Bone development is impaired in the ext2^-/-fish. A, Alizarin red stain for ossification in the craniofacial skeleton at 6dpf. Dermal bones: dentary (d), maxilla (mx), parasphenoid (p), endopterygoid (e), branchiostegal rays (br), opercle (op), cleithrum (cl), cartilage bones: hyomandibula (hm), ceratohyal (ch). ceratobranchial 5/pharyngeal arch (pa); and notochord (n), scale = 0.1 mm; B, The relative change in the expression of bone markers at 5dpf was evaluated by real time PCR and analysed by delta-delta-Ct in the homozygote ext2 mutants vs. wild type. The results represent an average from minimum four single embryos. Expression was normalized against slc25a5. Error bars indicate means with SEM. Expression of the ext2 was given as an example of a gene that was approximately 2-folds down-regulated and this under-expression was of biological relevance.

Homozygous ext2 mutant displays musco-skeletal phenotype. Whole mount immunolocalisation at 4dpf using MF-20 antibody for muscles (green) and collagen II for cartilage (red) shows thicker and shorter muscles fitting the malformed cartilaginous skeleton in the ext2^-/- fish. Muscles: intermandibularis anterior (ima), intermandibularis posterior (imp), adductor mandibulae (am), interhyoideus (ih), hyohyoideus (hh), levator arcus palatine (lap), adductor hyoideus (ah), dilator operculi (do), sternohyoideus (sh) and adductor operculi (ao). Note missing hh muscle and, marked with a star, increased musculature around heart (H) in ext2-/-. Scale = 0.1 mm.

Increased lipid levels in the ext2^-/-fish coincide with decreased bone formation. A, Oil red O stain (ORO) in fish at 6dpf; B, the levels of lipids represented as absorbance of bounded ORO in 6 days old proteoglycan mutants: ext2, slc35b2, gpc4 and uxs1 (for more description see Additional file 2); C, the level of transcripts in 5-days old fish determined by quantitative PCR and normalised to slc25a5 level. Primer sequences are given in Table 1; D, whole mount fabp11a mRNA in situ hybridisation showing prematurely developing adipocytes in the belly of the ext2^-/- fish. Error bars represent means with SEM.

Bone and lipid phenotypes in the ext2^-/-fish are partially rescued by inhibition of PPAR or activation of hedgehog signalling. A, Oil red O (ORO) stain for lipids in fish at 6dpf; B, Alizarin red stain for bones in fish at 6dpf. GW9662 (GW) and purmorphamine (PM) were added into fish water by 60hpf and fish were raised to 6dpf. Minimum 10 fish were scored from each group and experiment was repeated with similar results at least three times. Error bars represent means with SEM.

Muscle phenotype in the ext2^-/-fish. Muscles were detected with MF-20 antibody. Scale = 0.1 mm.