Expression ofsox3in the otic vesicle overlaps sensory and neurogenic domains. (A) Schematic depiction of the floor of the otic vesicle (anterior up, lateral to the left) showing expression domains of atoh1a in the utricular (ut) and saccular (sac) maculae, neurog1 in the neurogenic domain, and the medial marker pax2a. Dashed lines indicate the section planes shown in (B–D) and (E–G). (B–D) Expression of sox3 (B), pax2a (C) and atoh1a (D) in cross sections passing through the utricular macula at 24 hpf. (E–G) Expression of sox3 (E), pax2a (F) and neurog1 (G) in cross sections passing through the widest part of the neurogenic domain at 24 hpf. Otic vesicle borders are outlined in B–G.

Distinct roles forsox2andsox3in sensory and neural development. (A, B) Box-and-whisker plots of the total number of hair cells at 38 hpf (A) and mature SAG neurons at 36 hpf (B) in control, sox2-/-, sox3-/- and sox2-/-; sox3-/- double mutant embryos. Green lines represent means. Asterisks indicate statistically significant differences compared to controls (***P<0.001, Tukey's HSD test following ANOVA). (C–H) Dorsolateral views (anterior to left) of expression of atoh1a (C–E) and neurog1 (F–H) at 24 hpf in control embryos, hs:sox2/+ heterozygotes and hs:sox3/+ heterozygotes. Embryos were heat shocked at 12.5 hpf, 38 °C or 39 °C for 30 min, as indicated. (I–L) Expression of atoh1a (I, J) and neurog1 (K, L) at 24 hpf in cross sections through the middle of the otic vesicle in control embryos (I, K) and hs:sox3/+ heterozygotes (J, L). Embryos were heat shocked at 12.5 hpf, 38 °C for 30 min. Otic vesicle borders are outlined in C–L. (M, N) Quantification of the total number of hair cells (M) and mature SAG neurons (N) at 30 hpf in control and hs:sox2/+ embryos. (O, P) Quantification of the total number of hair cells (O) and mature SAG neurons (P) at 31 hpf in control and hs:sox3/+ embryos. Error bars represent standard deviation in M–P, and asterisks indicate statistically significant differences relative to controls (*P<0.05, ***P<0.001, student's t-test, n>13).

Effects of high-level misexpression ofsox2orsox3during early placode development. (A–H) Dorsolateral views (anterior to the left) of expression of atoh1a (A–D) and neurog1 (E–H) at 24 hpf in control (A, E), hs:sox2/hs:sox2 (B, F) and hs:sox3/hs:sox3 (C–H) embryos. (I–N) Expression of atoh1a (I–K) and neurog1 (L–N) at 24 hpf in cross sections (lateral to the left) through the middle of the otic vesicle in hs:sox2/hs:sox2 homozygotes (I, L) and hs:sox3/hs:sox3 homozygotes (J, K, M, N). hs:sox2/hs:sox2 homozygotes were heat shocked at 12.5 hpf, 39 °C for 60 min, whereas hs:sox3/hs:sox3 homozygotes were heat shocked at 12.5 hpf, 38 °C for 30 min. Otic vesicle borders are outlined.

Effects of misexpressingsox2orsox3at later stages. (A–H) Expression of atoh1a (A–D) and neurog1 (E–H) at 24 hpf in control (A, E), hs:sox3/+ (B, F), hs:sox3/hs:sox3 (C, G) and hs:sox2/hs:sox2 (D, H) embryos. Embryos were heat-shocked at 18 hpf with varying temperatures and durations as indicated. White arrows indicate otic expression of neurog1 in G. (I–N) Expression of atoh1a at 26 hpf (I–K) and 30 hpf (L–N) in control (I, L), hs:sox2/hs:sox2 (J, M) and hs:sox3/hs:sox3 (K, N) embryos following heat-shock (39 °C for 60 min) at 24 hpf. Black arrowhead indicates atoh1a expression in the saccular macula in N, which is strongly reduced compare to L. (O–T) Expression of neurog1 at 26 hpf (O–Q) and 30 hpf (R–T) in control (O, R), hs:sox2/hs:sox2 (P, S) and hs:sox3/hs:sox3 (Q, T) embryos following heat-shock (39 °C, 60 min) at 24 hpf. All images show dorsolateral views (anterior to the left) and otic vesicle borders are outlined.

