骨发育 bone development

Annu.Rev.Cell Dev.Biol.2000.16:191–220Copyright c
2000by Annual Reviews.All rights reserved B ONE D EVELOPMENT
Bjorn R.Olsen 1,Anthony M.Reginato 1,2,Wenfang Wang 1
1Harvard
Medical School,Department of Cell Biology,240Longwood Avenue,Boston,
Massachusetts 02115;e-mail:bjorn olsen@;
reginato anthony@;wenfang wang@
2Massachusetts General Hospital,Arthritis Unit,32Fruit Street,Boston,Massachusetts 02114
Key Words patterning,chondrogenesis,ossification,growth plate,joints
s Abstract Early development of the vertebrate skeleton depends on genes that pattern the distribution and proliferation of cells from cranial neural crest,sclero-tomes,and lateral plate mesoderm into mesenchymal condensations at sites of future skeletal elements.Within these condensations,cells differentiate to chondrocytes or osteoblasts and form cartilages and bones under the control of various transcription factors.In most of the skeleton,organogenesis results in cartilage models of future bones;in these models cartilage is replaced by bone by the process of endochondral ossifistly,through a controlled process of bone growth and remodeling the final skeleton is shaped and molded.Significant and exciting insights into all aspects of vertebrate skeletal development have been obtained through molecular and genetic studies of animal models and humans with inherited disorders of skeletal morphogen-esis,organogenesis,and growth.
CONTENTS
INTRODUCTION ................................................192SKELETAL MORPHOGENESIS .....................................193Craniofacial Bone Development ....................................193Development and Disorders of the Axial Skeleton ........................195Development and Disorders of the Limb Skeleton ........................197CHONDROCYTE DIFFERENTIATION AND ENDOCHONDRAL
BONE FORMATION .............................................200Chondrogenesis—the Role of Sox Genes ..............................200Chondrocyte Maturation and Control of Blood Vessel Invasion into Cartilage ....201Growth Plates and Bone Growth ....................................202OSTEOBLAST DIFFERENTIATION AND FUNCTION ....................205EXTRACELLULAR MATRIX IN SKELETAL DEVELOPMENT .............206CONCLUSIONS .................................................
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INTRODUCTION
The vertebrate skeleton,composed of cartilage and bone,is the product of cells from three distinct embryonic lineages.The craniofacial skeleton is formed by cranial neural crest cells,the axial skeleton is derived from paraxial mesoderm (somites),and the limb skeleton is the product of lateral plate mesodermal cells.Cells in these lineages migrate to the locations in the embryo where skeletal ele-ments will develop,form characteristic mesenchymal condensations of high cell density,and differentiate into osteoblasts or chondrocytes (Hall &Miyake 1992).In regions of the craniofacial skeleton and the clavicle,differentiation into os-teoblasts produces intramembranous bones directly,whereas differentiation into chondrocytes produces a framework of cartilage models (anlagen)of the future bones in the remaining skeleton.These cartilage models are subsequently replaced by bone and bone marrow through the process of endochondral ossification (for review,see Mundlos &Olsen 1997a).
What we currently know about the molecular and cellular basis of skeletal development is largely the result of experimental animal studies and investiga-tions of human bone disorders.From studies of transgenic mice and knockouts,correlations between specific embryological events and gene functions have been made.In addition,genetic studies of mice and humans with inherited disorders of skeletal development have led to the identification of novel genes and pathways required to build bone and cartilage and deepened our understanding of previously characterized molecular mechanisms.
Given the large number of murine and human inherited skeletal anomalies,the power and speed of current methods for linkage mapping,and the progress in genome sequencing,such genetic studies are increasingly important in identifying new or key genes and regulatory pathways in bone development.Therefore,in this review we have chosen to emphasize data that are relevant to a few selected genetic studies instead of attempting to give a complete discussion of all aspects of the molecular and cell biology of bone development.Also,since the postnatal physiological aspects of bone biology have recently been reviewed from a molec-ular genetic perspective (Karsenty 1999),we have elected to focus the discussion on early embryonic events.
