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	Figure 1. BPA treatment induces otolith abnormalities. A: BPA induced otolith malformation is dose dependent. Embryos were treated with different concentrations of BPA from 5 hpf onwards. Black bars represent embryos showing no otolith abnormalities, red bars represent treated embryos showing otolith abnormalities. (*: P < 0.01, Fisher test after Bonferroni correction). B: Anterior otolith (ao, upper left) and posterior otolith (po, upper middle) of control embryo and ao (lower left) and po (lower middle) of embryos treated with 70 μM BPA from 5 to 50 hpf showing otolith aggregation. In some rare cases a single otolith (upper right) or extra otolith (eo lower right) are observed. C: Upper panel: control embryo at 50 hpf (Upper right: close up of the otic vesicle). Lower panel: overall shape of BPA treated embryos form 6-50 hpf resembles control embryos, but display otolith aggregates (arrow). Lower right: close up of the otic vesicle in a BPA treated embryo form 24-50 hpf showing normal otoliths. D: After 75 hours of exposure, the concentration of BPA measured in embryos is 0.7 μg equivalent per mg fresh weight and 0.22 μg of BPF equivalent per mg fresh weight; see also Figure 2 C-E. E: BPA induces malformation of the otic vesicle in Xenopus embryos. Xenopus embryos were exposed to BPA (5 or 10 μM) from stage NF 18 to stage NF 40 (48 h) and examined at stage NF 45. The upper panel shows the morphology of the otoliths (or otoconia [73]) in control and BPA treated embryos. Note that 5 and 10 μM of BPA induce a progressive reduction in the size of the otoconia. The lower panel shows the semi-circular canals with a reduction in the distance between the two semi circular canals (arrowheads) induced by BPA. Similarly, the morphology of the developing semi circular canals is flattened under BPA treatment.
	Figure 2. Bisphenol effects are time and compound specific. A: Live pictures of the developing otic vesicle from 18 to 50 hpf in zebrafish embryo. B: BPA affects otolith in a restricted time window. Diagram showing the effect of 70 μM BPA treatments with different starting points or length of exposure. Upper panel: BPA pulse treatments (red bars) were performed from 10 hpf. At different stage of development (18, 24, 30 and 38 hpf) embryos were washed and developed in BPA free medium (gray bars). Otic vesicles were scored at 50 hpf. Lower panel: BPA acts prior 22 hpf to induce otolith defects. Treatments started prior or at 18 hpf lead to 100% of otolith defects. Treatment started from 20 hpf onwards lead to 85% of embryos with otolith defect. Treatment started at 22 hpf lead to only 2% of affected embryos. Treatments started later did not lead to any otolith defect. Red bars represent time when embryos were exposed to BPA. Gray bars represent time when embryos were not exposed to this compound. C: Various bisphenols affect otolith development and/or pigmentation. Embryos were treated from 5 to 72 hpf and malformed otolith or pigmentation defects scored (+++ indicates maximum defect, i.e. malformed otolith or total lack of pigment). Treatment of embryos with either BPA (70 μM) or BPE (70 μM) resulted in malformed otoliths. BPE also affected pigmentation, as did BPF (50 μM). BPC (70 μM) was without clear effects in these assays. D: Pictures of the effect of BPC, BPE and BPF on otolith development. All embryos were exposed to 70 μM of bisphenol. Note that only BPE gives an otolith phenotype similar to what in observed in BPA treated embryos with otolith aggregates marked by a black arrow. E: Chemical structures of BPA, BPC, BPE and BPF.
	Figure 3. Expression of markers of inner ear development in zebrafish embryos are affected by BPA treatment. (A,B)oc90 a gene require for otolith formation in zebrafish is up-regulated under BPA treatment at 24 hpf (n=30). In situ for control and BPA treated embryos were performed in the same tube with the tip of the tail removed for the control embryos. (C,D)aldh1a3 is detected in the anterior cristae (ac), the cranial epithelial projection (cp), the endolymphatic duct (ed), the posterior cristae (pc) and the anterior macula (am) in wild type zebrafish embryo at 50 hpf (M), its expression is reduced in ac, cp and am and is absent in ed and pc in BPA treated embryos from 6 hpf onward (D). (E,F) Expression of ugdh at 50 hpf in control otic vesicle (E) is detected in the ac, cp, ed and pc. In BPA treated embryos from 6 hpf onwards (D), ugdh expression is severely reduced and remains solely detected in the ac and the cp. (G,H) Expression of bmp4 in control embryos at at 50 hpf (G) in the anterior (ac) lateral (lc) in the posterior cristae (pc) and the endolymphatic duct (ed) remains unaffected after BPA treatment (H). (I,J) Confocal microscopy of acetulated tubulin antibody staining showing the presence of the ciliated macula (white arrow) in control embryos (I) and in BPA treated embryos (white arrow in J).
	Figure 4. BPA effect on otolith development is estrogen receptor-independent. Morphology of the inner ear of zebrafish embryos at 48 hpf following exposure to BPA with or without ER antagonists (ICI 182 780) or agonists (17-β estradiol, E2). Treatment with either 1 μM ICI (A) or 1 μM 17-β estradiol (B) from 5 to 48 hpf does not induce any malformation of the developing semi-circular canals nor otoliths. Moreover, the BPA-induced otolith phenotype is not rescued when embryos are co-treated with BPA 5 μM +ICI 1 μM (A) or BPA 5 μM +βE2 1 μM (B). Interestingly, co-treatment with BPA 5 μM +ICI 1 μM lead to in increased ratio of affected otolith than a treatment with BPA 5 μM alone (A). On the other hand co-treatment with BPA 5 μM +βE2 1 μM gives a similar ratio than a treatment with BPA 5 μM alone (B).
	Figure 5. BPA effect on otolith development is thyroid hormones independent. (A) Control otic vesicle at 50 hpf with two otoliths. (B) Treatment with 1 μM of T3, from 5 hpf onwards lead to a normal otolith development. (C) 70 μM BPA treated embryo from 5 hpf onwards lead to otolith aggregates. (D) Half dose of BPA, 35 μM, does not affect otolith development. (E) Co-treatment of half dose of BPA, 35 μM, supplemented with 1 μM of T3 does not affect otolith development. (F) Co-treatment of full dose of BPA, 70 μM supplemented with 1 μM of T3 lead to a similar otolith phenotype than BPA 70 μM alone (compare C with F).
	Figure 6. Chemical inhibition of Na/K ATPase rescues the BPA induced phenotype. (A) 50 hpf control embryo with two normal otolith. (B) Treatment with 1.8 mM ouabain from 6 hpf onwards leads to one misformed otolith. (C) Embryo treated with 70 μM BPA from 5 hpf onwards leads to otolith aggregates. (D) Co treatment with 70 μM BPA and 1.8 mM oubain from 6 hpf onwards leads to normal otolith. (E) Co treatment with 70 μM BPA and 0.9 mM oubain from 6 hpf onwards leads to a mild BPA like phenotype. In some cases one almost normal otolith is found (arrow). (F) Co treatment with 50 μM BPA and 1.8 mM oubain from 6 hpf onwards leads to two normal otoliths. (G)α1a1 morphants display a complete absence of otolith. (H) 70 μM BPA of α1a1 morphants did not rescue otolith formation and result in a complete absence of otolith. (I)α1a1 is expressed in the lower membrane of the otic vesicle at 24 hpf. (J) A similar expression of α1a1 is detected in BPA treated embryos.

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