Biochemical, Genetic, and Molecular Interactions in Development - part 2 potx

Chondrocyte Cell Fate Determination 27 SRY-related HMG- Campomelic dysplasia, Short limb dwarfism, large Targeted disruption. Perinatal lethality. Skeletal defects in box gene 9; SOX9, autosomal dominant anterior fontanelle, macrocephaly, all bones derived from endochondral ossification, include 17q24.3-q25.1 (114290) micrognathia, cleft palate, hypo- cleft secondary palate, hypoplasia and bending. Skeletal (608160) plastic thoracic cage, missing abnormalities similar to those found in campomelic dysplasia twelfth pair of ribs, hypoplastic, patients. Skeletal patterning was not affected. Premature poorly ossified cervical vertebrae, m ineralization of skeletal elements, including craniofacial small iliac wings, short phalanges region and vertebral column. Hypertrophic zone of growth for both hands and feet, anterior plate was thicker (208). bowing of tibia, short fibula, mildly bowed femur, absent ossification of proximal tibial, and distal femoral epiphysis T-box 5; Holt-Oram syndrome, Vertebral anomalies, thoracic Conditional knockout. Embryonic lethality because of mal- TBX5, 12q24.1 autosomal dominant scoliosis, absent or bifid thumb, formed heart tube. Elongated phalangeal segments of first (601620) (142900) triphalangeal thumb, carpal forelimb digit and hypoplastic falciformis bones in the bone anomalies, upper extremity wrist were present in multiple heterozygous mutant phocomelia, radial-ulnar mice (209). anomalies Transforming growth Camurati-Engelmann Sclerosis of skull base, mandible Targeted disruption. Lethality around weaning due to factor, beta-1; disease autosomal involvement, sclerosis of pos- massive inflammation lesions and tissue necrosis in TGFB1, 19q13.1 dominant (131300) terior part of vertebrae, scoliosis, many organs (210,211) . (190180) progressive diaphyseal widening, thickened cortices, narrowing of medullary canal Vitamin D3 receptor; Vitamin D-resistant rickets, Rickets Targeted disruption. Animals normal until after weaning. VDR, 12q12-q14 autosomal recessive By 7 wk, null mice develop alopecia, flat face and short (601769) (277440) nose. Severe bone malformation leading to growth retarda- tion and 40% loss of bone density. Early lethality around 15 wk (212). a Human gene description includes gene name, symbol, corresponding OMIM number, and locus. b Human disease description includes disease name and corresponding OMIM number. 27 28 Shum et al. Table 2 Human Genetic Disorders with As-Yet No Known Genetic Associations Disorder name OMIM number Gene location Brief description of skeletal defects Acrocallosal syndrome; 200990 12p13.3-p11.2 Macrocephaly, large anterior fontanel, prominent occiput and forehead, hypoplastic ACLS midface, cleft palate, tapered fingers, fifth finger clinodactyly, brachydactyly, postaxial polydactyly, bifid terminal phalanges of thumbs, toe syndactyly, duplicated halluces Chondrocalcinosis 1; CCAL1 600668 8q (CCAL1) CCAL1; chondrocalcinosis, severe degenerative osteoarthritis Chondrocalcinosis 2; CCAL2 118600 5p15(CCAL2) CCAL2; chondrocalcinosis, arthropathy, acute intermittent arthritis, ankylosis Chondoma; CHDM 215400 7q33 Sacrococcygeal chordoma Cohen syndrome; COH1 216550 8q22-q23 Microcephaly, maxillary hypoplasia, micrognathia, joint hyperextensibility, narrow hands and feet, mild shortening of metacarpals and metatarsals Craniometaphyseal dysplasia; 218400 6q21-q22 Cranial hyperostosis, facial palsy, prominent supraorbital ridges and mandible, CMDR square profile, diaphyseal sclerosis, metaphyseal dysplasia, metaphyseal broadening Otopalatodigital syndrome, 304120 Xq28 Prominent forehead, severe micrognathia, midface hypoplasia, cleft palate, sclerotic type II; OPD2 skull base, bowing of long bones, small to absent fibula, subluxed elbow, wrist, and knee, flexed, overlapping fingers, short, broad thumbs, postaxial polydactyly, syndactyly, second finger clinodactyly, hypoplastic, irregular metacarpals Craniosynostosis, 600593 4p16 Craniosynostosis, coned epiphyses of hands and feet, distal and middle phalangeal Adelaide type; CRSA hypoplasia, carpal bone malsegmentation, phalangeal, tarsonavicular and calcaneo- navicular foot fusions FG syndrome; FGS1 305450 Xq12-q21.31 Macrocephaly, large anterior fontanel caused by delayed closure, plagiocephaly, micrognathia, cleft palate, joint contractures, broad thumbs, clinodactyly, syndactyly, broad halluces Fibrodysplasia ossificans 135100 4q27-q31 Heterotopic ossification, especially of the neck, spine, and shoulder girdle, progressiva; FOP malformed cervical vertebrae, short broad femoral necks, malformed big toes, monophalangic big toes, short thumbs, fifth finger clinodactyly, severely restricted arm mobility Larsen syndrome; LRS1 150250 3p21.1-p14.1 Cleft palate, flattened frontal bone, small skull base, shallow orbits, dysplastic epiphyseal centers, cervical vertebrae hypoplasia, scoliosis, spondylolysis, short metacarpals and metatarsals, multiple carpal and calcaneal ossification centers with delayed coalescence 28 Chondrocyte Cell Fate Determination 29 Otopalatodigital syndrome, 311300 Xq28 Prominent occiput and supraorbital ridges, cleft palate, absent frontal and sphenoid type I; OPD1 sinuses, thick frontal bone and skull base, delayed closure of anterior fontanel, steep clivus, dense middle-ear ossicles, short, broad distal phalanges, especially thumbs, short third, fourth, fifth metacarpals, supernumerary carpal