TGF-beta signaling is essential for joint morphogenesis

Despite its clinical significance, joint morphogenesis is still an obscure process. In this study, we determine the role of transforming growth factor beta (TGF-beta) signaling in mice lacking the TGF-beta type II receptor gene (Tgfbr2) in their limbs (Tgfbr2(PRX-1KO)). In Tgfbr2(PRX-1KO) mice, the loss of TGF-beta responsiveness resulted in the absence of interphalangeal joints. The Tgfbr2(Prx1KO) joint phenotype is similar to that in patients with symphalangism (SYM1-OMIM185800). By generating a Tgfbr2-green fluorescent protein-beta-GEO-bacterial artificial chromosome beta-galactosidase reporter transgenic mouse and by in situ hybridization and immunofluorescence, we determined that Tgfbr2 is highly and specifically expressed in developing joints. We demonstrated that in Tgfbr2(PRX-1KO) mice, the failure of joint interzone development resulted from an aberrant persistence of differentiated chondrocytes and failure of Jagged-1 expression. We found that TGF-beta receptor II signaling regulates Noggin, Wnt9a, and growth and differentiation factor-5 joint morphogenic gene expressions. In Tgfbr2(PRX-1KO) growth plates adjacent to interphalangeal joints, Indian hedgehog expression is increased, whereas Collagen 10 expression decreased. We propose a model for joint development in which TGF-beta signaling represents a means of entry to initiate the process.     

TGF-β type II receptor/MCP-5 axis: at the crossroad between joint and growth plate development

Despite its clinical significance, the mechanisms of joint morphogenesis are elusive. By combining laser-capture microdissection for RNA sampling with microarrays, we show that the setting in which joint-forming interzone cells develop is distinct from adjacent growth plate chondrocytes and is characterized by downregulation of chemokines, such as monocyte-chemoattractant protein-5 (MCP-5). Using in vivo, ex vivo, and in vitro approaches, we show that low levels of interzone-MCP-5 are essential for joint formation and contribute to proper growth plate organization. Mice lacking the TGF-β-type-II-receptor (TβRII) in their limbs (Tgfbr2(Prx1KO)), which lack joint development and fail chondrocyte hypertrophy, show upregulation of interzone-MCP-5. In vivo and ex vivo blockade of the sole MCP-5 receptor, CCR2, led to the rescue of joint formation and growth plate maturation in Tgfbr2(Prx1KO) but an acceleration of growth plate mineralization in control mice. Our study characterized the TβRII/MCP-5 axis as an essential crossroad for joint development and endochondral growth.     

Long-term in vitro analysis of limb cartilage development: involvement of Wnt signaling

Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.     

Effect of TGF-beta 1, TGF-beta 2, and bFGF on chick cartilage and muscle cell differentiation

In insulin containing defined medium TGF-beta 1, TGF-beta 2, and bFGF all stimulate chondrogenic differentiation in high-density micromass cultures of distal limb bud mesenchyme cells of chick embryos. In addition bFGF inhibits myogenic differentiation, while TGF-beta 1 and TGF-beta 2 appear to have no effect. TGF-beta 1 and bFGF together act additively to enhance chondrogenesis, while TGF-beta blocks the bFGF inhibitory action on myoblasts, thus allowing them to differentiate. In the absence of insulin, the inhibitory effect of bFGF on muscle cell differentiation is reduced; cartilage differentiation in the presence of the above growth factors is also slightly reduced.     

Phorbol esters inhibit chondrogenesis in limb mesenchyme by mechanisms independent of PGE2 or cyclic AMP1

Effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) on chondrogenesis and concentrations of prostaglandin E2 (PGE2) and cyclic AMP (cAMP) were investigated in micromass cultures of chick limb mesenchyme derived from the distal tip of stage 25 limb buds. TPA completely inhibited chondrogenesis during the first 4 days of culture; however, a few small cartilage nodules formed by day 6. Relative to control cultures, both PGE2 and cAMP concentrations were altered by TPA treatment during the 6-day period of cell culture. Concentrations of both compounds increased in control cells during the first 24 h of culture and then declined during the remaining 5 days. In TPA-treated cells both PGE2 and cAMP levels increased progressively during the 6 days of days of cell culture, each being elevated at day 6 by twofold over control cells. The results suggest the presence of regulatory pathways important in chondrogenesis which occur independent of those initiated by PGE2 and the cAMP system.     

