Collagen XXVII Function in Growth Plate Figure 6. Ultrastructural analyses of the growth plate from the tibia of E18.5 day Col27del87 mouse. Transmission electron micrograph of proliferative zone chondrocytes from wild type (WT/WT) embryos and embryos homozygous for the mutation (M D87/M D87). The black granular material particularly evident in the cytoplasm of the MD87/MD87 sample is glycogen. Boxed areas are expanded below. Single-headed arrows indicate pericellular areas with relatively sparse staining present in the WT/WT but not mutant. Double headed arrows indicate the apparent alignment planes for the microfibrillar material deposited between the proliferative chondrocytes. Scale bar = 5.0 mm. doi:10.1371/journal.pone.0029422.g006 87 form of collagen XXVII did not alter the expression of key differentiation markers within the disorganized growth plate (Fig. S2) and proliferation rates were unaffected. The collagen XXVII mutation therefore appears to impact directly upon the organisation of the ECM of the proliferative zone rather than by affecting the signaling events controlling the differentiation process. The decrease in bone growth rate in the MDskel/MDskel mouse is therefore most likely a direct consequence of pericellular matrix disruption leading to disordered proliferation and failure to form columns efficiently. Similar changes in proliferative chondrocytes including a disruption in the structure of the pericellular matrix, occasional mis-oriented flattened chondrocytes between the columns of proliferative cells in older animals (Fig. 8B) and diffusion of collagen X into the lower part of the proliferative zone have been noted in the collagen IX knockout mouse [13 and Attila Aszodi personal communication]. These pathological features have been attributed to altered interactions between the proliferative chondrocyte and the pericellular matrix that are required for proliferative chondrocyte flattening and correct alignment of these cells into columns [14]. The collagen XXVII sequence does not contain proven integrin binding sites but does contain a GLOGEO and a GLOGDA sequence (where O is predicted to be hydroxyproline) which represent potential binding motifs based on homologies to known sites in the major fibrillar collagens (R. Farndale, personal communication). It is conceivable that the 87 amino acid deletion, which is C-terminal to the putative integrin-binging motifs and the resulting disruption in the triple helical structure of collagen XXVII may interfere with the chondrocyte’s ability to bind collagen XXVII. Alternatively, the disorder in the proliferative zone ECM may be caused by altered interactions of the mutant collagen XXVII with other matrix components. The knockdown of collagen XXVII isomers in zebrafish embryos produced the formation of notochord curves at the distal end of the tail and misshapen vertebrae [15] demonstrating a clear role for this collagen in skeletogenesis. The mutant mice expressing the del87 allele generated in this study did not show obviously misshapen vertebrae but exhibited a severe chondrodysplasia including a mild kyphosis, and a clear growth plate defect. These overlapping but distinct effects on skeletogenesis may reflect species differences or alternatively originate in the different strategies utilized (knockdown in the fish versus the expression of mutant forms of the protein in the current study). PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e29422