bones, fusion of hamate and capitate, toe syndactyly, anomalous fifth metatarsal, extracalcaneal ossification center Pituitary dwarfism II 262500 5p13-p12 Acrohypoplasia, short limbs, delayed bone age, markedly advanced osseous maturation for height and age Russell-Silver syndrome; 180860 7p11.2 Micrognathia, skeletal maturation retardation, craniofacial disproportion, delayed RSS fontanel closure, asymmetry of arms and/or legs, fifth finger clinodactyly, fifth digit middle or distal phalangeal hypoplasia, syndactyly of second and third toes Shwachman-diamond 260400 Costochondral thickening, irregular ossification at anterior rib ends, delayed skel etal syndrome maturation, slipped capital femoral epiphyses, metaphyseal chondrodysplasia of long bones Sotos syndrome 117550 5q35 Macrocephaly, frontal bossing, prognathism, advanced bone age, large hands and feet, disharmonic maturation of phalanges and carpal bones Spastic paraplegia 9; SPG9 601162 10q23.3-q24.1 Skeletal abnormalities, short fifth finger, clinodactyly, delayed bone age, shallow acetabulum, small carpal bones, dysplastic skull base Syndactyly, type I 185900 2q34-q36 Syndactyly, complete or partial webbing between third and fourth fingers, fusion of third and fourth finger distal phalanges, complete or partial webbing between the second and third toes Velocardiofacial syndrome 192430 22q11 Microcephaly, Pierre Robin syndrome, cleft palate 29 30 Shum et al. the many functions of BMPs is to induce cartilage, bone, and connective tissue formation in verte- brates (24,25). This osteochondro-inductive capacity of BMPs is highly promising for orthopedic applications, such as skeletal repair and regeneration, and in dental applications, such as the treat- ment of periodontal diseases (26–30). Since the discovery of BMPs over three decades ago, their abil- ity to induce ectopic bone and cartilage formation remains a topic of intense investigation. In particular, the characterization of the molecular mechanisms of BMP functions was reignited after the cloning of the activin receptor, the first TGF-` type receptor, in 1991 (31). Thereafter, the molecular pathways to differentiation have been meticulously dissected and exposed. BMP signals through heterodimeric serine–threonine kinase receptor complexes, containing type I and type II receptors, each class having a number of subtypes (32,33). Both type I and type II recep- tors are capable of low-affinity interaction with BMP but only when the ligand binds to both receptors can result in high-affinity heteromeric ligand–receptor complex formation capable of BMP-depen- dent signaling (34,35). Therefore, it is likely that the presence and number of different BMP receptors determine the cellular responses to the many ligands. Evidence suggests that the subtype BMPR-IB is essential for chondrogenesis for the entire developing skeletal system (36–39). However, target deletion studies of the BMPR-IB receptor suggest otherwise (39,40). In these animals, the BMPR-IB does appear to have an essential role to play during limb bud morphogenesis because the abnormali- ties are located in the appendicular skeletal elements and not in the axial skeletal structures. Moreover, in vitro studies show that BMPR-IB does not possess exclusive chondrogenic potential, suggesting that other BMP type I receptors may exert redundant functions during chondrogenesis (41–43). Taken together, the response to BMP signal is not solely defined by the identity of the type I receptor but additionally by elements in the signal transduction pathways that lie downstream of the receptor. These are the various cytoplasmic and nuclear transducers, both positive and negative. Downstream from the receptors, Smads are the predominant effectors of TGF-`/BMP signaling (44, 45). An important issue for BMP-dependent signaling is the type of Smad proteins involved in chondro- genic differentiation and whether the Smads alone are sufficient to direct differentiation. Smads func- tion as dimeric complexes and belong to three classes: regulatory, inhibitory, and common. The receptor- regulated Smads (R-Smads) are further subdivided into two groups. Smad1, Smad5, and Smad8 are directly phosphorylated and activated by BMP type I receptors. Smad2 and Smad3 are mediators of activin or TGF-` type I receptor signaling. A series of in vitro studies have shown that Smad1, Smad5, and Smad8 may be involved in osteochondrogenic differentiation (46–49). These findings suggest that different Smads or Smad combinations are engaged at different stages of mesenchymal cell differentia- tion into osteoblasts and chondrocytes. However, in vivo manipulations of Smads have not resulted in conclusive evidence because genetically engineered animal models targeted against Smads pro- duce embryonic lethality (50). Nevertheless, a glimpse of in vivo Smad function can be observed in Smad3 knockout animals, which manifest osteopenia and early onset osteoarthritis (51). The class of inhibitory Smads (I-Smads) includes Smad6 and Smad7. They have been shown to inhibit the effect of R-Smads by competing for binding to activated type I receptors (52–56). Indeed, I-Smads are potent inhibitors of skeletogenic differentiation (48,57,58). The common Smad4 (Co-Smad) associates with activated R-Smad complex, which translocates into the nucleus and participates in the regulation of target genes (59). Smad4 functions as a tumor suppressor gene, and mutations of the human SMAD4 lead to pancreatic carcinoma and