Runx2 and Runx3 are essential for chondrocyte maturation, and Runx2 regulates limb growth through induction of Indian hedgehog

The differentiation of mesenchymal cells into chondrocytes and chondrocyte proliferation and maturation are fundamental steps in skeletal development. Runx2 is essential for osteoblast differentiation and is involved in chondrocyte maturation. Although chondrocyte maturation is delayed in Runx2-deficient (Runx2(-/-)) mice, terminal differentiation of chondrocytes does occur, indicating that additional factors are involved in chondrocyte maturation. We investigated the involvement of Runx3 in chondrocyte differentiation by generating Runx2-and-Runx3-deficient (Runx2(-/-)3(-/-)) mice. We found that chondrocyte differentiation was inhibited depending on the dosages of Runx2 and Runx3, and Runx2(-/-)3(-/-) mice showed a complete absence of chondrocyte maturation. Further, the length of the limbs was reduced depending on the dosages of Runx2 and Runx3, due to reduced and disorganized chondrocyte proliferation and reduced cell size in the diaphyses. Runx2(-/-)3(-/-) mice did not express Ihh, which regulates chondrocyte proliferation and maturation. Adenoviral introduction of Runx2 in Runx2(-/-) chondrocyte cultures strongly induced Ihh expression. Moreover, Runx2 directly bound to the promoter region of the Ihh gene and strongly induced expression of the reporter gene driven by the Ihh promoter. These findings demonstrate that Runx2 and Runx3 are essential for chondrocyte maturation and that Runx2 regulates limb growth by organizing chondrocyte maturation and proliferation through the induction of Ihh expression.     

Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18

Gain of function mutations in fibroblast growth factor (FGF) receptors cause chondrodysplasia and craniosynostosis syndromes. The ligands interacting with FGF receptors (FGFRs) in developing bone have remained elusive, and the mechanisms by which FGF signaling regulates endochondral, periosteal, and intramembranous bone growth are not known. Here we show that Fgf18 is expressed in the perichondrium and that mice homozygous for a targeted disruption of Fgf18 exhibit a growth plate phenotype similar to that observed in mice lacking Fgfr3 and an ossification defect at sites that express Fgfr2. Mice lacking either Fgf18 or Fgfr3 exhibited expanded zones of proliferating and hypertrophic chondrocytes and increased chondrocyte proliferation, differentiation, and Indian hedgehog signaling. These data suggest that FGF18 acts as a physiological ligand for FGFR3. In addition, mice lacking Fgf18 display delayed ossification and decreased expression of osteogenic markers, phenotypes not seen in mice lacking Fgfr3. These data demonstrate that FGF18 signals through another FGFR to regulate osteoblast growth. Signaling to multiple FGFRs positions FGF18 to coordinate chondrogenesis in the growth plate with osteogenesis in cortical and trabecular bone.     

Bone morphogenetic protein rescues the lack of secondary cartilage in Runx2-deficient mice

Secondary cartilages including mandibular condylar cartilage have unique characteristics. They originate from alkaline phosphatase (ALP)-positive progenitor cells of the periosteum, and exhibit characteristic modes of differentiation. They also have a unique extracellular matrix, and coexpress type I, II and X collagens. We have previously shown that there is a total absence of secondary cartilages in Runx2-deficient (Runx2-/-) mice. To clarify whether Runx2 is essential for chondrocytic differentiation of secondary cartilages, we performed an organ culture system using mandibular explants derived from Runx2-/- mice at embryonic day 18.0. Since mRNA for bone morphogenetic protein 2 (BMP2) was strongly expressed in osteoblasts of condylar anlagen in wild-type mice, and was down-regulated in those of Runx2-/- mice, we chose to investigate BMP2 effects on secondary cartilage formation. Condensed mesenchymal cells of mandibular condylar anlagen in precultured explants were ALP-positive and expressed type I collagen and Sox9. After culture with recombinant human (rh) BMP2, chondrocytic cells showing ALP activity and expressing Sox5, Sox9, and type I and II collagens, appeared from mesenchymal condensation. This expression profile was comparable with the reported pattern of chondrocytes in mouse secondary cartilages. However, chondrocyte hypertrophy was not observed in the explants. These findings indicate that BMP2 partially rescued chondrocyte differentiation but not chondrocyte hypertrophy in secondary cartilage formation in Runx2-/- mice. Runx2 is required for chondrocyte hypertrophy in secondary cartilage formation, and it is likely that BMP2, which is abundantly secreted by osteoblasts in condylar anlagen, contributes to the early process of secondary cartilage formation.     

