Localization of erβ₂ mRNA with whole mount in situ hybridization in 96 hpf larvae. (A–C) Antisense probe was detected mainly in the brain and in the neuromasts (see black arrowheads). (D–F) With the sense probe, the signal was not detected in the neuromasts, but weakly in the brain. (A, B, D, E) Magnification 10x, scale bar = 200 μm. (C, F) Magnification 60x, scale bar = 50 μm.

Concentration-dependent endpoints. The morpholino against ERβ₂ (ERβ₂ MO) was used at concentrations ranging from 15 µM to 100 µM, as plotted on the Y axes. The X axes represent the age of observed larvae in hours post fertilization (hpf). The black arrows point to the time where the specific phenotype was observed. Four biological endpoints were observed: a curved tail at 100 µM, a lower hatching rate at 100 and 50 µM, a disturbed swimming behavior at all concentrations and a disruption in the neuromast development at all concentrations.

Neuromast detection with live staining (FM1-43) in 72 hpf larvae. (A-C) Larvae injected with coMO at 15 μM. (D-F) Larvae injected with ERβ₂ MO at 15 μM. (G-I) Larvae injected with ERβ₂ MO at 15 μM and cRNA at 300 ng. Arrowheads point to neuromasts with functional hair cells. Scale bar = 200 μM.

Number of neuromasts assessed using live staining (FM1-43) in wild-type (A-C) at 72 hpf and with GFP visualisation in transgenic line (ET4) (D). The boxes symbolize the interquartile space of each distribution; the median is indicated with a horizontal line within the box, and dots indicate extreme values (5th/95th percentile). The number of individuals recorded for elaboration of the box plots is indicated with N = number of individuals. Numbers of neuromasts are shown in (A) ERβ₂ MO- or coMO-injected wild-type larvae (both MOs at 15 µM); (B) ERβ₂ MO-injected wild-type larvae (15 µM) co-injected with cRNA (300 ng); (C) ERβ₂ MO-injected wild-type larvae co-injected with p53MO (both at 15 µM). (D) ET4 larvae injected with ERβ₂ MO or coMO (both at 15 µM). One-way ANOVA following the Tukey test were performed to test the differences between blank (uninjected) and injected embryos and significances were indicated with * when p ≤ 0.05.

Percentage of larvae that were swimming in circles after injection with the ERβ₂ MO or with a coMO at different concentrations (15, 25, 50, 100 µM) or uninjected (blank) at 72 hpf and 96 hpf. Values are reported as arithmetic means ± standard deviation of biological triplicates.

Detection of sustentacular cells and mantle cells with whole mount in situ hybridization against keratin15 (k15) (A, D) and claudinb (cldb) (B, C, E, F) in 72 hpf larvae injected with 15 μM ERβ₂ MO (A–C), and with 15 μM coMO (D–F). (A, B, D, E) Magnification 10x, scale bar = 500 μM, Arrowheads point to neuromasts. (C, F) Magnification 100x, scale bar = 10 μM. Arrowheads point to visible hair cells.

Localization of ngn1 mRNA with whole mount in situ hybridization in 72 hpf larvae. (A) In 50 μM ERβ₂ MO-injected embryos; (B, C) in 15 μM ERβ₂ MO injected embryos; (D) in 50 μM coMO injected embryos; (E, F) in 15 μM coMO injected embryos. (A, B, D, E) Magnification 10x, scale bar = 500 μm. (C) Magnification 60x, scale bar = 20 μm. (F) Magnification 100x. Scale bar = 20 μm. Arrowheads point to ngn1 expression in the tail.

Localization of notch3 (A–D) and notch1a (E, F) mRNA with whole mount in situ hybridization in 72 hpf larvae. (A, B) Notch3 expression in 15 μM ERβ₂ MO injected embryos. (C, D) Notch3 expression in 15 μM coMO injected embryos. (E) Notch1a expression in 15 μM ERβ₂ MO injected embryos. (F) Notch1a expression in 15 μM coMO injected embryos. (A, C) Scale bar = 200 μm. (B, D) Scale bar = 10 μm. (E, F) Scale bar = 20 μm. Arrowheads point to notch3 expression in neuromasts.