sox2andsox3do not directly specify neural or sensory fates. (A–F) Dorsolateral views (anterior to the left) showing otic expression of atoh1a at 16.5 hpf (A–C) and 18.5 hpf (D–F) in control (A, D), hs:sox2/hs:sox2 (B, E) and hs:sox3/hs:sox3 (C, F) embryos following heat-shock at 12.5 hpf. (G–L) Dorsolateral views showing otic expression of neurog1 at 16.5 hpf (G–I) and 18.5 hpf (J–L) in control (G, J), hs:sox2/hs:sox2 (H, K) and hs:sox3/hs:sox3 (I, L) embryos following heat-shock at 12.5 hpf. Otic vesicle borders are outlined in all images. (M, N) Quantification of the number of phospho-Histone H3 positive (PH3+) cells in the otic vesicle of control and hs:sox2/hs:sox2 (M) or hs:sox3/hs:sox3 (N) embryos at multiple time points following heat-shock at 12.5 hpf. Error bars represent standard deviation. No statistically significant differences between control and hs:sox2/hs:sox2 or hs:sox3/hs:sox3 embryos at any time point examined (student's t-test, n>15). In all panels, hs:sox2/hs:sox2 embryos were heat shocked at 39 °C for 60 min, whereas hs:sox3/hs:sox3 embryos were heat shocked at 38 °C for 30 min.

Axial patterning in the otic vesicle following early high-level misexpression ofsox2orsox3. Dorsolateral views (anterior to the left) showing expression of various regional markers at 24 hpf in the otic vesicle of control (A–J), hs:sox2/hs:sox2 (A’–J’) and hs:sox3/hs:sox3 (A’’–J’’) embryos following heat-shock at 12.5 hpf. hs:sox2/hs:sox2 embryos were heat shocked at 39 °C for 60 min, hs:sox3/hs:sox3 embryos were heat shocked at 38 °C for 30 min. Otic vesicle borders are outlined in all images.

Effects of early misexpression ofsox3in genetic mosaics. (A–D) Expression of atoh1a (A) and neurog1 (C) at 24 hpf in cross sections of wild-type hosts into which fluorescent dextran-labeled hs:sox3/hs:sox3 transgenic cells were transplanted. Mosaic embryos were heat shocked at 12.5 hpf, 38 °C for 30 min. (B, D) Fluorescent image of dextran and DAPI staining on the same sample shown in A and C respectively. Sections pass through the middle of the otic vesicle, just posterior to the utricular macula. Arrows indicate transgenic cells that ectopically express atoh1a or neurog1. Otic vesicle borders are outlined. (E, F) Quantification of the percentage of transgenic cells located outside endogenous sensory or neural domains that ectopically express atoh1a (E) or neurog1 (F).

Pax2a is required for prosensory but not proneural expansion. (A–H) Dorsolateral views (anterior to the left) showing otic expression of atoh1a (A–D) and neurog1 (E–H) at 24 hpf in control embryos (A, E), pax2a-morphants (C, G), hs:sox2/hs:sox2 homozygotes (B, F) and hs:sox2/hs:sox2 homozygotes injected with pax2a-mo (D, -H). Embryos were heat shocked at 12.5 hpf, 39 °C for 60 min. Otic vesicle borders are outlined in all images.

Roles foratoh1a-neurog1cross-repression but not Notch in sensory-neural segregation. (A, B) Expression of atoh1a at 30 hpf in a control (A) and hs:neurog1/+ (B) embryo following serial heat shock at 24 and 27 hpf, for 60 min at 39 °C. (C, D) Expression of neurog1 at 30 hpf in a control (C) and hs:atoh1a/+ (D) embryo following serial heat shock at 24 and 27 hpf, for 60 min at 39 °C. (E, F) Co-staining of atoh1a (red) and neurog1 (black) expression by two-color in situ hybridization in a control embryo (E) and a mib homozygote mutant (F) at 24 hpf. Otic vesicle borders are outlined in all images. The width of the utricular macula is marked by vertical lines. All images show dorsolateral views with anterior to the left.

Low-level misexpression of Fgf8 expands sensory potential in the early placode. (A–D) Dorsal views showing expression of pax8 (A, B, anterior up) and atoh1b (C, D, anterior to the left) at 10.8 hpf in control (A, C) and hs:fgf8/+ (B, D) embryos that were heat shocked at 10 hpf, 35 °C for 45 min. (E–G) Quantification of relative surface area of otic/epibranchial pax8 domain (E), atoh1b domain (F) and the number of atoh1b-expressing cells (G) in control and hs:fgf8/+ embryos following heat shock at 10 hpf, 35 °C for 45 min. Error bars represent standard error of the mean. Asterisks indicate statistically significant differences compare to control (*P<0.05, student's t-test, n>22).