an Alpha Inno-tech digital imaging system. The total poly(A)� mRNA contentof samples was determined by hybridizing aliquots of the samesamples used for determination of specific mRNA levels with32P-labeled oligo(dT)20 as described by Harley (1987).

RESULTS

Cux1 Is Highly Expressed at All of the Discrete Sitesof Incipient Joint Formation in the Developing LimbConcomitant with the Conversion of DifferentiatedChondrocytes into Interzone Tissue

The initial formation of the interzones at the onset of thesegmentation process that generates joints between theskeletal elements of the embryonic chick limb bud occurs

FIG. 1. Preparation of the Cux1 replication-defective retroviral expression vector. MG-1 is a derivative of the replication-defective vectorLXSN (Miller and Rosman, 1989) into which a CMV promoter and an enhanced green fluorescent protein (EGFP) cDNA had been clonedupstream of the SV40 promoter and neo gene cassette of the LXSN vector. GL-1 was prepared by excising a fragment containing the EGFPcDNA, SV40 promoter, and most of the neo gene with AgeI and RsrII. A 4.6-kb cDNA containing the full coding sequence of human Cux1was cloned by blunt-end ligation into the SalI site of an pIRES2/EGFP vector (Clontech), resulting in a construct containing Cux1 cDNAupstream of an IRES followed by an EGFP cDNA. The Cux1-IRES-EGFP fragment was excised by digestion with EcoRV and NotI, andcloned by blunt-end ligation into GL-1 downstream of the CMV promoter. This resulted in a Cux1 retroviral expression vector containingthe CMV promoter upstream of the Cux1 cDNA followed by an IRES and an EGFP cDNA. LTR, retroviral long terminal repeat; p(A),polyadenylation site. Arrows indicate transcriptional start sites and direction of transcription.



during stages 27–31. Cux1 expression was examined byNorthern blot analysis of RNA isolated from limb buds atthese stages. As shown in Fig. 2, a 993-bp cDNA fragmentfrom the 5� end of a chicken EST clone with homology tomouse and human Cux1 (see Materials and Methods) hy-bridizes to transcripts of about 12 and 2.8 kb in RNAisolated from stage 26/27, stage 29, and stage 30 embryonicchick limb buds. The 12-kb transcript is similar in size tothe full-length Cux1 transcripts containing the homeodo-main and DNA-binding cut repeats that are detectable inother vertebrate species (Neufeld et al., 1992; VandenHeuvel et al., 1996; Lievens et al., 1997). The 2.8-kb speciescorresponds to a highly conserved alternatively splicedproduct of the Cux1 gene called CASP that lacks DNA-binding motifs (Lievens et al., 1997).

The temporal and spatial pattern of Cux1 expressionduring the onset of joint formation was examined by in situhybridization. Identical expression patterns were detectedby using a probe that hybridizes to both full-length Cux1transcripts and its conserved alternatively spliced productCASP and probes that are specific for either full-lengthCux1 or CASP (see Materials and Methods).

As exemplified by the developing elbow joint shown inFigs. 3A and 3B, the onset of joint formation is character-ized by the conversion of differentiated chondrocytes thatexpress high levels of cartilage-characteristic type II colla-gen into the nonchondrogenic cells of the interzone whichexpress little type II collagen. At stage 25, before the

formation of the interzone, cartilage differentiation charac-terized by the expression of type II collagen has occurredthroughout the continuous Y-shaped humerus, radius, andulna rudiment, including the site at which the elbow jointwill subsequently form (Fig. 3A). At about stage 27, type IIcollagen expression greatly diminishes concomitant withthe formation of the interzone at the presumptive elbowjoint (Fig. 3B). As shown in Fig. 3C, Cux1 is highly ex-pressed in the developing elbow joint interzone concomi-tant with the downregulation of type II collagen expression.

During stages 28–35, Cux1 is highly expressed at virtu-ally all of the discrete sites of incipient joint formation inthe developing limb concomitant with the conversion ofdifferentiated chondrocytes into interzone tissue (Fig. 4). Asshown in Fig. 4, Cux1 is expressed in the developinginterzones of the presumptive elbow joint (Figs. 4A, 4C, and4E), the wrist joint (Figs. 4A, 4C, and 4E), the joints that areforming between the individual carpal bones (Fig. 4E), thecarpometacarpal joints (Figs. 4A, 4C, and 4E), and themetacarpophalangeal (Fig. 4A) and interphalangeal joints(Fig. 4G) of each of the digits. The striking localization ofCux1 expression at sites of joint formation suggests that itmay play an important role in regulating the onset of thesegmentation process that generates joints between theskeletal elements of the chick limb.