In the review,we first discuss skeletal morphogenesis,i.e.the migration of mesenchymal cells derived from the neural crest,sclerotomes,or lateral plate mesoderm to their ultimate locations,and their condensation into mesenchymal precursors of cartilage and bone (Figure 1).The genes involved in morphogenesis are frequently transcription factors regulating cellular determination and migration events (see Mundlos &Olsen 1997a).Mutations in such genes cause dysostoses,disorders that affect embryologically defined skeletal elements while the rest of the skeleton is normal.Important insights into skeletal patterning have been obtained from studies of dysostoses that affect the three major divisions of the vertebrate skeleton:craniofacial,axial,and appendicular.
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Figure 1Summary diagram showing how patterning genes (mostly transcription factors)reg-ulate the patterning of cells from cranial neural crest,somites,and lateral plate mesoderm in the craniofacial,axial,and limb anogenesis,the formation of cartilage and bone,is controlled by transcription factors,cytokines,growth factors,and extracellular matrix molecules.
We then discuss the control of chondrocyte and osteoblast differentiation and the formation of bones.This organogenesis phase of skeletal development is con-trolled by transcription factors,growth factors,cytokines,and extracellular matrix molecules (Figure 1).Mutations in such genes result in dysplasias,conditions that affect cartilage and bone tissues generally,or dysostoplasias,disorders caused by mutations in genes that have roles both in early patterning and in subsequent organogenesis (Mundlos &Olsen 2000).
Organogenesis of cartilage and bone includes the synthesis of specialized ex-tracellular matrices.We conclude our review with a discussion of some of the most critical matrix components and the consequences of mutations in the genes that encode them.
SKELETAL MORPHOGENESIS Craniofacial Bone Development
For the craniofacial skeleton several patterning genes have been identified as reg-ulators of differentiation and migration of neural crest cells.This is not surprising since most of the craniofacial bones are of neural crest origin (Bronner-Fraser
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1994,Noden 1991).These cells migrate from the dorsal aspect of the neural tube into the branchial arches and the frontonasal mass and contribute to a variety of tissues including cartilage and bone.Cell-tracing studies in mice and chick em-bryos have shown that crest cells from the caudal midbrain and rhombomeres 1and 2migrate into the first branchial arch,giving rise to maxilla,mandible,incus,malleus,and regions of the temporal bone;whereas cells in the second branchial arch,giving rise to stapes,styloid process of the temporal bone,and part of the hyoid bone,are derived from rhombomere 4(Noden 1983).
Neural crest cells also give rise to skull bones such as the frontal and parietal bones (Couly et al 1993,Le Douarin et al 1993).Commitment of neural crest cells to skeletal fates depends on their interactions with epithelial cells (Langille 1994).Cytokine-based signals from overlying epithelium activates signaling pathways and transcription factors in the underlying mesenchyme;the mesenchyme in turn secretes molecules that control growth and differentiation of the epithelium.
A large number of genes are consequently essential for craniofacial bone development (Winter 1996).These include several genes encoding homeobox-containing transcription factors such as goosecoid (gsc )(Rivera-Perez et al 1999),Barx1(Barlow et al 1999),Dlx1(Qiu et al 1997),Dlx2(Ferguson et al 2000,Qiu et al 1997,Thomas et al 2000),Dlx5(Acampora et al 1999,Ferguson et al 2000),Msx1(Satokata &Maas 1994,Vastardis et al 1996),Cart1(Zhao et al 1996),Hoxa1(Hunt et al 1998,Rossel &Capecchi 1999),Hoxa2(Hunt et al 1998),Hoxa3(Hunt et al 1998),Hoxb1(Hunt et al 1998,Rossel &Capecchi 1999);polycomb group genes such as rae28(Takihara et al 1997);basic helix-loop-helix transcription factors such as Twist (el Ghouzzi et al 1997,Howard et al 1997);the transcription factors AP-2(Schorle et al 1996,Zhang et al 1996)and Mf1(Kume et al 1998);and Pax genes encoding paired-box-containing transcription factors.Several cytokines and growth factors,including BMP4(St Amand et al 2000),fgf8(Trumpp et al 1999),Tgf-α(Miettinen et al 1999)and endothelin-1(Clouthier et al 1998),and their receptors (Burke et al 1998,Miettinen et al 1999)play important roles in craniofacial bone development.Finally,craniofacial skeletal development depends on controlled proteolytic processes in extracellular matrices mediated by members of the large family of matrix metalloproteases (MMPs)and their physio-logical inhibitors,the tissue inhibitors of metalloproteases (TIMPs)(Chin &Werb 1997).
