Identification of Synergistic Signals Initiating Inner Ear Development Raj K. Ladher, Kelly U. Anakwe, Austin L. Gurney, Gary C. Schoenwolf, and Philippa H. Francis-West

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Supplemental Figure 2. Fate mapping shows that the Fgf-19 expression domain correlates with otic development. Fgf-19-expressing mesoderm (and incidently the overlying neuroectoderm) at stage 7 and neuroectoderm (and the adjacent mesoderm) at stage 9 was labeled with DiI and rhodamine. The site of the injection at t = 0 was determined by fluorescent analysis, whereas the fate of the labeled tissue (stained brown) after 14 hours was determined by whole-mount staining and section analysis. Injections were performed rostral to the first somite (white line). At stage 7, the Fgf-19-expressing mesoderm and overlying neuroectoderm is or will be subjacent and adjacent to the otic placode/vesicle, respectively, 14 hours later. Likewise, the Fgf-19-expressing neuroectoderm at stage 9 is or will be adjacent to the otic placode/vesicle 14 hours later. Labeling the Fgf-19-expressing mesoderm at stage 7 and the Fgf-19-expressing neuroectoderm at stages 7 and 9 does not label the developing otic placode/vesicle (arrows), indicating that the placode arises lateral to the neural plate. This has been confirmed by independent labeling analyses (data not shown).

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Supplemental Figure 3. Fgf-19-expressing mesoderm can induce otic development in rostral ectoderm. Diagram showing the region of stage 5 ectoderm, Et (test ectoderm) and stage 7 mesendoderm [regions (a) through (d)] or stage 9+ mesoderm (a´), which were recombined in collagen gel culture. The Fgf-19-expressing regions are shaded. The otic placode arises from region (c). Whole-mount in situ hybridizations and antibody staining showing that only regions (a) (A) and (b) (B), which include Fgf-19-expressing mesoderm, and not regions (c) (C) or (d) (D), can induce the expression of Pax-2 and development of inner ear hair cells (B´), (C´) together with other otic markers (data not shown). Region (a) or (b): Pax-2, n = 5/5; Dlx-5, n = 4/4; Nkx5.1, n = 2/3; SOHo-1, [(a), n = 3/7 and (b), n = 5/7], inner hair cells, n = 4/4. The expression of these genes or development of inner hair cells was never induced in the equivalent recombinations with regions (c) and (d). Region (a´) also did not induce expression of Pax-2 [(E), n = 0/5] or Nkx-5.1 [(F), n = 0/2]. However a´ can induce Pax-2 [(G), n = 5/5] and Nkx-5.1 [(H), n = 2/2] when the overlying neural tube, N, which expresses Fgf-19, is included. Scale bars are 100 mm in (A) through (H) and 10 mm in(B´) and (C´).

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Supplemental Figure 4. FGF-19 can induce expression of otic markers in non-otic tissue. Whole-mount in situ hybridizations showing that FGF-19 but not control beads can induce the expression of the otic markers, Dlx-5, Fgf-3, Pax-2, and SOHo-1 in stage 5 ectoderm (Et, Fig. 2) and in region (d) at stage 7 (Fig. 2). Stage 5 ectoderm: Dlx-5, n = 6/7; Fgf-3, n = 3/4; Pax-2, n = 4/5, and SOHo-1, n = 5/8. Region (d): Dlx-5, n = 6/10; Fgf-3, n = 4/10, Pax-2, n = 4/5, and SOHo-1, n = 9/12.

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Supplemental Figure 5. Comparison of Fgf-19 and Wnt-8c expression. Whole-mount in situ hybridizations comparing Fgf-19 and Wnt-8c expression at stages 7 and 9. Section analysis indicates that initially Fgf-19 expression underlies Wnt-8c expression, which is confined to the neuroectoderm. From stage 9-, Fgf-19 and Wnt-8c expression overlap in the caudal hindbrain. hb, hindbrain; m, mesoderm; np, neural plate.

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Supplemental Figure 6. FGF-19 and Wnt-8c synergize to initiate otic development. Whole-mount in situ hybridizations showing that Wnt-8c alone can induce Fgf-3 expression, while FGF-19 and Wnt-8c synergize to induce the expression of other otic markers. Stage 7 region (c) explants (Fig. 2) do not express otic markers when cultured with beads soaked in a control solution or FGF-19. Similarly, with the exception of Fgf-3, Wnt-8c-expressing COS cells do not strongly induce otic markers Pax-2, Dlx-5, Nkx5.1, and SOHo-1. However, region (c) explants strongly express the otic markers, Pax-2 (n = 4/4), Dlx-5 (n = 3/3), Nkx 5.1 (n = 3/4), and SOHo-1 (n = 5/5) when cocultured with both Wnt-8c-expressing cells and beads soaked in FGF-19.

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