To further investigate the association of Cux1 with theonset of joint formation, we compared its expression inadjacent sections of the same limbs with that of Gdf5, asecreted signaling molecule that has been implicated in theregulating synovial joint formation (Storm and Kingsley,1996). As shown in Fig. 4 (E, F and G, H), during stages28–35, Cux1 and Gdf5 are coexpressed at all of the sites ofincipient joint formation in the developing limb. This

FIG. 2. Northern blot analysis of Cux1 expression in stage 26/27,stage 29, and stage 30 embryonic chick limb buds. A 993-bp cDNAfragment from the 5� end of a chicken EST clone with homology tomouse and human Cux1 (see Materials and Methods) hybridizes totranscripts of about 12 kb and 2.8 kb in RNA isolated from limbbuds at each stage. The 12-kb transcript is similar in size to thefull-length Cux1 transcripts containing the homeodomain andDNA-binding cut repeats that are detectable in other vertebratespecies (Neufeld et al., 1992; Vanden Heuvel et al., 1996; Lievens etal., 1997), and the 2.8-kb species corresponds to a highly conservedalternatively spliced product of the Cux1 gene called CASP thatlacks DNA-binding motifs (Lievens et al., 1997).

FIG. 3. Expression of Cux1 at the onset of elbow joint formation.(A) Type II collagen expression in a stage 25 wing bud. Note thatcartilage differentiation characterized by the expression of type IIcollagen occurs throughout the continuous Y-shaped humerus,radius, and ulna rudiment, including the site at which the elbowjoint will subsequently form. (B, C) Type II collagen (B) and Cux1(C) expression in adjacent sections of the same stage 27 wing bud.Type II collagen expression greatly decreases concomitant with theformation of the interzone at the presumptive elbow joint (B), andCux1 is expressed in the developing elbow joint interzone con-comitant with the downregulation of type II collagen expression.Bar, 200 �m.

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provides further support for a role for Cux1 in the regulatingthe onset of joint formation, and suggests the possibilitythat there may be regulatory relationships between Cux1and Gdf5 in the process.

Cux1 Suppresses Cartilage Differentiation in Vitro

As described above, Cux1 is highly expressed at all of thediscrete sites of incipient joint formation in the developinglimb where differentiated chondrocytes that stain withAlcian blue and express high amounts of type II collagen arebeing converted into the densely packed cells of the jointinterzone that exhibit little or no Alcian blue staining ortype II collagen expression. This suggests the hypothesisthat Cux1 may be involved in regulating the onset of jointformation by facilitating the conversion of differentiatedchondrocytes into the nonchondrogenic cells of the jointinterzone. To investigate this possibility further, we wishedto determine whether Cux1 could convert differentiatedlimb chondrocytes into nonchondrogenic cells in vitro.Accordingly, we examined the effect of retroviral misex-pression of Cux1 on the chondrogenic differentiation oflimb mesenchymal cells in high-density micromass cul-tures. In these studies, we utilized micromass culturesprepared from the mesenchymal cells in the distal tip ofstage 25 wing buds, since these cells are a relativelyhomogeneous population of undifferentiated chondrogenic

precursors which uniformly progress through the phases ofchondrogenic differentiation in micromass culture andform a virtually uniform sheet of cartilage with little or nononchondrogenic tissue detectable (Gay and Kosher, 1984).To misexpress Cux1, we used an MoMLV-derived replica-tion-defective retroviral expression vector containing theCMV promoter upstream of a cDNA containing the fullcoding sequence of human Cux1 followed by an IRES and acDNA encoding EGFP which serves as a reporter gene tomonitor the expression of the vector.

In our initial experiments, limb mesenchymal cells inmicromass culture were exposed to concentrated Cux1retroviral vector or to a control vector expressing EGFP butlacking the Cux1 cDNA insert for 3 h during the initialplating and attachment of the cells (see Materials andMethods). Widespread expression of EGFP detectable byfluorescence microscopy occurs 15–24 h after this initialinfection period, by which time the cultures will haveinitiated deposition of an Alcian blue-stainable cartilagematrix and upregulated expression of genes for type IIcollagen and other cartilage-specific matrix proteins(Kosher et al., 1986a,b; Kulyk et al., 1991).