juvenile intestinal polyposis, further illustrating the significance of TGF-` superfamily signaling and its regulation of cellular physiology (60). BMP signaling can be channeled through Smad-independent pathways, such as the extracellular signal-regulated kinase, Jun N-terminal kinase, Wnt, and p38 mitogen-activated protein kinase path- ways (61–65). Therefore, crosstalk between the signaling pathways during chondrogenic differentia- tion is inevitable. However, a detailed recount of these interactions is beyond the scope of this review. Finally, BMP and other growth factor signaling can coactivate chondrogenic differentiation. For exam- ple, fibroblast growth factor (FGF) signaling through mitogen-activated protein kinase promotes Chondrocyte Cell Fate Determination 31 chondrogenesis by increasing the level of Sox9 expression as well as increases its binding affinity on the type II collagen promoter (66). It is obvious that BMPs control of chondrogenesis is a highly regu- lated developmental process that involves multiple pathways and checkpoints. This combinatorial mode of signaling ensures fidelity in the patterning and timing of the cartilaginous template onto which most of the bony skeleton is produced. CRANIOFACIAL MORPHOGENESIS AND CRANIAL NEURAL CREST CELLS (CNCCS) CNCCs give rise to most of the craniofacial tissues (67–69). Interestingly, this cell population is derived from the dorsal cephalic neural tube. During embryogenesis, the ectoderm at the midline over- lying the notochord thickens to form the neural plate. Progressively, the flattened neural plate begins to bend, creating elevations, called the neural folds, with a central depression the neural groove. As neurulation proceeds, the bilateral neural folds oppose each other and fuse at the midline to form the closed neural tube. At the time of neural tube closure and at the junction of where the thickened neuro- ectoderm meets the non-thickened surface ectoderm, epithelial cells delaminate and emerge as mes- enchymal cells into the underlying space. These are the neural crest cells (70). Neural crest cells are formed along the entire length of the primary neural tube. CNCCs are formed from the neural tube at the level of the forebrain, midbrain, and hindbrain. Neural crest cells are multipotential, and they give rise to a number of cell lineages (71,72). Those arising from the cranial region have different sets of potentials when compared with those arising in the trunk. For example, trunk neural crest cells do not normally produce cartilage. However, recent evidence from lineage tracing and transplantation strategies suggest that some trunk crest cells are capable of differentiating into cranial cartilages when transplanted into the cranial region (73,74). From a number of studies using various lineage tracing approaches, we have learned that neural crest cells from the forebrain and midbrain contribute to the frontonasal mesenchyme for the formation of the upper and midface structures, including part of the cranial base, nasal, and otic capsules (75–77). CNCCs in the branchial arches are destined for skeletal, odontogenic, myogenic, neuronal, and con- nective tissue lineages of the lower face and neck regions. Following the cartilage lineage in particular, CNCCs in the first branchial arch contribute to form Meckel’s cartilage and the temporomandibular joint cartilage. The hyoid is derived from CNCCs in both the second and third arches, and the fourth and sixth arches in combination give rise to the thyroid, cricoid, arytenoid, corniculate, and cunei- form cartilage (68,72,75,78–80). BMP REGULATION OF CRANIOFACIAL CARTILAGE DEVELOPMENT AND APOPTOSIS The hindbrain is a segmented structure, each segment called a rhombomere (Fig. 1). In the verte- brate head, there are eight pairs of rhombomeres and each gives rise to segment-specific CNCCs. During the migratory phase of CNCC development, CNCCs converge into three major streams directed toward the branchial arches in an orderly and patterned manner (81,82). Therefore, an early step in the regulation of craniofacial cartilage differentiation is CNCC production and patterning within the hindbrain. Similar to setting up the overall body plan, the hindbrain is patterned by a series of homeo- box (Hox)- and homeobox-containing genes (83). The production of CNCCs from these rhombomeres is in part regulated by their Hox genes. In addition, cell fate determination in the CNCCs is an orches- trated process (84–86). CNCCs exert a “community effect” among themselves and cell–cell and/or cell–matrix signaling in the group can maintain their segmental identity (87,88). In addition to this “community” effect, it is also discovered that the isthmus, a region between the midbrain and hind- brain, serves as a patterning center for the rhombomeres and the CNCC derivatives. The isthmus expresses high levels of FGF8 that regulates the expression of the Hox genes in the rhombomeres. 