A distinct cohort of progenitor cells participates in synovial joint and articular cartilage formation during mouse limb skeletogenesis.

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

Conditional Kif3a ablation causes abnormal hedgehog signaling topography, growth plate dysfunction, and excessive bone and cartilage formation during mouse skeletogenesis.

The motor protein Kif3a and primary cilia regulate important developmental processes, but their roles in skeletogenesis remain ill-defined. Here we created mice deficient in Kif3a in cartilage and focused on the cranial base and synchondroses. Kif3a deficiency caused cranial base growth retardation and dysmorphogenesis, which were evident in neonatal animals by anatomical and micro-computed tomography (microCT) inspection. Kif3a deficiency also changed synchondrosis growth plate organization and function, and the severity of these changes increased over time. By postnatal day (P)7, mutant growth plates lacked typical zones of chondrocyte proliferation and hypertrophy, and were instead composed of chondrocytes with an unusual phenotype characterized by strong collagen II (Col2a1) gene expression but barely detectable expression of Indian hedgehog (Ihh), collagen X (Col10a1), Vegf (Vegfa), MMP-13 (Mmp13) and osterix (Sp7). Concurrently, unexpected developmental events occurred in perichondrial tissues, including excessive intramembranous ossification all along the perichondrial border and the formation of ectopic cartilage masses. Looking for possible culprits for these latter processes, we analyzed hedgehog signalling topography and intensity by monitoring the expression of the hedgehog effectors Patched 1 and Gli1, and of the hedgehog-binding cell-surface component syndecan 3. Compared with controls, hedgehog signaling was quite feeble within mutant growth plates as early as P0, but was actually higher and was widespread all along mutant perichondrial tissues. Lastly, we studied postnatal mice deficient in Ihh in cartilage; their cranial base defects only minimally resembled those in Kif3a-deficient mice. In summary, Kif3a and primary cilia make unique contributions to cranial base development and synchondrosis growth plate function. Their deficiency causes abnormal topography of hedgehog signaling, growth plate dysfunction, and un-physiologic responses and processes in perichondrial tissues, including ectopic cartilage formation and excessive intramembranous ossification.

Temporomandibular joint formation and condyle growth require Indian hedgehog signaling.

The temporomandibular joint (TMJ) is essential for jaw function, but the mechanisms regulating its development remain poorly understood. Because Indian hedgehog (Ihh) regulates trunk and limb skeletogenesis, we studied its possible roles in TMJ development. In wild-type mouse embryos, Ihh expression was already strong in condylar cartilage by embryonic day (E) 15.5, and expression of Ihh receptors and effector genes (Gli1, Gli2, Gli3, and PTHrP) indicated that Ihh range of action normally reached apical condylar tissue layers, including polymorphic chondroprogenitor layer and articular disc primordia. In Ihh(-/-) embryos, TMJ development was severely compromised. Condylar cartilage growth, polymorphic cell proliferation, and PTHrP expression were all inhibited, and growth plate organization and chondrocyte gene expression patterns were abnormal. These severe defects were partially corrected in double Ihh(-/-)/Gli3(-/-) mutants, signifying that Ihh action is normally modulated and delimited by Gli3 and Gli3(R) in particular. Both single and double mutants, however, failed to form an articular disc primordium, normally appreciable as an independent condensation between condylar apex and neighboring developing temporal bone in wild-type. This failure persisted at later stages, leading to complete absence of a normal functional disc and lubricin-expressing joint cavities. In summary, Ihh is very important for TMJ development, where it appears to regulate growth and elongation events, condylar cartilage phenotype, and chondroprogenitor cell function. Absence of articular disc and joint cavities in single and double mutants points to irreplaceable Ihh roles in formation of those critical TMJ components.

Indian and sonic hedgehogs regulate synchondrosis growth plate and cranial base development and function.