As shown in Fig. 5, by day 4 of culture, virtually all of thecells in the Cux1-infected and control cultures exhibitEGFP expression (Figs. 5C and 5D). In addition, Cux1transcripts are expressed at high levels in the Cux1-infectedcultures, but are not expressed in the control cultures

FIG. 4. Expression of Cux1 at all of the discrete sites of incipient joint formation in the developing limb. (A, B and C, D) Cux1 and typeII collagen expression in adjacent sections of the same limbs at stage 28/29 (A, B) and stage 29/30 (C, D). Cux1 is expressed in the interzoneof the developing elbow joint (e), the wrist joint (w), the carpometacarpal joints (cm), and the metacarpophalangeal joints (mp). (E, F) Cux1and Gdf5 expression in adjacent sections of the same stage 30 limb. Cux1 and Gdf5 are coexpressed at all of the sites of incipient jointformation, including the elbow joint (e), wrist joint, carpal joints, and carpometacarpal joints. (G, H) Coexpression of Cux and Gdf5 in theinterphalangeal joints in adjacent sections of the same stage 35 limb. Bar, 200 �m.



infected with the retroviral vector expressing EGFP butlacking the Cux1 cDNA insert (data not shown). On day 4,the cells of control cultures have uniformly deposited arefractile extracellular cartilage matrix detectable by phasemicroscopy (Fig. 5A). In contrast, relatively little refractilecartilage matrix is detectable in the Cux1-infected cultures,but rather large numbers of densely packed apparentlynonchondrogenic cells are present that are not surroundedby a refractile matrix (Fig. 5B). The suppression of chondro-genic differentiation detectable by phase microscopy isreflected in a striking decrease in the accumulation ofAlcian blue-stained cartilage in the Cux1-infected cultures.

As shown in Fig. 5, whereas a uniform, darkly stainingAlcian blue-positive cartilage matrix is present throughoutthe control cultures, little or no Alcian blue-staining matrixis detectable in the Cux1-infected cultures. The suppres-sion of chondrogenesis in the Cux1-infected cultures de-tectable by phase microscopy and Alcian blue staining isaccompanied by about a threefold reduction in the steadystate levels of transcripts for cartilage-characteristic type IIcollagen and the core protein of the cartilage proteoglycanaggrecan (Fig. 6). Thus, Cux1 elicits a striking diminutionin Alcian blue staining and downregulation of cartilage-specific gene expression as occurs at the onset of normal

FIG. 5. Suppression of limb cartilage differentiation in vitro by Cux1. (A, B) Phase photomicrographs of day 4 micromass cultures of limbmesenchymal cells infected for 3 h at the onset of culture with the Cux1 retroviral expression vector (Cux1-RV) (B) or with a control vectorexpressing EGFP but lacking the Cux1 cDNA insert (EGFP-RV) (A). Control cultures have uniformly deposited a refractile pericellularcartilage matrix (A), whereas relatively little refractile cartilage matrix is detectable in the Cux1 infected cultures, which contain largenumbers of densely packed apparently nonchondrogenic cells. (C, D) EGFP expression detectable by fluorescence microscopy in theEGFP-RV control (C) and Cux1-infected (D) cultures shown in (A) and (B). EGFP is expressed by virtually all the cells of the control (C) andCux1-infected (D) cultures. (E, F) Accumulation of Alcian blue-stainable cartilage matrix by EGFP-RV control (E) and Cux1-infected (F)cultures. A uniform, darkly staining Alcian blue-positive cartilage matrix is present throughout the control cultures, whereas little Alcianblue-staining matrix is detectable in the Cux1-infected cultures. Bar, in (A–D), 200 �m; in (E, F), 8 mm.

50 Lizarraga et al.


joint formation. The total poly(A)� RNA content of Cux1-infected cultures is 92% (n � 4) that of control cultures,indicating that Cux1 is not a general repressor of transcrip-tion. Cux1 does not induce the expression of transcripts foreither Gdf5 or Wnt14, signaling molecules that have beenimplicated in regulating the onset of joint formation (datanot shown).

In the preceding experiments, the Cux1 retroviral expres-sion vector was supplied to the limb mesenchymal cells fora 3-h period at the initiation of culture, which is 15–24 hbefore the cells undergo overt chondrogenic differentiationcharacterized by deposition of an Alcian blue stainingmatrix and upregulation of cartilage-specific gene expres-sion. Taking into account the time it should take for thevirus to be internalized and integrated into the genome, weassume it would take 15–24 h for ectopic expression ofCux1 to occur, which is after overt cartilage differentiationwill have taken place. This is important, since we wantedto reproduce a situation comparable to that which occurs invivo, where, at sites of incipient joint formation, Cux1 isexpressed by chondrocytes after the onset of their differen-tiation. Although our prediction of the timing of ectopicCux1 expression is consistent with the timing of expression