Given the space limitations for this review,a detailed discussion of all these genes and their specific roles in craniofacial bone development is not possible.We have therefore selected (somewhat arbitrarily)one gene,Pax3,as an example,and discuss how information about its role has been derived from both animal and human studies.
Abnormalities that affect neural crest cells and therefore patterning of cran-iofacial bones include several types of Waardenburg syndrome (Read &Newton 1997).Waardenburg syndrome types 1and 3are caused by mutations in the tran-scription factor PAX3(Hoth et al 1993,Tassabehji et al 1992).PAX3is expressed in the dorsal region of the neural tube at a time when neural crest cells are formed
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and start their migration (Dahl et al 1997).A variety of mutations in PAX3,includ-ing in-frame deletions,premature terminations,and splice site mutations resulting in loss of function,result in craniofacial bone and soft tissue abnormalities,com-bined with partial albinism,hearing loss,and increased risk of spina bifida,cleft lip/palate,and scapular anomalies.Pigmentation abnormalities are variable,in part depending on the nature of the mutation.Some mutations,such as the replacement of an asparagine residue with histidine in the paired domain,are associated with a craniofacial and hand phenotype (small maxilla,absent or small nasal bone,ulnar deviation of hands)without pigment abnormalities (Hoth et al 1993).
The PAX3gene is one of the nine PAX genes that encode a homeodomain,as well as a pairbox sequence (Dahl et al 1997).Both are DNA-binding domains,but whereas the homeodomain activates transcription of target genes,the paired do-main acts as a transcriptional repressor.In the mouse,point mutations or deletions of Pax3cause the splotch phenotype (Epstein et al 1991).Heterozygotes show white spotting;homozygotes die before or at birth (depending on the mutations)with neural tube defects and exencephaly.Interestingly,staining for chondroitin and heparan sulfate proteoglycans is more intense in splotch homo-and heterozy-gotes than in wild-type embryos,raising the possibility that abnormalities in ex-tracellular matrices may contribute to the tissue consequences of Pax3mutations (Trasler &Morriss-Kay 1991).
Another Pax gene,Pax9,is also expressed in the neural crest–derived mes-enchyme involved in craniofacial and tooth development.In addition,it is ex-pressed in the developing axial and limb skeleton (see below)and in the endodermally derived epithelium of the pharyngeal pouches (Peters et al 1998).Mice that are homozygous for Pax9null alleles show various craniofacial abnor-malities and no teeth (Peters et al 1998),and a frameshift mutation in the paired domain of PAX9has been reported in a family with oligodontia (lacking most permanent molars)(Stockton et al 2000).
Development and Disorders of the Axial Skeleton
The early patterning of the axial skeleton is controlled by genes that regulate the segmentation of paraxial mesoderm into somites and their subsequent differentia-tion into sclerotomes (Tam &Trainor 1994).The somites give rise to the vertebrae and the dorsolateral portion of the ribs,the dermis of the dorsal skin,and the skele-tal muscle of the body wall and the limbs.Somitogenesis occurs bilaterally,in a precisely timed rostro-caudal sequence.The process is driven by a molecular seg-mentation clock that involves oscillation of hairy 1expression (Palmeirim et al 1997).This in turn drives the expression of lunatic fringe (Forsberg et al 1998,Mc-Grew et al 1998),which controls changes in Notch activity and Delta expression.The importance of the Notch-Delta pathway for somitogenesis is illustrated by the irregular size and asymmetry of somites seen in several mouse knockouts that affect the pathway.These knockouts,including Mesp2(Saga et al 1997),Notch1(Conlon et al 1995),Dll1(Hrabe de Angelis et al 1997),lunatic fringe (Evrard
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Figure 2Diagram showing the migration of sclerotomal cells from the ventromedial area of the somites toward the notochord in response to sonic hedhehog expression by the notochord and floor plate.Modified from Mundlos &Olsen 2000.
et al 1998,Zhang &Gridley 1998),and Dll3(Kusumi et al 1998),and the pudgy mutation in mice (disruption of Dll3),result in vertebral and rib defects that are similar to many axial abnormalities in humans.These genes are therefore excel-lent candidates for human disorders of segmentation in the vertebral column.In fact,one form of such a disorder has been mapped to the locus of Dll3(Turnpenny et al 1999),and mutations in Dll3have been found (Bulman et al 2000).