Core binding factor beta functions in the maintenance of stem cells and orchestrates continuous proliferation and differentiation in mouse incisors

Rodent incisors grow continuously throughout life, and epithelial progenitor cells are supplied from stem cells in the cervical loop. We report that epithelial Runx genes are involved in the maintenance of epithelial stem cells and their subsequent continuous differentiation and therefore growth of the incisors. Core binding factor β (Cbfb) acts as a binding partner for all Runx proteins, and targeted inactivation of this molecule abrogates the activity of all Runx complexes. Mice deficient in epithelial Cbfb produce short incisors and display marked underdevelopment of the cervical loop and suppressed epithelial Fgf9 expression and mesenchymal Fgf3 and Fgf10 expression in the cervical loop. In culture, FGF9 protein rescues these phenotypes. These findings indicate that epithelial Runx functions to maintain epithelial stem cells and that Fgf9 may be a target gene of Runx signaling. Cbfb mutants also lack enamel formation and display downregulated Shh mRNA expression in cells differentiating into ameloblasts. Furthermore, Fgf9 deficiency results in a proximal shift of the Shh expressing cell population and ectopic FGF9 protein suppresses Shh expression. These findings indicate that Shh as well as Fgf9 expression is maintained by Runx/Cbfb but that Fgf9 antagonizes Shh expression. The present results provide the first genetic evidence that Runx/Cbfb genes function in the maintenance of stem cells in developing incisors by activating Fgf signaling loops between the epithelium and mesenchyme. In addition, Runx genes also orchestrate continuous proliferation and differentiation by maintaining the expression of Fgf9 and Shh mRNA.     

FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis

Fibroblast growth factor (FGF) signaling is involved in skeletal development of the vertebrate. Gain-of-function mutations of FGF receptors (FGFR) cause craniosynostosis, premature fusion of the skull, and dwarfism syndromes. Disruption of Fgfr3 results in prolonged growth of long bones and vertebrae. However, the role that FGFs actually play in skeletal development in the embryo remains unclear. Here we show that Fgf18 is expressed in and required for osteogenesis and chondrogenesis in the mouse embryo. Fgf18 is expressed in both osteogenic mesenchymal cells and differentiating osteoblasts during calvarial bone development. In addition, Fgf18 is expressed in the perichondrium and joints of developing long bones. In calvarial bone development of Fgf18-deficient mice generated by gene targeting, the progress of suture closure is delayed. Furthermore, proliferation of calvarial osteogenic mesenchymal cells is decreased, and terminal differentiation to calvarial osteoblasts is specifically delayed. Delay of osteogenic differentiation is also observed in the developing long bones of this mutant. Conversely, chondrocyte proliferation and the number of differentiated chondrocytes are increased. Therefore, FGF18 appears to regulate cell proliferation and differentiation positively in osteogenesis and negatively in chondrogenesis.     

The perichondrium plays an important role in mediating the effects of TGF-beta1 on endochondral bone formation.