32 Shum et al. Transplantation experiments that include or exclude the isthmus yield different outcomes. The inclu- sion of the isthmus during grafting allows the rhombomeres and CNCCs to maintain their original iden- tity, whereas the exclusion of the isthmus renders CNCCs responsive to environmental cues (89). In addition to the isthmus, CNCCs can be patterned by signals from the endoderm to give rise to distinct pieces of craniofacial cartilages. Interestingly, this is only limited to CNCCs above the level of the second rhombomere, the so-called Hox-negative cells. CNCCs expressing Hox genes are not respon- sive to endodermal induction (90). Although each rhombomere can give rise to CNCCs, it is observed that those of rhombomeres 3 and 5 contribute to a minority of the population. A large number of CNCCs within the rhombomere undergo apoptosis, and only a small population migrate out. These cells join the major streams and, thus, lateral to rhombomeres 3 and 5, the area appears relatively free of CNCCs (91–98). This may serve to gauge the number of CNCCs being produced and to better delimit the migratory streams and their eventual destination. Evidence suggests that CNCC apoptosis is regulated by BMP and Wnt signaling. BMP4 is expressed coincidentally within rhombomeres 3 and 5. BMP4 induces the expres- sion of Msx2 in these rhombomeres, and ectopic expression of Msx2 increases the number of apopto- tic CNCCs (5,13,93,99). The lack of BMP signaling in even-numbered rhombomeres may be attributed to the presence of the BMP antagonist, noggin (100).Taken together, these experiments suggest that CNCC apoptosis is regulated by signals from BMP4 and is mediated by Msx2. Wnt signaling is signif- icant in this cascade because of the expression of cSFRP2 in rhombomeres that have limited apoptosis. cSFRP2 is an antagonist of the Wnt signaling and overexpression of cSFRP2 inhibits BMP4 expression and rescues CNCC from apoptotic elimination. Consistently, inhibition of cSFRP2 or overexpression of Wnt1 results in ectopic CNCC apoptosis (101). However, another Wnt family member; Wnt6, has been recently shown to be necessary and sufficient for the induction of neural crest formation (102). The use of different Wnt genes in combination that regulate CNCC formation is an elegant example of the complexity of the system. As CNCCs migrate from the neural tube towards the forming face, they converge into major streams, migrating toward the respective branchial arches. Migration is largely governed by adhesive proper- ties between cells and substrate, and a number of factors have defining roles in this developmental event (103). During migration, the cells remain in an undifferentiated state such that they are allowed to reach their destination before they expand further and undergo overt differentiation. Localization studies reveal that premigratory CNCCs and a subpopulation of migrating CNCCs may already be par- tially committed to the cartilage lineage by virtue of their expression of the key cartilage transcription factor, Sox9 (104,105). However, these cells do not differentiate yet. Differentiation of these cells may be suppressed by the coexpression of Msx2 in the Sox9-expressing cells. Msx2 may serve to main- tain these cells in an undifferentiated state until migration is completed. Overexpression of dominant- negative forms of Msx2 in these migratory cells inhibits normal Msx2 functions and leads to preco- cious cartilage differentiation (105). The mandible and maxilla arise from the anterior and posterior processes of the first branchial arch, respectively. These structures receive extensive contributions of CNCCs from the posterior midbrain and rhombomeres 1 and 2 of the anterior hindbrain. In addition to the lineages found in the other branchial arches, CNCCs in the first arch also differentiate into tooth structures that are unique to this arch (106,107). Meckel’s cartilage formed within the mandibular process has a unique pattern. It consists of an anterior, triangular piece at the midline, bilateral rod-shaped pieces that regress to form the sphenom- andibular ligament, and posterior pieces that give rise to the malleus, incus, and temporomandibular joint cartilage. The formation of Meckel’s cartilage is regulated by the mandibular epithelium through epithelial-mesenchymal interactions (108,109). The instructive signal from the epithelium can be substituted by epidermal growth factor (EGF), which sustains mesenchymal proliferation and delays chondrocyte differentiation (110,111). Removal of the epithelium results in increased but dysmorphic cartilage formation (112,113). Indeed, EGF and EGF receptors are endogenous to the mandibular Chondrocyte Cell Fate Determination 33 process (114,115). Antisense oligonucleotide inhibition of EGF in the mandibular process results in ectopic cartilage formation. In contrast, exogenous EGF reduces and disrupts cartilage formation (46,115). Furthermore, targeted disruption of EGF receptor in the mouse results in Meckel’s carti- lage deficiency as well (116). These defects are attributed to changes in matrix metalloproteinases expression and its regulation of cartilage morphogenesis. Expression of matrix metalloproteinases is regulated by EGF, and they function in multiple tissue morphogenesis, including that of the anterior segment of the developing Meckel’s cartilage (117). Within the mesenchyme, cartilage formation is further delimited by the expression of the tran- scription factor Msx2, which is excluded from regions with chondrogenic potential (118). Antisense oligonucleotide inhibition of Msx2 expression in the mandible results in disruption of Meckel’s car- tilage formation (119). Furthermore, adenoviral expression of ectopic Msx2 also abrogates cartilage formation (120). Interestingly, endogenous Msx2 expression is regulated by BMP expression and that ectopic BMP signaling can alter Msx2 expression domain, leading to cartilage dysmorphogenesis (120–122). Msx2 can also inhibit ectopic cartilage formation that