Patterning and differentiation of cells within the somites are controlled by sig-nals from the notochord and neural tube floorplate,the surface ectoderm,and the neural tube.These signals induce epithelial-mesenchymal transformation and proliferation of the ventromedial somitic cells and their migration toward the noto-chord (Figure 2).These cells form the sclerotome and become the cartilage in the vertebral bodies and the dorsolateral portion of the ribs.Sonic hedgehog (Shh)is the major signal from the notochord/floorplate that induces sclerotome formation,but subsequent differentiation of chondrocytes can occur in the absence of Shh.In vitro experiments suggest that the function of Shh is to make sclerotomal cells competent to respond to bone morphogenetic proteins (BMPs)and differentiate into chondrocytes (Murtaugh et al 1999).No sclerotomes develop in mice that are homozygous for Shh null alleles,and there is complete absence of vertebrae and the dorsolateral portion of the ribs (Chiang et al 1996).The mice also show com-plete absence of the distal limb skeleton;this provides a striking demonstration of the crucial role of Shh in patterning of the distal part of the limbs (see below).Under the influence of Shh,two related Pax genes,Pax1and Pax9,are upregu-lated in sclerotomal cells (Peters et al 1999).The expression of Pax1precedes that of Pax9.It is initially seen in all cells but later is at the highest level in the pos-terior ventromedial region of the sclerotomes.Pax9expression is more intense in
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the posterior ventrolateral region.As chondrogenesis proceeds,the expression of both Pax genes is downregulated,but it continues in cells in the perichondrium and in intervertebral regions.Studies of natural mutations in Pax1in undulated mouse mutants (Chalepakis et al 1991),as well as of mice with targeted Pax1null alleles (Wilm et al 1998),suggest that homozygosity for loss-of-function mutations in Pax1results in abnormalities in the vertebral column,sternum,and scapula that are not embryonic lethal.In contrast,mice that are homozygous for null alleles of Pax9die shortly after birth;they lack the derivatives of both the 3rd and 4th pha-ryngeal pouches (thymus,parathyroid,ultimobranchial bodies).They also have a cleft palate and other craniofacial defects and no teeth,as well as extra digits in fore-and hindlimbs (Wallin et al 1994),consistent with Pax9expression in those regions of the embryo.It is possible that lack of axial bone defects in Pax9null mice is the result of compensation by Pax1because Pax1and Pax9double-null mutants have no ventral vertebral bodies or intervertebral discs (Peters et al 1999).Patterning of the axial skeleton along the rostrocaudal axis,i.e.the definition of the identity of individual vertebrae,is accomplished by the expression of homeotic (Hox )genes in overlapping domains along the vertebral column (Burke et al 1995,Favier &Dolle 1997).Ectopic expression or targeted deletion of Hox genes in the mouse leads to addition or deletion of vertebral elements or changes into shapes resembling other elements (Chen et al 1998,Zakany et al 1997).
Development and Disorders of the Limb Skeleton
The skeletal tissues in the limb are produced by cells derived from the lateral plate mesoderm (Cohn &Tickle 1996).Other tissues,such as nerves,blood vessels,and muscles,are formed by cells from somites that migrate into the growing limb buds.The patterning of the mesenchyme in the limb and the ultimate shaping of the limb bones are due to a series of interactions between the mesenchyme and the overlying epithelium (Ng et al 1999).The proximodistal outgrowth is directed by fibroblast growth factor signals from the specialized epithelial cells in the apical ectodermal ridge (AER)covering the tip of the limb bud (Ng et al 1999);anteroposterior patterning is directed by sonic hedgehog produced by a small group of cells in the posterior zone of polarizing activity (ZPA)(Riddle et al 1993);and dorsoventral patterning depends on secretion of Wnt7a (Parr &McMahon 1995)and expression of radical fringe by the dorsal ectoderm and expression of the homeobox-containing transcription factor engrailed in the ventral ectoderm (Laufer et al 1997a,Loomis et al 1998,Rodriguez-Esteban et al 1997).Wnt7a induces and maintains expression of the LIM-homeodomain protein Lmx1b in dorsal limb mesenchymal cells;a mutation in Wnt7a is the basis for the mouse mutant postaxial hemimelia (Parr et al 1998).Ectopic expression of Lmx1in ventral mesenchyme generates double-dorsal limbs in chick embryos (Riddle et al 1995),and mutations in LMX1B in humans are the causes of the so-called nail-patella syndrome,a disorder characterized by dysplasia of nails and hypoplasia/aplasia of the patella,combined with renal anomalies (Dreyer et al 1998,V ollrath et al 1998).Since the limbs develop in a proximodistal sequence,the cartilage anlagen of the
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Figure 3Diagram showing how cartilage elements in the developing forelimb are formed in a proximodistal sequence with increasing developmental time (arrow),such that the humerus anlage is formed first,the metacarpal anlagen (I-V)last.The elements formed arise through a series of bifurcations and segmentations of mesenchymal condensations along the posterior (ulnar)axis of the limb.The limb genes discussed in the review are listed outside the diagram.On the right are genes controlled by sonic hedgehog and genes related to BMP signaling.At the top are genes expressed by the apical ectodermal ridge;at the bottom are transcription factor genes associated with establishment of limb identity.On the left are genes associated with specification of dorsal-ventral properties.Modified from Shubin &Alberch 1986.