of the EGFP reporter gene (see above), we neverthelesswanted to be absolutely certain that Cux1 would suppresscartilage differentiation when expressed after the initiationof the process. Accordingly, we performed experiments inwhich micromass cultures were not supplied with the Cux1retroviral vector until 24 h after the initiation of culture, bywhich time lightly staining Alcian blue cartilage matrixhad already been uniformly deposited by the cultures (seeFig. 7A). Indeed, in these experiments, Cux1 misexpressionwould not be expected to occur until 36–48 h of culture(i.e., 15–24 h after exposure to the retrovirus), by whichtime a uniform darkly staining Alcian blue cartilage matrixhas accumulated (Fig. 7B). As shown in Fig. 7C, micromasscultures supplied with the control retroviral vector at 24 hprogressively continue to accumulate Alcian blue-stainablecartilage matrix for the next 3 days. In contrast, 3 days afterbeing supplied with the Cux1 retroviral vector at 24 h, littleAlcian blue-staining cartilage matrix is detectable (Fig. 7D).Thus, this experiment clearly demonstrates that Cux1

FIG. 6. Cux1 suppresses cartilage-specific gene expression invitro. Relative steady state levels of type II collagen and aggrecantranscripts in day 4 micromass cultures infected with the Cux1retroviral expression vector (Cux1-RV) or with the EGFP controlvector (EGFP-RV). The amount of each specific mRNA/totalpoly(A)� RNA in the Cux1-infected cultures is presented as anamount relative to that in the EGFP-RV control cultures, whichwas arbitrarily set to 100. Values are the means of four determina-tions � SEM.

FIG. 7. Conversion of differentiated chondrocytes in vitro byCux1. (A) A 24-h untreated control micromass culture exhibitinguniform lightly staining Alcian blue-positive cartilage matrix. Thisis the time that cultures were supplied with either the EGFPcontrol or Cux1 retroviral expression vectors for 5 h. (B) A 48-huntreated control culture exhibiting a uniform darkly stainingAlcian blue cartilage matrix. This corresponds roughly to the timeat which high expression of Cux1 and/or EGFP would be occurringwhen the vectors are supplied at 24 h. (C, D) 96-h cultures thatwere supplied with the EGFP control (EGFP-RV) (C) or the Cux1(Cux1-RV) (D) retroviral vectors at 24 h. The EGFP-RV controlcultures have continued to accumulate Alcian blue-stainable car-tilage matrix, whereas the Cux1-infected cultures exhibit littleAlcian blue-stainable cartilage matrix. Bar, 7 mm.



converts differentiated chondrocytes into nonchondrogeniccells.

DISCUSSION

The Role of Cux1 in Regulation of the Onsetof Joint Formation

Joint formation is one of the fundamental aspects of limbpattern formation playing a critical role in determining thesize, shape, and number of individual skeletal elements(Shubin and Alberch, 1986). The segmentation process thatgenerates joints between the skeletal elements is initiatedby the conversion of differentiated chondrocytes at the sitesof presumptive joint formation into the densely packedcells of the joint interzone. This conversion is accompaniedby a loss of Alcian blue-staining cartilage matrix at sites ofpresumptive joint formation and a downregulation in ex-pression of cartilage-characteristic type II collagen andother cartilage-specific genes.

The results of the present study suggest that Cux1, ahomolog of the Drosophila Cut gene which encodes atranscription factor containing a homeodomain and otherDNA binding motifs, plays an important role in regulatingthe onset of joint formation. Cux1 is highly expressed at allof the discrete sites of incipient joint formation in thedeveloping limb concomitant with the conversion of differ-entiated chondrocytes into interzone tissue. Moreover,misexpression of Cux1 in micromass cultures of limbmesenchymal cells that had undergone cartilage differen-tiation results in a conversion of the differentiated chon-drocytes into densely packed nonchondrogenic cells whichexhibit loss of Alcian blue-stainable cartilage matrix anddownregulation of cartilage-specific gene expression as oc-curs during the onset of normal joint formation. Theseresults suggest that Cux1is involved in regulating the onsetof joint formation by facilitating the conversion of differen-tiated chondrocytes into the nonchondrogenic cells of thejoint interzone.

Significantly, a recent study indicates that expression ofCux1 can disrupt the differentiation of cartilaginous skel-etal elements in vivo (Tavares et al., 2000). Adenoviral-mediated misexpression of Cux1 in the developing chicklimb in ovo results in severe skeletal defects, including anabsence of cartilage elements and gaps and/or severe reduc-tions in Alcian blue staining (Tavares et al., 2000). Al-though these effects were suggested to result from impairedAER activity (Tavares et al., 2000), they are consistent withour in vitro micromass culture studies demonstrating thatCux1 misexpression can directly suppress limb cartilagedifferentiation.