is induced by BMP4 as a feedback reaction. However, the competence of the mesenchyme to respond to BMP4 is dependent on local signals and the key cartilage transcription factor Sox9, functions in antagonistic combination with Msx2 to regulate cartilage formation (120). LIMB MORPHOGENESIS AND LIMB MESENCHYME The limb cartilage develops from paired primordial buds that appear on the embryo’s lateral sur- face at specific levels along its anterior posterior body axis. At the early stages of limb development, the buds exhibit a paddle shape and consist of undifferentiated mesenchymal cells derived from the lateral plate and somitic mesoderm, and overlying ectoderm. At the distal tip of the bud, the ectoderm forms a specialized thickened epithelial structure, known as the apical ectodermal ridge (AER). Pat- terning along the proximal–distal axis depends in part on signaling molecules from the AER (123, 124). Instrumental to this process is the family of FGFs (125–130). The classic model of limb pat- terning involves the determination of positional values along the proximal–distal axis specified by instructive signaling from the AER to the subridge mesenchyme, known as the progress zone (131). However, recent revolutionary interpretation of limb patterning describes the specification of distinct proximal–distal segments of the limb early in development, with subsequent development involving expansion of these mesenchymal progenitor before differentiation (128,132). The anterior–posterior axis of the limb is patterned by the zone of polarizing activity (ZPA), which is located at the posterior margin of the limb (124,133,134). The major morphogen from this organizing center is the sonic hedge- hog (Shh) gene (135), which maintains anterior–posterior patterning in conjunction with other gene products, such as the HoxD gene (136), and participate in regulatory feedback signaling with the AER (137). Dorsal–ventral patterning is governed by ectodermally expressed Wnt7a and engrailed-1 pro- teins and their coregulation of Lmx1b gene expression at the dorsal mesenchyme (138,139). Therefore, patterning along the three axes is interlinked with each other. The limb cartilage elements form in a temporal proximal-to-distal sequence but are initially con- tiguous (36). Through the gradual recruitment of cells, the primary condensation of the stylopod (humorous/femur) forms first, the zeugopod (radius-ulna/tibia-fibula) forms second, and the autopod (carpals/tarsals and phalanges) forms last. There is considerable mixing of cells along the proximal– distal axis within each future segment but not between segments. Positional information is expressed by determinants of the Hox family of genes. The first part of the limb in which a subset of Hoxa and Hoxd genes are activated is the posterior limb (140,141). Subsequently, the expression domains extend anteriorly, in the distal part. In the final stage of limb morphogenesis, the mesenchyme in the distal region of the limb bud (autopod) can have two different fates, chondrogenesis or apoptosis, depend- ing on whether they are incorporated into the digital ray or into the interdigital regions. There is now considerable evidence to indicate that BMPs are essential mediators in specifying mesenchymal cells 34 Shum et al. undergoing either apoptosis or chondrogenesis and in the determination of digit identity (3,6,142–144). This point will be elaborated further in the next section. Finally, in regions of the mesenchymal conden- sation where joints from, condensed chondroprogenitors do not differentiate into chondrocytes but instead become tightly packed and adopt a fate of apoptosis as part of the normal program (25,145,146). Therefore, the orchestration of the apoptotic and chondrogenic response results in the formation and delineation of the limb cartilaginous template. Failure of either process results in limb malformations such as syndactyly or polydactyly of soft or hard tissues. BMP REGULATION OF LIMB CARTILAGE DEVELOPMENT AND APOPTOSIS BMPs are instrumental to the formation of the limb and are intimately involved in multiple stages of limb development, including patterning, outgrowth, AER regression, digit formation, digit identity, and interdigital apoptosis. To function in multiple developmental events, BMPs engage in signaling networks during limb morphogenesis and operate in concert with other key morphoregulatory factors, such as FGFs and Shh. Furthermore, several BMPs are already present during early development. BMP2, BMP4, and BMP7 are expressed in the limb mesenchyme in overlapping patterns before the formation of precartilagenous condensation (20). The specificity of BMPs for multistep action during limb morphogenesis is also reflected by different expression profile of the receptor subtypes trans- ducing the BMP signal (38). BMPs at an early stage regulate mesenchymal condensation into carti- lage nodules, as well as the induction of the AER (147). At later stages, BMPs are responsible for the maturation of limb cartilage and the regression of the AER (148). In vitro evidence supports that exog- enous BMP enhances chondrogenesis in limb mesenchyme after the condensation step (149). Through their function in the maintenance of the AER and consequential regulation of limb outgrowth along the proximal–distal axis, BMPs also relay information and participate in interdependent developmen- tal processes, such as patterning along the dorsal–ventral axis (150). There is little genetic