Versican Facilitates Chondrocyte Differentiation and Regulates Joint Morphogenesis

Versican/PG-M is a large chondroitin sulfate proteoglycan in the extracellular matrix, which is transiently expressed in mesenchymal condensation areas during tissue morphogenesis. Here, we generated versican conditional knock-out mice Prx1-Cre/Vcan^flox/flox, in which Vcan is pruned out by site-specific Cre recombinase driven by the Prx1 promoter. Although Prx1-Cre/Vcan^flox/flox mice are viable and fertile, they develop distorted digits. Histological analysis of newborn mice reveals hypertrophic chondrocytic nodules in cartilage, tilting of the joint, and a slight delay of chondrocyte differentiation in digits. By immunostaining, whereas the joint interzone of Prx1-Cre/Vcan^+/+ shows an accumulation of TGF-β, concomitant with versican, that of Prx1-Cre/Vcan^flox/flox without versican expression exhibits a decreased incorporation of TGF-β. In a micromass culture system of mesenchymal cells from limb bud, whereas TGF-β and versican are co-localized in the perinodular regions of developing cartilage in Prx1-Cre/Vcan^+/+, TGF-β is widely distributed in Prx1-Cre/Vcan^flox/flox. These results suggest that versican facilitates chondrogenesis and joint morphogenesis, by localizing TGF-β in the extracellular matrix and regulating its signaling.     

Inactivation of Patched1 in the Mouse Limb Has Novel Inhibitory Effects on the Chondrogenic Program

The bones of the vertebrate limb form by the process of endochondral ossification, whereby limb mesenchyme condenses to form an intermediate cartilage scaffold that is then replaced by bone. Although Indian hedgehog (IHH) is known to control hypertophic differentiation of chondrocytes during this process, the role of hedgehog signaling in the earlier stages of chondrogenesis is less clear. We have conditionally inactivated the hedgehog receptor Ptc1 in undifferentiated limb mesenchyme of the mouse limb using Prx1-Cre, thus inducing constitutively active ligand-independent hedgehog signaling. In addition to major patterning defects, we observed a marked disruption to the cartilage elements in the limbs of Prx1-Cre:Ptc1^c/c embryos. Using an in vitro micromass culture system we show that this defect lies downstream of mesenchymal cell condensation and likely upstream of chondrocyte differentiation. Despite early increases in levels of chondrogenic genes, soon after mesenchymal condensation the stromal layer of Prx1-Cre:Ptc1^c/c-derived micromass cultures is characterized by a loss of cell integrity, which is associated with increased cell death and a striking decrease in Alcian blue staining cartilage nodules. Furthermore, inhibition of the hedgehog pathway activation using cyclopamine was sufficient to essentially overcome this chondrogenic defect in both micromass and ex vivo explant assays of Prx1-Cre:Ptc1^c/c limbs. These data demonstrate for the first time the inhibitory effect of cell autonomously activated hedgehog signaling on chondrogenesis, and stress the importance of PTC1 in maintaining strict control of signaling levels during this phase of skeletal development.     

Expression of Cre Recombinase in the developing mouse limb bud driven by a Prxl enhancer

We have used a Prx1 limb enhancer to drive expression of Cre Recombinase in transgenic mice. This regulatory element leads to Cre expression throughout the early limb bud mesenchyme and in a subset of craniofacial mesenchyme. Crossing a murine line carrying this transgene to a reporter mouse harboring a floxed Cre-reporter cassette revealed that recombinase activity is first observed in the earliest limb bud at 9.5 dpc. By early to mid bud stages at 10.5 dpc recombination is essentially complete in all mesenchymal cells in the limb. Expression of the Cre recombinase was never detected in the limb bud ectoderm. The use of Prx1-Cre mice should facilitate analysis of gene function in the developing limb.     

Conditional deletion of Tgfbr2 in hypertrophic chondrocytes delays terminal chondrocyte differentiation

Transforming growth factor β (Tgfb) signaling plays an important role in endochondral ossification. Previous studies of mice in which the Tgfb type II receptor gene (Tgfbr2) was deleted in the limb bud mesenchymal cells or differentiated chondrocytes showed defects in the development of the long bones or the axial skeleton, respectively. Here, we generated mouse embryos in which the Tgfbr2 gene was ablated in hypertrophic chondrocytes. These mice exhibited delays in both the hypertrophic conversion of proliferating chondrocytes and the subsequent terminal chondrocyte differentiation. The expression domains of Col10a1, Matrix metalloproteinase 13, and Osteopontin were small, and the expression of Vascular endothelial growth factor and Platelet endothelial cell adhesion molecule was downregulated. The calcification of the bone collar in the mutant mice was markedly delayed and the periosteum was thin, possibly because of the downregulation of Indian hedgehog expression. We conclude that Tgfb signaling in hypertrophic chondrocytes positively regulates terminal chondrocyte differentiation, angiogenesis in calcified cartilage, and osteogenesis in the bone collar, at least partly through Indian hedgehog signaling in vivo.     

