In situ hybridization analysis of Pax8, Dlx5, and Pax2 in control and double mutant embryos at pre-otic field (A – F) and placode (G – L) stages. (A – F) Arrowheads point to the expression in the pre-otic field for the genes indicated. (G, H) Otic placode staining of Pax8 is outlined (white dashed line). Epibranchial placode (e) staining adjacent to the otic domain is indicated. (I – L) Dlx5 and Pax2 expression in the otic placode is indicated (brackets). Expansion of gene expression domains is highlighted (asterisk). Anterior is to the right. Scale bar, 100 μm.

(A, B) Average anterior to posterior lengths of the otic placode in double mutants and controls, measured on sagittal embryonic sections at 9 – 11 s and 12 – 13 s. (C, D) Sagittal sections from bin 41–60% in a 12 – 13 s control and double mutant. Anterior to posterior extent of the otic placode is bracketed. Anterior is to the left. (E) Average cell densities per bin, as in (B). (F – I) Surface views of control and double mutant embryos stained with E-cadherin antibody, at the somite stages indicated. Dorsal is at the top. *, p < 0.05. **, p = 0.01 by ANOVA; error bars represent standard error of sample means. Scale bar, C, D 50 μm.

(A – H) In situ hybridization analysis of Spry1 (A – D) and Spry2 (E – H) in transverse embryonic sections at the stages indicated. Otic tissue is bracketed. (I, J) In situ hybridization on whole mount embryos for Spry1 and Spry2 at the pre-otic field stage. (K, L) Higher-magnification images of Spry1 expression in the pre-otic field (K) and otic placode (L). The cellular region in which Spry1 transcript is observed (yellow bracket) and not observed (purple bracket) is indicated. Abbreviations: (fb) forebrain, (hb) hindbrain, (mb) midbrain, (oc) otic cup, (op) otic placode, (p-of) pre-otic field, (pe) pharyngeal endoderm. Anterior (for I, J) is to the left.

(A, B) Transverse embryonic sections co-immunolabeled for pH3 (red) and PAX2 (green), counterstained with a nuclear dye (blue). (C) The average number of pH3+ cells within the PAX2 domain on both sides (left and right) of the embryo over a 80 μm distance from anterior to posterior. (D, E) Transverse embryonic sections immunolabeled for activated caspase-3 (green), counterstained with a nuclear dye (blue). (F) The average number of ectodermal, activated caspase-3+ cells over a 160 μm distance from anterior to posterior, encompassing the pre-otic field. (G – L) Foxi2 expression analyzed by in situ hybridization. (G, J) Lateral views of whole embryos, anterior to the right. The otic placode, which does not express Foxi2, is outlined in the control embryo (white dashed circle) and black bracketed in the Spry1^−/−; Spry2^−/− mutant. (H, K) Dorsal views of the same embryos shown in G, J, respectively. The white bracket indicates the region in the Spry1^−/−; Spry2^−/− mutant lacking Foxi2 expression. (I, L) Sagittal sections through the otic placode in the plane indicated by a white dashed line in H, K, respectively. The otic placode is indicated with a black bracket. Arrows point to patches of cells expressing Foxi2. Anterior is the left (I and L). Error bars (C, F) represent standard deviations. Scale bar, I, L, 50 μm; G, H, J, K, 100 μm.

Dorsal views of whole mount embryos analyzed using in situ hybridization to visualize the rhombomeric organization of the hindbrain from rhombomeres 3 to 5 (r3 – r5) for the probes and genotypes indicated. (B, J) Bracket indicates the extent of the Wnt8a expression domain in double mutants. Scale bar, A – H, I – K 100 μm.

In situ hybridization analysis of Dlx5 expression to visualize the otic placode (A – D, lateral views) and of Wnt8a expression in the hindbrain (E – G, dorsal views). (A) The normal domain of Dlx5 expression is indicated with an arrowhead in a Spry1^−/+; Spry2^−/+; Fgf10^+/+ control embryo. (B) The expanded domain of Dlx5 expression is black bracketed in a Spry1^−/−; Spry2^−/−; Fgf10^+/+ mutant embryo. (C, D) Spry1^−/−; Spry2^−/−; Fgf10^−/+ embryos demonstrating complete rescue of the size of the Dlx5 domain (asterisk, C) and intermediate rescue (white bracket, D). (E) The normal domain of Wnt8a expression in rhombomere 4 (r4) in a Spry1^−/+; Spry2^−/+; Fgf10^+/+ control embryo. (F) The expanded domain of Wnt8s expression is black bracketed in a Spry1^−/−; Spry2^−/−; Fgf10^+/+ mutant embryo. (G) The domain of Wnt8a expression in a Spry1^−/−; Spry2^−/−; Fgf10^−/+ embryo is indicated (asterisk). (A – D) Anterior is to the right.

(A-C) Model of Spry1 and Spry2 function in the pre-otic field for the genotypes indicated. The ectodermal region in which both Spry1 and Spry2 are expressed is indicated by blue shading; expression of Wnt8a is indicated by purple shading. The effective FGF-responsive region is indicated by the hatched oval.