evidence to support the role of BMPs in limb development because target mutation in animal studies of BMPs and their receptors result in early lethality or lack of phenotype directly related to cartilage formation (14,20,151). However, experiments using retroviral-mediated misexpression to simulate loss of func- tion result in limbs that show a lack of Alcian blue stain cartilage elements (37,42). However, infec- tion of the chick limb with retrovirus encoding BMP2, BMP4, or constitutively active receptor type I to simulate BMP gain of function results in fusion and hyperplasia of the cartilage elements (37,152). Mouse models show that BMP receptor type IB appears to be the necessary mediator of BMP-induced chondrogenesis (39,40), although overexpression of the receptor or constitutive activation of the receptor can also cause apoptosis (37,153). In addition to driving chondrogenesis, BMPs are key regulators of interdigital apoptosis that leads to the delineation of the digits. Among them, BMP2, BMP4, and BMP7 are expressed in the inter- digital regions before and during the occurrence of apoptosis, suggesting a role in cell death (20). Implantation of BMP4-soaked beads in interdigital regions accelerates interdigital cell death. In addi- tion, BMP4 can also cause ectopic cell death when applied at the tip of the developing digit pad (154). The apoptotic effect of BMP4 can be antagonized by FGF2 (155). Similarly, BMP2 and BMP7 are potent apoptotic signals for the undifferentiated limb mesenchyme but not for the ectoderm or the differentiating chondrogenic cells (156). Perturbations of BMP signaling through manipulation of BMP receptors also result in aberrations in interdigital apoptosis. For example, overexpressing domi- nant-negative BMP receptors in chick leg bud via replication-competent retrovirus to block endog- enous BMP signals results in inhibition of apoptosis in the interdigital mesenchyme, which leads to webbed chick feet (37).Taken together, these results indicate that BMP signaling is necessary for the apoptotic cascade in the interdigital mesenchyme. Interestingly, in parallel with craniofacial apoptosis as described in previous sections, Msx2 is also a mediator of BMP-induced interdigital apoptosis Chondrocyte Cell Fate Determination 35 (157). However, it is still unclear as to how Msx2 expression is instructive or permissive to apoptosis. Therefore, the totality of limb development and the emergence of its intricate design are dependent in part on BMP signaling in the larger context of many other growth and transcription factor signaling networks. Of particular interest are the role of retinoic acid and its interactions with BMP signaling and their coregulation of limb development. Retinoic acid is an endogenous morphogen at physiological levels and a teratogen in excess. Endogenous retinoids serve to pattern the hindbrain and the limb bud (158). Excessive retinoids lead to retinoic acid embryopathy characterized by craniofacial abnormalities (159). There are three distinct aspects of how retinoic acid modulates BMP signals. First, retinoic acid is well known for its ability to pattern the limb bud by virtue of its ability to substitute for the ZPA and for its upregulation of Shh that is endogenous to the ZPA (160,161). In tandem, retinoic acid also upregulates BMP expression that is needed for anterior–posterior patterning event (162,163). Second, retinoic acid regulates interdigital apoptosis by activating BMP expression and activities (164). Third, retinoic acid can also enhance chondrogenesis mediated by both BMP-dependent and BMP-indepen- dent pathways (164,165). Therefore, the regulation of chondrogenesis and apoptosis by BMP may rest on the ability of retinoic acid to divert BMP signaling to one pathway vs another, or the regulation by retinoic acid on distinct cofactors of BMP signaling for different pathways. SUMMARY AND FUTURE CHALLENGES Studies from classical developmental models suggest that cell fate determination is a progressive process that is dependent on combinatorial signaling of a repertoire of growth and differentiation fac- tor networks. Signaling is modulated by restricted expression profiles of factors organized in precise temporal and spatial arrays. Signaling is gauged by checkpoints where rate-limiting factors determine the threshold for progression. Because BMPs are multifunctional factors, the challenge is to identify the molecular basis for chondrogenic differentiation of mesenchymal cells. Functional studies should establish the mechanisms of lineage commitment and diversification, and provide a platform for molec- ular manipulations with predictable lineage outcomes. This knowledge will provide the molecular basis for tissue engineering and biomimetics of mesenchymal cells. ACKNOWLEDGMENTS We are grateful to Dr. Rocky Tuan for his support and encouragement. We have benefited from a long-standing scientific partnership with Dr. Glen Nuckolls. We have been blessed with outstand- ing visiting and postdoctoral scientists who had contributed to our knowledge base. Finally, we are indebted to Dr. Harold Slavkin, who continues to be an inspiration. This work was supported by NIH funding Z01AR41114. REFERENCES 1. Shum, L. and Nuckolls, G. (2002) The life cycle of chondrocytes in the developing skeleton. Arthritis Res. 4, 14994–15106. 2. Lee, S., Christakos, S., and Small, M. B. (1993) Apoptosis and signal transduction: clues to a molecular mechanism. Curr. Opin. Cell. Biol. 5, 286–291. 