Endochondral bone formation is complex and requires the coordination of signals from several factors and multiple cell types. Thus, chondrocyte differentiation is regulated by factors synthesized by both chondrocytes and cells in the perichondrium. Previously, we showed that expression of a dominant-negative form of the transforming growth factor beta (TGF-beta) type II receptor in perichondrium/periosteum resulted in increased hypertrophic differentiation in growth plate chondrocytes, suggesting a role for TGF-beta signaling to the perichondrium in limiting terminal differentiation in vivo. Using an organ culture model, we later demonstrated that TGF-beta1 inhibits chondrocyte proliferation and hypertrophic differentiation by two separate mechanisms. Inhibition of hypertrophic differentiation was shown to be dependent on Parathyroid hormone-related peptide (PTHrP) and expression of PTHrP mRNA was stimulated in the perichondrium after treatment with TGF-beta1. In this report, the hypothesis that the perichondrium is required for the effects of TGF-beta1 on growth and/or hypertrophic differentiation in mouse metatarsal organ cultures is tested. Treatment with TGF-beta1 inhibited expression of type X collagen mRNA in metatarsal cultures with the perichondrium intact. In contrast, hypertrophic differentiation as measured by expression of Type X collagen was not inhibited by TGF-beta1 in perichondrium-free cultures. TGF-beta1 added to intact cultures inhibited BrdU incorporation in chondrocytes and increased incorporation in the perichondrium; however, TGF-beta1 treatment stimulated chondrocyte proliferation in metatarsals from which the perichondrium had been enzymatically removed. These results suggest that the TGF-beta1-mediated regulation of both chondrocyte proliferation and hypertrophic differentiation is dependent upon the perichondrium. Thus, one or several factors from the perichondrium might mediate the way chondrocytes respond to TGF-beta1.     

Expression of p53 and hsp70 in relation to apoptosis during Meckel’s cartilage development in the mouse

The process of Meckel’s cartilage development was examined with regard to expression of p53, a tumor suppressor gene product and hsp70, a stress protein (heat-shock protein), in association with the occurrence of programmed cell death (apoptosis). Balb C mice embryos from embryonic days E13, E14, E15, E16, E17, E18 and 1- and 3-day-old pups were used. P53-positive cells were detected first at E15, and were found in the perichondrium of the distal part of Meckel’s cartilage. During the degeneration process chondrocytes also became p53-positive. In contrast to p53, the expression of hsp70 was high and widespread in the early stages of development (E13–E15); however, it decreased with age, except for Meckel’s cartilage, where hsp70 was found in the cytoplasm or nuclei of the hypertrophic cells. Apoptosis was first detected at E14–E15 in the perichondrium of the distal parts of Meckel’s cartilage. The number of apoptotic bodies increased with age and the ongoing resorption of Meckel’s cartilage. From the present study it can be concluded that expression of p53 and hsp70 varied during the development of Meckel’s cartilage and that both proteins showed nuclear location in hypertrophic cells. No direct spatial or temporal correlation was observed between the expression of p53 and hsp70 and the occurrence of apoptotic bodies.     

Ontogeny of the tessellated skeleton: insight from the skeletal growth of the round stingray Urobatis halleri

The majority of the skeleton of elasmobranch fishes (sharks, rays and relatives) is tessellated: uncalcified cartilage is overlain by a superficial rind of abutting, mineralized, hexagonal blocks called tesserae. We employed a diversity of imaging techniques on an ontogenetic series of jaw samples to investigate the development of the tessellated skeleton in a stingray (Urobatis halleri). We compared these data with the cellular changes that characterize cartilage calcification in bony skeletons. Skeletal growth is characterized by the appearance of tesserae as well as changes in chondrocyte shape, arrangement and density. Yolk sac embryos (35–56 mm disc width, DW) have untessellated lower jaw tissue wrapped in perichondrium and densely packed with chondrocytes. Chondrocyte density decreases dramatically after yolk sac absorption (histotroph stage: 57–80 mm DW) until the formation of tesserae, which are first visible using our techniques as thin (~60 µm), sub‐perichondral plaques. During the histotroph stage, flattened chondrocytes align parallel to the perichondrium at the tissue periphery, where we believe they are incorporated into developing tesserae to form the cell‐rich laminae observed within tesserae; in older animals peripheral cells in the uncalcified phase are rounder and less uniformly oriented. By parturition (~75 mm DW), cell density and the number of adjoining chondrocyte pairs (an indicator of cell division) have dropped to less than a third of their initial values; these remain low and tesserae continue to grow in size. The tessellated skeleton is a simple solution to the conundrum of growth in an endoskeleton with external mineralization and no remodeling. Although we see parallels with endochondral ossification (e.g. chondrocytes decreasing in density with age), the lack of chondrocyte hypertrophy and the fact that mineralization is sub‐perichondral (not the case in mammalian cartilage) suggest that the similarities end there.