limb bones are produced in the same order,with humerus/femur formed first and the phalangeal anlagen added last.The cartilages are formed as continuous rods that through a series of bifurcations and segmentations give rise to the characteristic limb skeleton (Shubin &Alberch 1986)(Figure 3).Segmentation and associated apoptotic cell death result in the formation of joints.The development of joints is controlled by BMP and BMP homologues,as well as their extracellular antagonists such as noggin,that bind BMP and reduce signaling through their cell surface receptors (Laufer et al 1997b,McMahon et al 1998,Storm &Kingsley 1999,Zou et al 1997).
Several conditions that include shortening of fingers and toes (brachydactylies),either alone or in combination with short bones in the lower limbs (acrome-somelic dysplasia),provide insights into the roles of BMPs and related molecules in limb bone growth and joint formation.Brachydactyly type C is caused by mutations in CDMP1(cartilage-derived morphogenetic protein 1;also called GDF5for growth and differentiation factor 5)that result in functional haploin-sufficiency (Polinkovsky et al 1997).In mice,homozygosity for functional null alleles of Gdf5leads to brachypodism (Storm et al 1994).Homozygosity for CDMP1mutations in humans causes acromesomelic dysplasias (Thomas et al 1996,1997).These are disorders characterized by short stature in combination with shortening of forearms and lower legs as well as the long bones of hands and feet.With homozygosity for CDMP1null alleles,the result is the clinical condition called acromesomelic dysplasia Hunter-Thompson type.In the more severe Grebe type,affected individuals have a null allele on one chromosome
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and an allele with a dominant-negative mutation on the other chromosome.The dominant-negative effect appears to be the result of the ability of the mutant protein to form heterodimers with other BMPs and prevent their secretion.Interference with BMP signaling is also the cause of inherited absence of proximal interpha-langeal joints,fusion of wrist and ankle bones,and conductive deafness.This disorder,called proximal symphalangism,has been shown to be caused by muta-tions in the BMP-binding molecule noggin.Noggin mutations are also associated with multiple dysostoses syndrome,a disorder characterized by fusion of sev-eral joints (elbows,hips,intervertebral)in addition to joints in the hands and feet (Gong et al 1999).Not surprisingly,mice that are homozygous for targeted noggin alleles have multiple defects,including fusion of limb bones (McMahon et al 1998).
Abnormalities of limb bones associated with either anomalies of the heart or breast tissues are seen in Holt-Oram and the ulnar-mammary syndromes.The two conditions are caused by mutations in the transcription factors TBX5and TBX3,respectively (Bamshad et al 1997,Basson et al 1994,Li et al 1997),which are members of the T-box transcription factor family whose canonical member is Brachyury or T ,a transcription factor involved in the development of posterior mesoderm during gastrulation.TBX5is expressed in developing forelimbs but not hindlimbs (Chapman et al 1996),whereas TBX4is expressed only in the developing leg (Isaac et al 1998).It is currently believed that the two factors regulate limb outgrowth and identity (Gibson-Brown et al 1996,1998;Ohuchi et al 1998;Takeuchi et al 1999).The homeodomain transcription factor Pitx1is an upstream regulator of Tbx4(Logan &Tabin).Mice with Pitx1null alleles have abnormalities in tibia,fibula,patella,and tarsal bones so that they look more like the skeletal elements of forelimbs,as well as cleft palate and changes in the pituitary (Lanctot et al 1999,Szeto et al 1999).Misexpression of Pitx1in the wing bud of chick embryos causes the wings to become more like legs (Logan &Tabin 1999),and misexpression of Tbx4and Tbx5in wing buds and leg buds of chick embryos changes the identity,at least partially,of the limbs (Rodriguez-Esteban et al 1999,Takeuchi et al 1999).