3. Chen, Y. and Zhao, X. (1998) Shaping limbs by apoptosis. J. Exp. Zool. 282, 691–702. 4. Graham, A., Koentges, G., and Lumsden, A. (1996) Neural crest apoptosis and the establishment of craniofacial pattern: an honorable death. Mol. Cell Neurosci. 8, 76–83. 5. Graham, A., Francis-West, P., Brickell, P., and Lumsden, A. (1994) The signalling molecule BMP4 mediates apoptosis in the rhombencephalic neural crest. Nature 372, 684–686. 6. Tang, M. K., Leung, A. K., Kwong, W. H., Chow, P. H., Chan, J. Y., Ngo-Muller, V., Li, M., and Lee, K. K. (2000) Bmp-4 requires the presence of the digits to initiate programmed cell death in limb interdigital tissues. Dev. Biol. 218, 89–98. 7. Ros, M. A., Piedra, M. E., Fallon, J. F., and Hurle, J. M. (1997) Morphogenetic potential of the chick leg interdigital mesoderm when diverted from the cell death program. Dev. Dyn. 208, 406–419. 36 Shum et al. 8. de Crombrugghe, B., Lefebvre, V., Behringer, R. R., Bi, W., Murakami, S., and Huang, W. (2000) Transcriptional mechanisms of chondrocyte differentiation. Matrix Biol. 19, 389–394. 9. Wagner, T., Wirth, J., Meyer, J., Zabel, B., Held, M., Zimmer, J., et al. (1994) Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 79, 1111–1120. 10. Foster, J. W., Dominguez-Steglich, M. A., Guioli, S., Kowk, G., Weller, P. A., Stevanovic, M., et al. (1994) Campo- melic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature 372, 525–530. 11. Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Osf2/Cbfa1: a transcriptional activator of osteoblast differentiation. Cell 89, 747–754. 12. Rodriguez-Esteban, C., Tsukui, T., Yonei, S., Magallon, J., Tamura, K., and Izpisua Belmonte, J. C. (1999) The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398, 814–818. 13. Takahashi, K., Nuckolls, G. H., Tanaka, O., Semba, I., Takahashi, I., Dashner, R., Shum, L., and Slavkin, H. C. (1998) Adenovirus-mediated ectopic expression of Msx2 in even-numbered rhombomeres induces apoptotic elimination of cranial neural crest cells in ovo. Development 125, 1627–1635. 14. Dunn, N. R., Winnier, G. E., Hargett, L. K., Schrick, J. J., Fogo, A. B., and Hogan, B. L. (1997) Haploinsuffi- cient phenotypes in Bmp4 heterozygous null mice and modification by mutations in Gli3 and Alx4. Dev. Biol. 188, 235–247. 15. Zehentner, B. K., Haussmann, A., and Burtscher, H. (2002) The bone morphogenetic protein antagonist Noggin is regulated by Sox9 during endochondral differentiation. Dev. Growth Differ. 44, 1–9. 16. Urist, M. R. (1965) Bone: formation by autoinduction. Science 150, 893–899. 17. Ducy, P. and Karsenty, G. (2000) The family of bone morphogenetic proteins. Kidney Int. 57, 2207–2214. 18. Wozney, J. M. (1998) The bone morphogenetic protein family: multifunctional cellular regulators in the embryo and adult. Eur. J. Oral. Sci. 106, 160–166. 19. Graff, J. M. (1997) Embryonic patterning: to BMP or not to BMP, that is the question. Cell 89, 171–174. 20. Hogan, B. L. (1996) Bone morphogenetic proteins in development. Curr. Opin. Genet. Dev. 6, 432–438. 21. Mehler, M. F., Mabie, P. C., Zhu, G., Gokhan, S., and Kessler, J. A. (2000) Developmental changes in progenitor cell responsiveness to bone morphogenetic proteins differentially modulate progressive CNS lineage fate. Dev. Neurosci. 22, 74–85. 22. Reddi, A. H. (2001) Interplay between bone morphogenetic proteins and cognate binding proteins in bone and carti- lage development: noggin, chordin and DAN. Arthritis Res. 3, 1–5. 23. Balemans, W. and Hul, W. V. (2002) Extracellular regulation of BMP signaling in vertebrates: a cocktail of modulators. Dev. Biol. 250, 231–250. 24. Hoffmann, A. and Gross, G. (2001) BMP signaling pathways in cartilage and bone formation. Crit. Rev. Eukaryot. Gene Exp. 11, 23–45. 25. Kingsley, D. M. (2001) Genetic control of bone and joint formation. Novartis Found. Sympos. 232, 213–222; discussion 222–234, 272–282. 26. Yoon, S. T. and Boden, S. D. (2002) Osteoinductive molecules in orthopaedics: basic science and preclinical studies. Clin. Orthop. Feb, 33–43. 27. King, G. N. (2001) The importance of drug delivery to optimize the effects of bone morphogenetic proteins during periodontal regeneration. Curr. Pharm. Biotechnol. 2, 131–142. 28. Li, R. H. and Wozney, J. M. (2001) Delivering on the promise of bone morphogenetic proteins. Trends Biotechnol. 19, 255–265. 29. Wikesjo, U. M., Sorensen, R. G., and Wozney, J. M. (2001) Augmentation of alveolar bone and dental implant osseo- integration: clinical implications of studies with rhBMP-2. J. Bone Joint. Surg. Am. 83-A Suppl 1, S136–S145. 30. Reddi, A. H. (2001) Bone morphogenetic proteins: from basic science to clinical applications. J. Bone Joint. Surg. Am. 83-A Suppl 1, S1–S6. 31. Mathews, L. S. and Vale, W. W. (1991) Expression cloning of an activin receptor, a predicted transmembrane serine kinase. Cell 65, 973–982. 32. Miyazono, K., Kusanagi, K., and Inoue, H. (2001) Divergence and convergence of TGF-beta/BMP signaling. J. Cell. Physiol. 187, 265–276. 33. Kawabata, M., Imamura, T., and Miyazono, K. (1998) Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev. 9, 49–61. 34. Nohe, A., Hassel, S., Ehrlich, M., Neubauer, F., Sebald, W., Henis, Y. I., and Knaus, P. (2002) The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J. Biol. Chem. 277, 5330–5338. 35. Knaus, P. and Sebald, W. (2001) Cooperativity of binding epitopes and receptor chains in the BMP/TGFbeta super- family. J. Biol. Chem. 382, 1189–1195. 