Fig. S7

Treatment of wild-type embryos with cAMP agonists results in semicircular canal projection defects. (A-L) Live DIC images of 5dpf wild-type embryos incubated with DMSO, 25μM forskolin or 100μM IBMX between 60 and 90hpf show a gradation in phenotype. A low dose (25μM) of forskolin has little effect on the ear (B,E,H,K). However, treatment with 100μM IBMX can give rise to a severe phenotype, with a swollen ear and unfused projections (C,F,I,L). This is similar to the ‘severe’ category for the tb233c allele, but the swelling is not as extreme as in tk256a or fr24 mutants. The dorsolateral septum (black arrowheads, J-L) and the ventral pillar (asterisks, D-F) have failed to form correctly in the presence of 100mM IBMX. (M-O) In situ hybridisation shows an upregulation of the ECM marker vcana in unfused canal projections (white arrows, O). Lateral views. Scale bars: A-C, G-I 200μm; D-F, J-L, M-O 50μm. (P) Graphical representation of the dataset. Ears swell as a graded response to both forskolin and IBMX concentration. Data were analysed with a 333 chi-square contingency table for each drug; P<0.001 for both drugs. N numbers are shown in parentheses.