Collagen XXVII Function in Growth Plate Figure 4. Gross phenotypic analyses of the Col27del87 mouse. A. Mice heterozygous for the Col27del87 allele were mated and offspring genotyped at 3 weeks of age revealing that litters contained wild type (WT/WT) and heterozygous (WT/MD87) mice but none homozygous (MD87/MD87) for the Col27del87 allele. However, at E18.5, M D87/MD87 embryos were present in the normal Mendelian range. B. E18.5 MD87/MD87 embryos were smaller, had a domed head and shortened snout and developed a hernia at the umbilicus as a result of breaking the cord. WT/WT and WT/MD87 littermates were of normal size and appearance. Scale bar = 3 mm. C. H+E stained sections of the lungs from E18.5 embryos carrying the Col27del87 allele revealed that M D87/MD87 mice have hypercellular lungs when compared to WT/WT and WT/M D87 littermates. Scale bar = 100 mm. D. Gross analysis of the skeleton from Col27del87 mice reveals that M D87/M D87 mice were smaller than their WT/WT and WT/M D87 littermates. Scale bar = 3 mm. E. M D87/M D87 mice had either a fused 4th and 5th sternebrae or a cleft through the xiphoid complex up to the 4th sternebrae. doi:10.1371/journal.pone.0029422.g004 secreted into the extracellular compartment [11,12]. However, collagen XXVII seems to be able to function relatively normally with this missense mutation in that whether heterozygous or homozygous for the mutation, animals grew normally with little disruption of growth plate architecture or collagen XXVII secretion. The only obvious skeletal abnormality in the MG/C/ MG/C animals was the fusion of two sternebrae, a relatively common type of defect that was also apparent in the MD87/MD87 mice (see below). The relatively milder than anticipated effects of the Gly to Cys mutation in collagen XXVII are likely due to two factors. Firstly, collagen XXVII has two conserved interruptions in the triple helical domain which indicates that the function of this fibrillar collagen is not dependent upon a perfect Gly –X –Y repeat in the collagenous domain [4,5]. Hence collagen XXVII may have some capacity to accommodate small interruptions without significant effects upon folding and secretion. Secondly, collagen XXVII appears to assemble into very thin fibrils in the ECM that are independent of the thicker fibrils formed by the classical fibrillar collagens [7,8]. Again these thin fibrils may be better able to accommodate mutant collagen trimers. In contrast, the phenotypic effects of an 87 amino acid deletion within the C-terminal portion of the type XXVII collagen triple helical domain were severe in mice homozygous for the mutation (MD87/MD87 ) which died at birth due to a lung defect accompanied by chondrodysplasia and midline defects including a similar sterna defect to that described above in the MG/C/MG/C mice. Collagen XXVII clearly plays a subtle role in the development and fusion events forming the sternum and involving the midline but extensive histological examination failed to reveal any overt reason for the abdominal weakness and the diaphragm was intact in the MD87/MD87 embryos. The growth plate proliferative zone defect caused by the 87 amino acid deletion in the collagenous domain of collagen XXVII was apparent from E16.5 in the MD87/MD87 embryos and was characterized postnatally in the MDskel/MDskel mouse. The first apparent changes in the E16.5 day proliferative zone involved a reduced tendency of these chondrocytes to flatten and organise into vertical columns accompanied by a disruption to the pericellular matrix (Fig. 5) that was also apparent by EM (Fig. 6). Postnatally, the characteristic features of proliferative zone chondrocytes (column formation and cell flattening) became more apparent in the MDskel/MDskel mice but accompanied by obvious signs of a proliferative zone defect including misaligned cells and a disruption of the pericellular matrix. Furthermore, the diffusion of collagen X into the lower portion of the proliferative zone of the mutant growth plate again suggests a disruption of the ECM in the proliferative zone (Fig. 8). It is of note that the expression of the del PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e29422