Supporting Information Figure S1 Transmission EM of growth plate from newborn Col27G1516C mouse. 2 mm thick epoxy-embedded sections were stained with toluidine blue and examined by light microscopy (LM) for orientation purposes. 70 nm sections were stained as described in methods and examined by transmission EM. (TIF) Figure S2 Isotopic in situ hybridisation of molecules involved in the major signalling pathways controlling growth plate differentiation. In the tibia of E18.5 day embryos homozygous for the Col27del87 allele, collagen XXVII (M, S), PPR (N, T), collagen X (O, U), FGFR3 (P, V), IHH (Q, W), PTCH1 (R, X) appear unaffected when compared to the wild type littermates (A, G; B, H; C, I; D, J; E, K; F, L; respectively). G- References 1. Boot-Handford RP, Tuckwell DS (2003) Fibrillar collagen: The key to vertebrate evolution? A tale of molecular incest. Bioessays 25: 142–151. 2. Wenstrup RJ, Florer JB, Brunskill EW, Bell SM, Chervoneva I, et al. (2004) Type V collagen controls the initiation of collagen fibril assembly. J Biol Chem 279: 53331–53337. 3. Kadler KE, Baldock C, Bella J, Boot-Handford RP (2007) Collagens at a glance. J Cell Sci 120: 1955–8. 4. Pace JM, Corrado M, Missero C, Byers PH (2003) Identification, characteriza- tion and expression analysis of a new fibrillar collagen gene, COL27A1. Matrix Biol 22: 3–14. 5. Boot-Handford RP, Tuckwell D, Plumb DA, Farrington Rock C, Poulsom R (2003) A novel and highly conserved collagen [proa1(XXVII)] with a unique expression pattern and unusual molecular characteristics establishes a new clade within the vertebrate fibrillar collagen family. J Biol Chem 278: 31067–77. 6. Koch M, Laub F, Zhou P, Hahn RA, Tanaka S, et al. (2003) Collagen XXIV, a vertebrate fibrillar collagen with structural features of invertebrate collagens: selective expression in developing cornea and bone. J Biol Chem 278: 43236–44. 7. Plumb DA, Dhir V, Mironov A, Ferrara L, Poulsom R, et al. (2007) Collagen XXVII is developmentally-regulated and forms thin fibrillar structures distinct from those of classical vertebrate fibrillar collagens. J Biol Chem 282: 12791–12795. 8. Hjorten R, Hansen U, Underwood RA, Telfer HE, Fernandes RJ, et al. (2007) Type XXVII collagen at the transition of cartilage to bone during skeletogenesis. Bone 41: 535–42. 9. Jenkins E, Moss JB, Pace JM, Bridgewater LC (2005) The new collagen gene COL27A1 contains SOX9-responsive enhancer elements. Matrix Biol 24: 177–184. 10. Mayo JL, Holden DN, Barrow JR, Bridgewater LC (2009) The transcription factor Lc-Maf participates in Col27a1 regulation during chondrocyte matura- tion. Exp Cell Res 315: 2293–2300. 11. Bateman JF, Boot-Handford RP, Lamande´ SR (2009) Genetic diseases of connective tissues: cellular and extracellular effects of ECM mutations. Nat Rev Genet 10: 173–183. 12. Marini JC, Forlino A, Cabral WA, Barnes AM, San Antonio JD, et al. (2007) Consortium for osteogenesis imperfecta mutations in the helical domain of type I Collagen XXVII Function in Growth Plate R, dark field; A-F:S-X, bright field. Bar = 100 mm. Wild type (WT/WT), Homozygous (M/M). (TIF) Figure S3 Tibial growth plates from perinatal MDskel / MDskel mice. H&E stained growth plates from phenotypically normal control (+ /MDskel) and MDskel /MDskel mice. Growth plates from 3 week old equivalents (included in Fig. 8) are shown for comparison. (TIF) Author Contributions Conceived and designed the experiments: RBH. Performed the experi- ments: DAP LF TT AM RBH. Analyzed the data: DAP LF TT MDB KEK RBH. Contributed reagents/materials/analysis tools: LK. Wrote the paper: RBH MDB. collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans. Hum Mutat 28: 209–221. 13. Dreier R, Opolka A, Grifka J, Bruckner P, Gra¨ssel S (2008) Collagen IX- deficiency seriously compromises growth cartilage development in mice. Matrix Biol 27: 319–329. 14. Aszodi A, Hunziker EB, Brakebusch C, Fa¨ssler R (2003) b1 integrins regulate chondrocyte rotation, G1 progression, and cytokinesis. Genes Dev 17: 2465–2479. 15. Christiansen HE, Lang MR, Pace JM, Parichy DM (2009) Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth. PLoS One 4: e8481. 16. Ishida Y, Yamamoto A, Kitamura A, Lamande´ SR, Yoshimori T, et al. (2009) Autophagic elimination of misfolded procollagen aggregates in the endoplasmic reticulum as a means of cell protection. Mol Biol Cell 20: 2744–2754. 17. Talts JF, Brakebusch C, Fa¨ssler R (1999) Integrin gene targeting. Methods Mol Biol 129: 153–187. 18. Leighton MP, Nundall S, Starborg T, Meadows RS, Suleman F, et al. (2007) Decreased chondrocyte proliferation and dysregulated apoptosis in the cartilage growth plate are key features of a murine model of epiphyseal dysplasia caused by a matn3 mutation. Hum Mol Genet 16: 1728–1741. 19. Pirog-Garcia K, Meadows R, Knowles L, Heinegard D, Thornton DJ, et al. (2007) Reduced cell proliferation and increased apoptosis are significant pathological mechanisms in a murine model of mild pseudoachondroplasia resulting from a mutation in the C-terminal domain of COMP. Hum Mol Genet 16: 2072–88. 20. Sakai K, Hiripi L, Glumoff V, Brandau O, Eerola R, et al. (2001) Stage- and tissue-specific expression of a Col2a1-Cre fusion gene in transgenic mice. Matrix Biol 19: 761–767. 21. Kimmel CA, Trammell C (1981) A rapid procedure for routine double staining of cartilage and bone in fetal and adult animals. Stain Technol 56: 271–2. 22. Rajpar MH, McDermott B, Kung L, Eardley R, Knowles L, et al. (2009) Targeted Induction of Endoplasmic Reticulum Stress Induces Cartilage Pathology. PLoS Genet 5(10): e1000691. 23. Gagnon ML, Angerer LM, Angerer RC (1992) Posttranscriptional regulation of ectoderm-specific gene expression in early sea urchin embryos. Development 114: 457–467. PLoS ONE | www.plosone.org 12 December 2011 | Volume 6 | Issue 12 | e29422