36. Sandell, L. J. and Adler, P. (1999) Developmental patterns of cartilage. Front. Biosci. 4, D731–D742. 37. Zou, H., Wieser, R., Massague, J., and Niswander, L. (1997) Distinct roles of type I bone morphogenetic protein receptors in the formation and differentiation of cartilage. Genes Dev. 11, 2191–2203. 38. Cheifetz, S. (1999) BMP receptors in limb and tooth formation. Crit. Rev. Oral Biol. Med. 10, 182–198. 39. Yi, S. E., Daluiski, A., Pederson, R., Rosen, V., and Lyons, K. M. (2000) The type I BMP receptor BMPRIB is required for chondrogenesis in the mouse limb. Development 127, 621–630. 40. Baur, S. T., Mai, J. J., and Dymecki, S. M. (2000) Combinatorial signaling through BMP receptor IB and GDF5: shaping of the distal mouse limb and the genetics of distal limb diversity. Development 127, 605–619. [...]... Restricting Bmp-4 mediated apoptosis in hindbrain neural crest Dev Dyn 22 0, 27 6 28 3 101 Ellies, D L., Church, V., Francis-West, P., and Lumsden, A (20 00) The WNT antagonist cSFRP2 modulates programmed cell death in the developing hindbrain Development 127 , 528 5– 529 5 1 02 Garcia-Castro, M I., Marcelle, C., and Bronner-Fraser, M (20 02) Ectodermal Wnt Function As a Neural Crest Inducer Science 29 7, 848–851... al (20 00) Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2 Dev Dyn 21 7, 401–414 121 Ekanayake, S and Hall, B K (1997) The in vivo and in vitro effects of bone morphogenetic protein -2 on the development of the chick mandible Int J Dev Biol 41, 67–81 122 Barlow, A J and Francis-West, P H (1997) Ectopic application of recombinant BMP -2 and BMP-4 can change patterning... neuronal development in the chick hindbrain Nature 337, 424 – 428 82 Trainor, P A., Sobieszczuk, D., Wilkinson, D., and Krumlauf, R (20 02) Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways Development 129 , 433–4 42 83 Hunt, P., Wilkinson, D., and Krumlauf, R (1991) Patterning the vertebrate head: murine Hox 2 genes mark distinct subpopulations of premigratory and. .. protein -2 - mediated chondrogenesis of murine C3H10T1 /2 mesenchymal cells J Biol Chem 27 7, 30870 20 878 63 Mulder, K M (20 00) Role of Ras and Mapks in TGFbeta signaling Cytokine Growth Factor Rev 11, 23 –35 64 Williams, J G (20 00) STAT signalling in cell proliferation and in development Curr Opin Genet Dev 10, 503–507 65 Zhang, Y and Derynck, R (1999) Regulation of Smad signalling by protein associations and. .. new patterning role for cranial mesoderm Nat Cell Biol 2, 96–1 02 89 Trainor, P A., Ariza-McNaughton, L., and Krumlauf, R (20 02) Role of the isthmus and FGFs in resolving the paradox of neural crest plasticity and prepatterning Science 29 5, 128 8– 129 1 90 Couly, G., Creuzet, S., Bennaceur, S., Vincent, C., and Le Douarin, N M (20 02) Interactions between Hox-negative cephalic neural crest cells and the foregut... hypertrophic chondrocytes (9) Finally, BMP -2 , BMP-4, and BMP-7 are expressed in the precartilaginous mesenchyme (28 ) and were shown to enhance chondrogenesis in vitro (29 ) and in vivo in chick limb buds (30) Regulation of BMP -2 and BMP-4 expression by Ihh (31) further indicates that the interplay between these various signals in vivo increases the intricacy by which chondrocyte growth and differentiation is... the TGF- family, BMPs are key regulators of organogenesis in early embryonic development because they also play an active role in regulating cartilage formation and differentiation during later stages BMP -2 , BMP-3, BMP-4, BMP-5, and BMP-7 all are expressed in the perichondrial cells (26 ) Although BMP-4 and BMP-7 are expressed at low levels by chondrocytes undergoing maturation (27 ), BMP-6 and BMP-7 are... during tooth and mandibular morphogenesis Development 127 , 1671–1679 108 Hall, B K (1980) Tissue interactions and the initiation of osteogenesis and chondrogenesis in the neural crestderived mandibular skeleton of the embryonic mouse as seen in isolated murine tissues and in recombinations of murine and avian tissues J Embryol Exp Morphol 58, 25 1 26 4 109 Tyler, M S and Hall, B K (1977) Epithelial influences... Bonnin, M A., and Le Douarin, N M (1997) Hox gene induction in the neural tube depends on three parameters: competence, signal supply and paralogue group Development 124 , 849–859 87 Schilling, T F., Prince, V., and Ingham, P W (20 01) Plasticity in zebrafish hox expression in the hindbrain and cranial neural crest Dev Biol 23 1, 20 1 21 6 88 Trainor, P and Krumlauf, R (20 00) Plasticity in mouse neural crest... promoters of the TGF- -regulated genes contain either AP-1 sites, such as the PAI-1 (55), TIMP-1 (56), TGF- ß1 (57), c-Jun (58), and 2( I) collagen (59) or CREB/ATF binding sites, such as fibronectin (60), cyclinD1 (37), and cyclin A (38) The presence of multiple DNA binding sites for different transcription factors allows for transcriptional crosstalk between Smads and the MAPKs during TGF- -induced gene . 11, 23 –45. 25 . Kingsley, D. M. (20 01) Genetic control of bone and joint formation. Novartis Found. Sympos. 23 2, 21 3 22 2; discussion 22 2 23 4, 27 2 28 2. 26 . Yoon, S. T. and Boden, S. D. (20 02) Osteoinductive. morphogenetic proteins and cognate binding proteins in bone and carti- lage development: noggin, chordin and DAN. Arthritis Res. 3, 1–5. 23 . Balemans, W. and Hul, W. V. (20 02) Extracellular regulation. of neuronal development in the chick hindbrain. Nature 337, 424 – 428 . 82. Trainor, P. A., Sobieszczuk, D., Wilkinson, D., and Krumlauf, R. (20 02) Signalling between the hindbrain and paraxial tissues