Mechanism by Which Cux1 May Regulatethe Onset of Joint Formation

A key event in the onset of joint formation is a down-regulation in the expression of type II collagen and other

cartilage matrix protein genes, such as aggrecan. Signifi-cantly, this downregulation of cartilage-specific gene ex-pression correlates with the high level of Cux1 expressionthat occurs in the developing interzone at the onset of jointformation. Furthermore, Cux1 can indeed downregulatetype II collagen and aggrecan transcript expression in limbchondrocytes in micromass culture. It is therefore notewor-thy that vertebrate Cux1 homologs, in general, appear tofunction as transcriptional repressors, and, in particular,inhibit expression of tissue-specific genes in multiple lin-eages. In cotransfection assays, vertebrate Cux1 homologsrepress the activity of promoter/enhancer constructs ofgenes for muscle-specific �-cardiac myosin heavy chain(Andres et al., 1992), erthyroid-specific �-globin (Superti-Furga et al., 1989), myeloid-specific cytochrome b heavychain (Skalnik et al., 1991), neural-specific NCAM (Val-arche et al., 1993), and bone-specific osteocalcin (van Gurpet al., 1999) (see also Higgy et al., 1997; Dufort and Nepveu,1994; Benan et al., 1997). Thus, it has been suggested thatCux genes, in general, act as developmentally regulatedrepressors of tissue-specific gene expression. Cux1 ho-mologs can inhibit gene transcription by interfering withthe interaction of tissue-specific transcription activatorswith their target sequences (Andres et al., 1992; Mailley etal., 1996) and can actively repress the transcription ofalready active tissue-specific genes (Mailley et al., 1996).These observations suggest that the primary function ofCux1 at the onset of joint formation may be to repress theexpression of the type II collagen gene or other cartilage-specific genes, thus facilitating the formation of the jointinterzone tissue.

Although Cux1 downregulates type II collagen and aggre-can expression in limb chondrocytes in micromass cul-tures, it does not induce the expression of either Wnt14 orGdf5, signaling molecules that have been implicated inregulating joint formation (Hartmann and Tabin, 2001;Storm and Kingsley, 1996; Thomas et al., 1996, 1997). Thissuggests the possibility that Cux1 may be a downstreamtarget gene of these secreted signaling molecules. Alter-nately, Cux1 might function in an separate or parallelpathway at the onset of joint formation. It will be ofparticular interest to determine whether Cux1 is a down-stream target of Wnt14 signaling at the onset of jointformation, since Wnt14, like Cux1, suppresses chondro-genic differentiation (Hartmann and Tabin, 2001). In anycase, it is likely that Cux1 functions in conjunction withother signaling molecules and transcription factors whichmay regulate various and distinct aspects of joint forma-tion. In particular, Cux1 may function by repressing theexpression of cartilage-specific genes, while other factors,such as Wnt14 and/or Gdf5, may act as positive regulatorsof the expression of markers of early joint formation.Understanding the precise roles and relationships betweenCux1 and other factors in the regulation of joint formationwill require considerable further investigation.

52 Lizarraga et al.


SUMMARY

The results of this study suggest that the homeodomain-containing transcription factor Cux1 plays an importantrole in regulating the onset of the segmentation process thatgenerates joints between the skeletal elements of the devel-oping limb by facilitating the conversion of differentiatedchondrocytes into the nonchondrogenic cells of the jointinterzone. Understanding the mechanisms and moleculesinvolved in joint formation during embryonic developmentmay provide insights into the nature of and possible thera-peutic treatments for degenerative joint diseases.

ACKNOWLEDGMENTS

We thank Ellis Neufeld for human Cux1 cDNA; William Jeffer-ies for chicken CASP cDNA; A. M. C. Brown for chicken Wnt14cDNA; Frank Luyten for chicken Gdf5 cDNA; and David Dorskyfor the MG-1 retroviral vector. This work was supported by NIHGrant HD22610 (to R.A.K. and W.B.U.).

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Received for publication September 26, 2001Revised December 5, 2001

Accepted December 5, 2001Published online January 23, 2002

54 Lizarraga et al.


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Studies on the Role of Cux1 in Regulation of the Onset of ... · Studies on the Role of Cux1 in Regulation of the Onset of Joint Formation in the Developing Limb Gail Lizarraga,*