The importance of sonic hedgehog signaling for anteroposterior patterning of the limb skeleton is dramatically illustrated by several disorders in which an in-creased number of digits,polydactyly,are seen in variable association with fu-sion of soft interdigital tissues or bones,syndactylies.In Greig cephalopolysyn-dactyly,deletions or truncations of the transcription factor gene GLI3,a zinc finger transcriptional repressor downstream of sonic hedgehog signaling (Marigo et al 1996),result in broad thumbs,polydactyly,syndactyly,and craniofacial anoma-lies (V ortkamp et al 1991).Pallister-Hall syndrome,with a phenotype that overlaps that of Greig syndrome,and isolated postaxial polydactyly with an extra digit on the ulnar or fibular side of the hands or feet have been shown to be caused by frameshift mutations in GLI3(Kang et al 1997,Radhakrishna et al 1997).In mice,mutations in Gli3cause the phenotypes extra-toes (xt )(Hui &Joyner 1993)and polydactyly Nagoya (Pdn )(Thien &Ruther 1999).
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One of the target genes of sonic hedgehog signaling is Sall1,a zinc finger transcription factor and vertebrate homologue of Drosophila spalt ,expressed in all known hedgehog signaling areas of the embryo,including the notochord,limb buds,and urogenital ridge-derived structures (Ott et al 1996).This explains why Townes-Brocks syndrome,caused by mutations in SALL1,is characterized by craniofacial anomalies,hand anomalies with polydactyly,as well as renal and anal anomalies (Kohlhase et al 1998).
Sonic hedgehog also controls a posterior-anterior nested expression pattern of Hoxd genes in the developing lower arms (legs)and hands (feet).A striking il-lustration of the role of Hoxd genes in the limbs is provided by synpolydactyly,where mutations in HOXD13,the most 5 gene in the HOXD cluster on chromo-some 2,result in syndactyly between fingers three and four and toes four and five,and duplication of a finger in the syndactylous web in the hand (Muragaki et al 1996).All mutations,as well as the mutation in a mouse model (Spdh )of the human condition,are in-frame expansions of a 15-residue polyalanine tract in the N-terminal region of HOXD13(Goodman et al 1997,Johnson et al 1998).It is likely that the mutations result in gain-of-function in direct proportion to the size of the expansion.Frameshift mutations in HOXD13that are expected to lead to null alleles have also been described;these mutations cause the same hand phenotype as the polyalanine expansions,but a different foot abnormality,with a rudimentary digit between metatarsals one and two (Goodman et al 1998).
CHONDROCYTE DIFFERENTIATION AND ENDOCHONDRAL BONE FORMATION Chondrogenesis—the Role of Sox Genes
The role of the transcription factor SOX9in chondrogenesis was first recognized through the discovery that mutations in SOX9cause the rare and severe dwarfism campomelic dysplasia (CD)in humans (Foster et al 1994,Wagner et al 1994).CD patients show bowing and angulation of long bones,scapular and pelvic hy-poplasia,abnormalities of the vertebral column,a decreased number of ribs,and a small chondrocranium resulting in several craniofacial anomalies.These skele-tal abnormalities are frequently associated with XY sex reversal (Mansour et al 1995),consistent with a role for SOX9in testis differentiation (Morais da Silva et al 1996).Mutations in the SOX9coding region are scattered along the protein sequence and are believed to result in haploinsufficiency (Meyer et al 1997).These skeletal abnormalities are consequences of a defect in chondrocyte differ-entiation within mesenchymal condensations and deficient synthesis of cartilage matrix.Sox9transcripts are detected in all prechondrogenic mesenchymal con-densations as early as 8.5to 9.5days of mouse embryonic development,and the expression peaks in cartilage primordia at 11.5to 14.5days (Ng et al 1997,Wright et al 1995).Like collagen type II,encoded by Col2a1,Sox9is expressed at high levels in all prechondrocytes and chondrocytes.Consistent with XY reversal,Sox9
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