Abstract

Fibronectin (FN) is a ubiquitous matrix glycoprotein essential for the physiological development of various tissues. Mutations in FN cause a form of skeletal dysplasia, emphasizing the importance of FN in cartilage and bone development. However, the relevance and functional role of FN during skeletal development remains elusive. We employed conditional knockout mouse models for the cellular FN isoform in cartilage (cFNKO), the plasma FN isoform in hepatocytes (pFNKO), and a double knockout (FNdKO) to determine the relevance of these two principal FN isoforms in postnatal skeletal development spanning from P1 to 2 months of age. We identified a unique topological FN deposition pattern in the mouse limb with prominent levels at the resting and hypertrophic chondrocyte zones and in the trabecular bone. Circulating pFN did not enter the growth plate and was limited to the primary ossification center, whereas cartilage-specific cFN was detected as the major isoform in epiphyseal cartilage. Deletion of either one of the isoforms in single knockouts (cFNKO or pFNKO) only led to subtle changes in some of the analyzed parameters. Complete ablation of both cFN in the growth plate and circulating pFN in plasma resulted in significantly reduced postnatal body weight, body length, and bone length in the FNdKO mice. Assessment of the FNdKO adult bone microarchitecture using micro-CT revealed significantly reduced trabecular bone volume, trabecular network, bone mineral density, and increased bone marrow adiposity. Analysis of chondrogenesis in FNdKO mice showed changes in the proliferating and hypertrophic growth plate zones, consistent alterations in chondrogenic markers such as collagen type II and type X, reduced apoptosis of hypertrophic chondrocytes, and downregulation of bone formation markers. FNdKO mice also displayed decreased levels of transforming growth factor-{beta}1 (TGF{beta}1) and downstream phospho-AKT levels, which are critical regulators of chondrogenesis and bone formation. In conclusion, the data demonstrate that FN is essential for proper chondrogenesis and postnatal bone development. Simultaneous deletion of both FN isoforms in the developing cartilage leads to critical TGF{beta}-mediated alterations in chondrogenic differentiation, resulting in bone and skeletal defects. Significance/HighlightsO_LIFN is highly expressed during mouse limb development with increased deposition in resting and hypertrophic chondrocyte zones and the primary ossification center. C_LIO_LICartilage-specific cFN and circulating pFN are distinctly distributed during embryonic and postnatal bone development, with chondrocyte-specific cFN present in the growth plate and pFN limited to trabecular bone and the bone marrow. C_LIO_LIDeletion of both cFN and pFN leads to reduced bone growth during early postnatal development. C_LIO_LIDeletion of cFN and pFN leads to reduced trabecular bone formation, bone mineralization, and increased bone marrow adiposity in 2-month adult mice. C_LIO_LIAbsence of both FN isoforms in the FNdKO mouse model leads to altered chondrogenesis and reduced bone formation. C_LIO_LIFN regulates chondrogenesis via TGF{beta}-mediated phospho-AKT signaling. C_LI