Treatment with all-trans-retinoic acid decreases levels of endogenous TGF-beta(1) in the mesenchyme of the developing mouse inner ear

Background: Previous studies have shown that in utero exposure of the mouse embryo to high doses of all-trans-retinoic acid (atRA) produces defects of the developing inner ear and its surrounding cartilaginous capsule, while exposure of cultured periotic mesenchyme plus otic epithelium to high doses of exogenous atRA results in an inhibition of otic capsule chondrogenesis. Methods: In this study, we examine the effects of atRA exposure on the endogenous expression of transforming growth factor-beta(1) (TGF-beta(1)), a signaling molecule that mediates the epithelial-mesenchymal interactions that guide the development of the capsule of the inner ear. Results: Our results demonstrate a marked reduction in immunostaining for TGF-beta(1) in the periotic mesenchyme of atRA-exposed embryos of age E10.5 and E12 days in comparison with control specimens. Consistent with these in vivo findings, high-density cultures of E10.5 periotic mesenchyme plus otic epithelium, treated with doses of atRA that suppress chondrogenesis, showed significantly decreased levels of TGF-beta(1), as compared with TGF-beta(1) levels in untreated control cultures. Furthermore, we demonstrate a rescue of cultured periotic mesenchyme plus otic epithelium from atRA-induced chondrogenic suppression by supplementation of cultures with excess TGF-beta(1). Conclusions: Our results support the hypothesis that TGF-beta(1) plays a role in mechanisms of atRA teratogenicity during inner ear development.     

TGF-beta mediated Msx2 expression controls occipital somites-derived caudal region of skull development

Craniofacial development involves cranial neural crest (CNC) and mesoderm-derived cells. TGF-beta signaling plays a critical role in instructing CNC cells to form the craniofacial skeleton. However, it is not known how TGF-beta signaling regulates the fate of mesoderm-derived cells during craniofacial development. In this study, we show that occipital somites contribute to the caudal region of mammalian skull development. Conditional inactivation of Tgfbr2 in mesoderm-derived cells results in defects of the supraoccipital bone with meningoencephalocele and discontinuity of the neural arch of the C1 vertebra. At the cellular level, loss of TGF-beta signaling causes decreased chondrocyte proliferation and premature differentiation of cartilage to bone. Expression of Msx2, a critical factor in the formation of the dorsoventral axis, is diminished in the Tgfbr2 mutant. Significantly, overexpression of Msx2 in Myf5-Cre;Tgfbr2flox/flox mice partially rescues supraoccipital bone development. These results suggest that the TGF-beta/Msx2 signaling cascade is critical for development of the caudal region of the skull.     

Evidence for histogenic interactions during in vitro limb chondrogenesis

The requirement for homotypic cell interaction was studied by making chimeric micromass cultures containing various proportions of chick and quail limb mesenchyme. Cultures made from limb mesenchyme from embryos of Hamburger and Hamilton stages 23–24 produce large clumps of cartilage cells, identified by the accumulation of an extracellular matrix which stains with alcian blue at pH 1 and by the ability of cells to take up ³⁵SO₄ rapidly, as demonstrated autoradiographically. Dissociated mesenchyme from stage 19 embryos did not produce cartilage in micromass cultures, but only precartilage cell aggregates. Micromass cultures prepared from mixtures of mesenchyme cells obtained from stage 19 and stages 23–24 embryos contained decreasing numbers of cartilage nodules as the proportion of stage 19-derived mesenchyme increased. At the same time the number of aggregates was not affected. When the ratio of stage 19- to stage 24-derived cells was 3:1 or greater, no nodules were detected. The actual number of cells from each stage was verified by using mixtures of quail and chick cells, which are microscopically distinguishable. Additional evidence suggests that the stage 19-derived mesenchyme inhibits chondrogenesis by passively preventing stage 24-derived cells from interacting. The results presented are consistent with the suggestions that (1) homotypic cell interaction plays a role in limb chondrogenesis and (2) the capacity to interact in the required manner is acquired after the embryos have reached stage 19. These phenomena might be involved in the normal histogenesis of cartilage tissue.     

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.     

Epithelial control of periotic mesenchyme chondrogenesis

Morphogenesis of the cartilaginous otic capsule is directed by interactions between the epithelial anlage of the membranous labyrinth (otocyst) and its associated periotic mesenchyme. Utilizing a developmental series of high-density (micromass) cultures of periotic mesenchyme to model capsule chondrogenesis, we have shown that the early influence of otic epithelium in cultures of 10.5- or 14-gestation day (gd) periotic mesenchyme results in initiation or suppression of chondrogenesis, respectively. Furthermore, we have shown that introduction of otic epithelium at two distinct times during in vitro development to cultures of 10.5-gd mesenchyme cells results first in an initiation and then in an inhibition of their chondrogenic response. These influences of epithelial tissue on chondrogenic differentiation by periotic mesenchyme are not tissue specific but are characterized by temporal selectivity. The ability of otic epithelium to influence chondrogenesis and the competence of the periotic mesenchyme to respond to its signals are dependent upon the developmental stage of both tissues. This study provides conclusive evidence that otic epithelium acts as a developmental "switch" during otic capsule morphogenesis, signaling first the turning on and then the turning off of chondrogenic programs in the responding cephalic mesenchyme.     

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.