The synchondroses consist of mirror-image growth plates and are critical for cranial base elongation, but relatively little is known about their formation and regulation. Here we show that synchondrosis development is abnormal in Indian hedgehog-null mice. The Ihh(-/-) cranial bases displayed reduced growth and chondrocyte proliferation, but chondrocyte hypertrophy was widespread. Rather than forming a typical narrow zone, Ihh(-/-) hypertrophic chondrocytes occupied an elongated central portion of each growth plate and were flanked by immature collagen II-expressing chondrocytes facing perichondrial tissues. Endochondral ossification was delayed in much of the Ihh(-/-) cranial bases but, surprisingly, was unaffected most posteriorly. Searching for an explanation, we found that notochord remnants near incipient spheno-occipital synchondroses at E13.5 expressed Sonic hedgehog and local chondrocytes expressed Patched, suggesting that Shh had sustained chondrocyte maturation and occipital ossification. Equally unexpected, Ihh(-/-) growth plates stained poorly with Alcian blue and contained low aggrecan transcript levels. A comparable difference was seen in cultured wild-type versus Ihh(-/-) synchondrosis chondrocytes. Treatment with exogenous Ihh did not fully restore normal proteoglycan levels in mutant cultures, but a combination of Ihh and BMP-2 did. In summary, Ihh is required for multiple processes during synchondrosis and cranial base development, including growth plate zone organization, chondrocyte orientation, and proteoglycan production. The cranial base appears to be a skeletal structure in which growth and ossification patterns along its antero-posterior axis are orchestrated by both Ihh and Shh.

Differential expression of type XII collagen in developing chicken metatarsal tendons.

Type XII collagen is a fibril-associated collagen with multiple functional domains. The purpose of this work was to determine its role in regulating tendon matrix assembly. The temporal and spatial expression patterns of both collagen and mRNA were analysed in developing chicken metatarsal tendons using immunofluorescence microscopy, in situ hybridization and real-time quantitative PCR. Temporally, type XII collagen was present during all stages of development (day 14-hatch). However, spatially, type XII collagen expression shifted from the entire tendon at day 14, when the tendon is immature and fascicles are not well developed, to the interfacial matrix (endotendinium) associated with developing fascicles. This shift was obvious beginning at day 17, becoming prominent at day 19. Associated with this shift was a gradual decrease in type XII collagen reactivity in the tendon proper (non-sheath). By hatching, the reactivity was sequestered almost exclusively to the sheaths with some reactivity remaining at the fibroblast-matrix interface within the fascicle. In situ hybridization indicated that fibroblasts in the tendon expressed type XII collagen mRNA homogeneously at day 14. However, by hatching, when the tendon matures, type XII collagen is restricted primarily to the sheath cells. Quantitative PCR analyses, of NC3 splice variants, demonstrated highest expression levels for the short splice variant mRNA at days 14-17, followed by a significant decrease at day 19 with levels remaining constant to adult. Long variant mRNA expression was highest at day 14 then decreased and was constant from day 17 to adult. These changing patterns may be related to the spatial shift in type XII collagen expression to the sheaths. Differential temporal and spatial expression patterns indicate that type XII collagen functions to integrate the developing tendon matrices and fascicles into a functional unit.

The roles of types XII and XIV collagen in fibrillogenesis and matrix assembly in the developing cornea.

Corneal transparency depends on the architecture of the stromal extracellular matrix, including fibril diameter, packing, and lamellar organization. The roles of collagen types XII and XIV in regulation of corneal fibrillogenesis and development were examined. The temporal and spatial expression patterns were analyzed using semi-quantitative RT-PCR, in situ hybridization, Western analysis, and immunohistochemistry. Expression of types XII and XIV collagens in cornea development demonstrated that type XII collagen mRNA levels are constant throughout development (10D-adult) while type XIV mRNA is highest in early embryonic stages (10D-14D), decreasing significantly by hatching. The spatial expression patterns of types XII and XIV collagens demonstrated a homogeneous signal in the stroma for type XIV collagen, while type XII collagen shows segregation to the sub-epithelial and sub-endothelial stroma during embryonic stages. The type XII collagen in the anterior stroma was an epithelial product during development while fibroblasts contributed in the adult. Type XIV collagen expression was highest early in development and was absent by hatching. Both types XII and type XIV collagen have different isoforms generated by alternative splicing that may alter specific interactions important in fibrillogenesis, fibril-fibril interactions, and higher order matrix assembly. Analysis of these splice variants demonstrated that the long XII mRNA levels were constant throughout development, while the short XII NC3 mRNA levels peaked early (12D) followed by a decrease. Both type XIV collagen NC1 splice variants are highest during early stages (12D-14D) decreasing by 17D of development. These data suggest type XII collagen may have a role in development of stromal architecture and maintenance of fibril organization, while type XIV collagen may have a role in regulation of fibrillogenesis.