Abstract. As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen and cease synthesizing type I collagen. Changes in the cytoplasmic levels of type I and type II collagen mRNAs during the course of limb chondrogenesis in vivo and in vitro were examined using cloned cDNA probes. A striking increase in cytoplasmic type II collagen m R N A occurs coincident with the crucial condensation stage of chondrogenesis in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed before depositing a cartilage matrix. Thereafter, a continuous and progressive increase in the accumulation of cytoplasmic type II coUagen m R N A occurs which parallels the progressive accumulation of cartilage matrix by ceils. The onset of overt chondrogenesis, however, does not involve activation of the transcription of the type II collagen gene. Low levels of type II collagen m R N A are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages of limb development, well before the accumulation of detectable levels of type II collagen. Type I collagen gene expression during chondrogenesis is regulated, at least in part, at the translational level. Type I collagen mRNAs are present in the cytoplasm of differentiated chondrocytes, which have ceased synthesizing detectable amounts of type I collagen. T HE onset of cartilage differentiation in the developinglimb is characterized by a transient cellular condensation or aggregation process in which prechondrogenic mesenchymal cells become closely juxtaposed to one another before initiating cartilage matrix deposition. During this process, a cell-cell interaction, cell shape change, or some other event occurs which is necessary to trigger the chondrogenic differentiation of the cells (15). The critical condensation process may be initiated, at least in part, by a progressive decrease in the accumulation of extracellular hyaluronate (20,35). Fibronectin and type I collagen have been implicated in the cell-cell interaction occurring during condensation (7, 21, 34). Prostaglandin-mediated elevations in cAMP levels during condensation are involved in regulating chondrogenesis (3, 6, 11, 14, 16-18, 32, 33). A change in the shape of the cells from a flattened mesenchymal morphology to a rounded configuration also plays an important role in the process (2, 41).Although considerable insight has been gained into the extracellular influences and intracellular regulatory molecules that are involved in regulating limb chondrogenesis, virtually nothing is known about the molecular mechanisms by which these factors directly influence the changes in gene activity that occur during cartilage differentiation. Prechondrogenic limb mesenchymal cells synthesize type I collagen (36). As the cells differentiate into chondrocytes, they initiate the synthesis of cartilage-characteristic type II collagen and cease synthesizing type I collagen (36). Thus the conversion of mesenchymal chondrogenic progenitor cells into chondrocytes i...

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The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

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A partial cDNA that encodes a newly discovered member of the syndecan family of integral membrane proteoglycans, which we have termed syndecan 3, has been isolated from an embryonic chicken limb bud cDNA library. Syndecan 3 is distinct from but structurally related to syndecan and fibroglycan, two previously characterized members of this family of membrane-intercalated proteoglycans. Syndecan 3 contains a cytoplasmic domain potentially associated with the cytoskeleton that is 85% identical in amino acid sequence to the cytoplasmic domain of syndecan. Syndecan 3 also possesses a hydrophobic transmembrane domain and an extracellular domain containing several clustered potential glycosaminoglycan attachment sites. Like syndecan, the ectodomain of syndecan 3 has a single dibasic protease-susceptible site adjacent to the transmembrane domain, which might be involved in shedding the ectodomain from the cell surface. A striking feature of syndecan 3 is an extensive (182 amino acid) threonine, serine, and proline (T+S+P)-rich domain that closely resembles T+S+P-rich regions in several mucin-like proteins in which O-linked oligosaccharides are bound to the threonine and serine residues. Syndecan 3 is expressed in high amounts during a critical phase of chicken limb chondrogenesis in which limb mesenchymal cells condense, round up, and interact with one another before depositing a cartilage matrix. The multiple functional domains of syndecan 3 provide potential sites for mediating the adhesive cell-matrix interactions and cytoskeletal reorganization involved in this critical condensation process.

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During early stages of chick limb development, the homeobox-containing gene Msx-2 is expressed in the mesoderm at the anterior margin of the limb bud and in a discrete group of mesodermal cells at the midproximal posterior margin. These domains of Msx-2 expression roughly demarcate the anterior and posterior boundaries of the progress zone, the highly proliferating posterior mesodermal cells underneath the apical ectodermal ridge (AER) that give rise to the skeletal elements of the limb and associated structures. Later in development as the AER loses its activity, Msx-2 expression expands into the distal mesoderm and subsequently into the interdigital mesenchyme which demarcates the developing digits. The domains of Msx-2 expression exhibit considerably less proliferation than the cells of the progress zone and also encompass several regions of programmed cell death including the anterior and posterior necrotic zones and interdigital mesenchyme. We have thus suggested that Msx-2 may be in a regulatory network that delimits the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed. In the present study we show that ectopic expression of Msx-2 via a retroviral expression vector in the posterior mesoderm of the progress zone from the time of initial formation of the limb bud severely impairs limb morphogenesis. Msx-2-infected limbs are typically very narrow along the anteroposterior axis, are occasionally truncated, and exhibit alterations in the pattern of formation of skeletal elements, indicating that as a consequence of ectopic Msx-2 expression the morphogenesis of large portions of the posterior mesoderm has been suppressed. We further show that Msx-2 impairs limb morphogenesis by reducing cell proliferation and promoting apoptosis in the regions of the posterior mesoderm in which it is ectopically expressed. The domains of ectopic Msx-2 expression in the posterior mesoderm also exhibit ectopic expression of BMP-4, a secreted signaling molecule that is coexpressed with Msx-2 during normal limb development in the anterior limb mesoderm, the posterior necrotic zone, and interdigital mesenchyme. This indicates that Msx-2 regulates BMP-4 expression and that the suppressive effects of Msx-2 on limb morphogenesis might be mediated in part by BMP-4. These studies indicate that during normal limb development Msx-2 is a key component of a regulatory network that delimits the boundaries of the progress zone by suppressing the morphogenesis of the regions of the limb mesoderm in which it is highly expressed, thus restricting the outgrowth and formation of skeletal elements and associated structures to the progress zone. We also report that rather large numbers of apoptotic cells as well as proliferating cells are present throughout the AER during all stages of normal limb development we have examined, indicating that many of the cells of the AER are continuously undergoing programmed cell death at the same time that new AER cells are being generated by cell prol...