FGF signalling is required for ossification and osteoblast maturation.

(A-D) Continuous inhibition of FGF signalling from 48hpf to 120hpf in hs:dnfgfr1 larvae results in loss of ossification (B) but cartilage formation is relatively unaffected (D). (E-H) Expression of mature osteoblast markers col1a2 and col10a1 is severely reduced in hs:dnfgfr1 larvae (F,H). High magnification images of the opercle are shown to the right of panels E-H. Abbreviations: bs = branchiostegal ray, cl = cleithrum, op = opercle. Scale bar = 200μM.

FGF signalling regulates expression of osx.

(A-H) Larvae were treated at 48hpf, fixed at 51hpf and expression of several bone markers was analysed by in situ hybridisation. Arrowheads point to the opercle in the high magnification images to the right of each panel. Expression of runx2a (A-D) and runx2b (E-H) is unchanged after inhibition of FGF signalling using hs:dnfgfr1 larvae as well as over activation in hs:fgf3 transgenic fish. Expression of runx2b is slightly reduced after SU5402 treatment (F). (I-L) Expression of osx is decreased after inhibition of FGF signalling and increased after over activation of FGF signalling as shown by in situ hybridization. Abbreviations: cl = cleithrum, op = opercle. Scale bar = 200μM. (M) Quantification for presence or absence of osx staining in the opercle from I-L. Expression was normalised to the control. (N) qPCR performed in parallel to I-L confirm effects of FGF signalling on osx expression. Treatments were normalised to gapdh. Treatment with SU5402 showed a significant reduction (p<0.05 Student's t-test).

Wnt/β-Catenin signalling regulates ossification and development of mature osteoblasts.

(A-F) Continuous inhibition (hs:dkk1) or over activation (hs:wnt8) of Wnt/β-Catenin signalling from 72-120hpf. Inhibition of Wnt/β-Catenin signalling results in reduced ossification as shown by Alizarin red staining (B) whereas cartilage formation is largely unaffected (E). Overactivation of Wnt/β-Catenin signalling results in increased ossification and precocious ossification of the hyomandibula (C). (G-L) Analysis of osteoblast markers after treatment from 72-108hpf. Expression of mature osteoblast markers col1a2 and col10a1 is slightly reduced when Wnt/β-Catenin signalling is inhibited (H,K) and enhanced by increased Wnt/β-Catenin signalling (I,L). Abbreviations: hm = hyomandibula, op = opercle, te = teeth. Scale bar = 50μM.

Wnt/β-Catenin signalling regulates osx expression.

(A-I) Larvae were treated at 48hpf and fixed 12hours later. Expression of runx2a (A-C) and runx2b (D-F) is unchanged after inhibition (hs:dkk1) or over activation (hs:wnt8) of Wnt/β-Catenin signalling. (G-L) Expression of osx is reduced in hs:dkk1 and increased in hs:wnt8 larvae 12 hours after the treatment. Arrowheads point to the opercle in the high magnification images to the right of each panel. Abbreviations: cl = cleithrum. Scale bar = 50μM. (J) Quantification for presence or absence of osx staining in the opercle from G and H. Expression was normalised to the control. (K) qPCR performed in parallel on g and h confirms the effects of Wnt/β-Catenin signalling on osx expression (p<0.01 using Student's t-test). Treatments were normalised to gapdh.

FGF and Wnt/β-Catenin signalling interact to regulate osteoblast differentiation.

(A-D) Larvae were heat shocked at 48hpf, treated with SU5402 at 50hpf and fixed at 52hpf. Expression of osx is reduced after SU5402 (C) treatment and increased in hs:wnt8 larvae (B). Combined SU5402 and hs:wnt8 treatment still results in reduced expression of osx (D). (E-H) Larvae were heat shocked at 49hpf and fixed at 52hpf. Expression of osx is reduced in hs:dkk1 larvae (F) and increased in hs:fgf3 larvae (G). Expression is reduced in the combined treatment (H). (I-T) fgf3, sprouty1, sprouty4 and sef expression is inhibited by dkk1 over expression (K, N, Q, T) and activated by over expression of wnt8 (J, M, P, S). Arrowheads point to the opercle in the high magnification images to the right of each panel. Scale bars = 200μM.

Inhibition and over activation of FGF signalling have optimal effects 3 hours after the treatment.

(A-H) Inhibition of FGF signalling, using SU5402 treatment or hs:dnfgfr1 larvae resulted in strong down regulation of pea3 and erm expression 3 hours after the treatment (B, C, F, G). Over activation of FGF signalling in hs:fgf3 larvae resulted in strong upregulation of pea3 and erm after 3 hours (D, H). (I-P) 6 hours after the treatment, inhibition of FGF signalling still resulted in slight down regulation of pea3 and erm expression (J, K, N, O) whereas expression was unchanged in hs:fgf3 larvae (L, P). Scale bar = 200μM.

Neither FGF nor Wnt signalling affect col1a2 expression.

(A-C) Larvae were treated at 60hpf and fixed 3 hours later. Expression of col1a2 is unaffected 3 hours after inhibition (SU5402) or over activation (hs:fgf3) of FGF signalling. (D-F) Larvae were treated at 60hpf and fixed 12 hours later. Inhibition (hs:dkk1) or over activation (hs:wnt8) of Wnt/β-Catenin signalling also does not affect expression of col1a2. Arrowheads point to the opercle in the high magnification images to the right of each panel. Abbreviations: cl = cleithrum. Scale bar in F = 200μM.

Wnt/β-Catenin signalling regulates wif1 and axin2 expression.

(A-P) Larvae were treated from 48hpf to 56hpf. Expression of the known Wnt/β-Catenin targets wif1 and axin2 is strongly reduced 8 hours after inhibition (hs:dkk1; D, H, L, P) or increased after over activation (hs:wnt8; B, F, J, N) of Wnt/β-Catenin signalling.

Expression of the Ets factors elk1, ets1a and ets2.

(A-C) All three factors are expressed in regions around the developing bone at 54hpf, with ets1a and ets2 showing the highest expression.

Expression of Wnt/β-Catenin target genes is unaffected by FGF signalling.

(A-P) Larvae were heat shocked or treated with SU5402 at 48hpf and fixed at 51hpf. Expression of wif, axin2, lef1 and tcf7 is unaffected by inhibition (hs:dnfgfr1) or over activation (hs:fgf3) of FGF signalling. Scale bar = 200μM.