Distinct functions of BMP4 and GDF5 in the regulation of chondrogenesis

Bone morphogenetic protein 4 (BMP4) and growth/differentiation factor 5 (GDF5) are closely related protein family members and regulate early cartilage patterning and differentiation. In this study, we compared the functional outcome of their actions systematically at various stages of chondrogenesis in mouse embryonic limb bud mesenchyme grown in micromass cultures. Overall, both growth factors enhanced cartilage growth and differentiation in these cultures. Uniquely, BMP4 not only accelerated the formation and maturation of cartilaginous nodules, but also induced internodular mesenchymal cells to express cartilage differentiation markers. On the other hand, GDF5 increased the number of prechondrogenic mesenchymal cell condensation and cartilaginous nodules, without altering the overall pattern of differentiation. In addition, GDF5 caused a more sustained elevated expression level of Sox9 relative to that associated with BMP4. BMP4 accelerated chondrocyte maturation throughout the cultures and sustained an elevated level of Col10 expression, whereas GDF5 caused a transient increase in Col10 expression. Taken together, we conclude that BMP4 is instructive to chondrogenesis and induces mesenchymal cells toward the chondrogenic lineage. Furthermore, BMP4 accelerates the progression of cartilage differentiation to maturation. GDF5 enhances cartilage formation by promoting chondroprogenitor cell aggregation, and amplifying the responses of cartilage differentiation markers. These differences may serve to fine-tune the normal cartilage differentiation program, and can be exploited for the molecular manipulation in biomimetics.     

BMPs are required at two steps of limb chondrogenesis: formation of prechondrogenic condensations and their differentiation into chondrocytes

Formation of the long bones requires a cartilage template. Cartilage formation (chondrogenesis) proceeds through determination of cells and their aggregation into prechondrogenic condensations, differentiation into chondrocytes, and later maturation. Several studies indicate that members of the bone morphogenetic protein (BMP) family promote cartilage formation, but the exact step(s) in which BMPs are involved during this process remains undefined. To resolve this issue, we have used a retroviral vector to misexpress the BMP antagonist Noggin in the embryonic chick limb. Unlike previous reports, we have characterized the resulting phenotype in depth, analyzing histological and early chondrogenic markers, as well as the patterns of cell death and proliferation. Misexpression of Noggin prior to the onset of chondrogenesis leads to the total absence of skeletal elements, as previously reported (J. Capdevila and R. L. Johnson, 1998, Dev. Biol. 197, 205-217). Noggin inhibits cartilage formation at two distinct steps. First, we demonstrate that mesenchymal cells do not aggregate into prechondrogenic condensations, and additional results suggest that these cells persist in an undifferentiated state. Second, we show that differentiation of chondroprogenitors into chondrocytes can also be blocked, concurrent with expanded expression of a presumptive joint region marker. In addition, we observed alterations in muscle and tendon morphogenesis, and the potential role of BMPs in these processes will be discussed. Our studies therefore provide in vivo evidence that BMPs are necessary for different steps of chondrogenesis: chondroprogenitor determination and/or condensation and subsequent differentiation into chondrocytes.     

L-Sox5 and Sox6 proteins enhance chondrogenic miR-140 microRNA expression by strengthening dimeric Sox9 activity

Sox9 plays a critical role in early chondrocyte initiation and promotion as well as repression of later maturation. Fellow Sox family members L-Sox5 and Sox6 also function as regulators of cartilage development by boosting Sox9 activation of chondrocyte-specific genes such as Col2a1 and Agc1; however, the regulatory mechanism and other target genes are largely unknown. MicroRNAs are a class of short, non-coding RNAs that act as negative regulators of gene expression by promoting target mRNA degradation and/or repressing translation. Analysis of genetically modified mice identified miR-140 as a cartilage-specific microRNA that could be a critical regulator of cartilage development and homeostasis. Recent findings suggest Sox9 promotes miR-140 expression, although the detailed mechanisms are not fully understood. In this study we demonstrate that the proximal upstream region of pri-miR-140 has chondrogenic promoter activity in vivo. We found an L-Sox5/Sox6/Sox9 (Sox trio) response element and detailed binding site in the promoter region. Furthermore, detailed analysis suggests the DNA binding and/or transactivation ability of Sox9 as a homodimer is boosted by L-Sox5 and Sox6. These findings provide new insight into cartilage-specific gene regulation by the Sox trio.     

Sox9 neural crest determinant gene controls patterning and closure of the posterior frontal cranial suture

Cranial suture development involves a complex interaction of genes and tissues derived from neural crest cells (NCC) and paraxial mesoderm. In mice, the posterior frontal (PF) suture closes during the first month of life while other sutures remain patent throughout the life of the animal. Given the unique NCC origin of PF suture complex (analogous to metopic suture in humans), we performed quantitative real-time PCR and immunohistochemistry to study the expression pattern of the NCC determinant gene Sox9 and select markers of extracellular matrix. Our results indicated a unique up-regulated expression of Sox9, a regulator of chondrogenesis, during initiation of PF suture closure, along with the expression of specific cartilage markers (Type II Collagen and Type X Collagen), as well as cartilage tissue formation in the PF suture. This process was followed by expression of bone markers (Type I Collagen and Osteocalcin), suggesting endochondral ossification. Moreover, we studied the effect of haploinsufficiency of the NCC determinant gene Sox9 in the NCC derived PF suture complex. A decrease in dosage of Sox9 by haploinsufficiency in NCC-derived tissues resulted in delayed PF suture closure. These results demonstrate a unique development of the PF suture complex and the role of Sox9 as an important contributor to timely and proper closure of the PF suture through endochondral ossification.