Schlosser G , Awtry T , Brugmann SA , Jensen ED , Neilson K , Ruan G , Stammler A , Voelker D , Yan B , Zhang C , Klymkowsky MW , Moody SA .

GM54001 NIGMS NIH HHS , NS23158 NINDS NIH HHS , R01 GM054001-07 NIGMS NIH HHS , R01 GM054001 NIGMS NIH HHS , R01 NS023158 NINDS NIH HHS

	Fig. 1. Placodal distribution of Sox3-immunopositive cells in relation to Six1, NeuroD, Delta1 and Ngnr1 expression in transverse sections through stage 26 embryos. Each section is shown in bright field (A1–I1: gene expression), red fluorescence (A2–I2: Sox3 protein), and a superposition of these two with blue DAPI fluorescence (A3–I3). White asterisks indicate placodally derived cranial ganglia (main body of ganglia out of level of section in panels B, C and G). Black asterisks mark endodermal Sox3 nuclei. (A–C) Six1 expression domains in otic vesicle (A), anterodorsal lateral line (B) and facial epibranchial (C) placodes encompass Sox3 immunopositive nuclei (arrows). (D–F) NeuroD is expressed in ganglia derived from otic vesicle (D), anterodorsal lateral line (E) and facial epibranchial placodes (F) immediately abutting Sox3-positive placodal cells (arrows). (G–I) Delta1 expression in the otic vesicle (G), and the anterodorsal lateral line and glossopharyngeal epibranchial placodes and ganglia (H upper and lower part, respectively) and Ngnr1 expression in the facial epibranchial placode (I) overlap with some (black arrows) but not all (white arrows) Sox3-cells. Bar in I: 50 μm (for all panels).
	Fig. 2. Effects of Eya1 and Six1 knockdown on markers of neurogenesis and placodal ectoderm. (A) Immunoblot showing that Eya1MO1 but not control MO blocks synthesis of Eya1 protein. (B) Co-injecting Eya1MO2 or Eya1MO1+2 with myc-tagged Eya1 mRNA significantly restores NeuroD and prevents ectopic p27Xic1 expression (χ2 test; *: p < 0.05, **: p < 0.001) in three independent rescue experiments. (C–Q) Neural plate stage embryos after unilateral injection (lower half) of Eya1MO1+2 (C–L) or Six1MO1+2 (M–Q). In all cases, where myc-GFP was co-injected as lineage tracer (C–K), embryos are shown superimposed with green fluorescent channel. Asterisks indicate reductions, arrows and arrowheads indicate increased marker gene expression. For Delta1 after Eya1MO1+2 injections two different phenotypes are depicted (J, K). R: Tail bud stage embryo after unilateral injection (R1, R3, R5: injected side; R2, R4, R6: control side) of Eya1MO1+2 reveals reduction of NeuroD expression in all neurogenic placodes or derivative ganglia. R3 and R4 depict a section at the level indicated (white line) with boxed areas shown magnified in R5 and R6, respectively, superimposed with green (myc-GFP co-injected with Eya1MO1+2) and blue (DAPI) fluorescence. Residual NeuroD expression is confined to cells receiving little or no MO. Bar in R4: 100 μm (for R3, R4). Abbreviations: e: eye; epVII+AV: facial epibranchial and anteroventral lateral line placode; epIX: glossopharyngeal epibranchial placode; gVII/AV/AD: ganglion of the facial, anteroventral and anterodorsal lateral line nerve; gVIII: vestibulocochlear ganglion; nt: neural tube; pAD: anterodorsal lateral line placode; pM: middle lateral line placode; pOl: olfactory placode; pP: posterior lateral line placode; pPr: profundal placode; pV: trigeminal placode; vOt: otic vesicle.
	Fig. 3. Effects of Eya1 and Six1 overexpression on markers of neurogenesis and placodal ectoderm at neural plate stages. Embryos after unilateral injection (lower half) of Eya1 (A–J), Six1 (K–N) or Eya1+Six1 (O) mRNA. In all cases, where myc-GFP was co-injected as lineage tracer (A–D, F–I, K, L, N, O), embryos are shown superimposed with green fluorescent channel. Black asterisks indicate reductions in the placodal or non-neural ectoderm. Arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm; black arrowhead identifies deformed blastopore due to gastrulation defects (O). For Sox3 (C, D) and p27Xic1 (I, J) after Eya1 injections two different phenotypes are depicted.
	Fig. 4. Effects of Eya1, Six1 and Eya1+Six1 overexpression on various neurogenesis markers at tail bud stages (stage 26). Injected (A1–N1) and control (A2–N2) sides of tail bud stage embryos after unilateral injection of Eya1 (A–H), Six1 (I–L) or Eya1+Six1 (M, N) mRNAs. In all cases, where myc-GFP was co-injected as lineage tracer (A–G, M–N), embryos are shown superimposed with green fluorescent channel. Black asterisks indicate reductions, while black arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm. Yellow asterisks identify expression in pharyngeal pouches. For Sox3 (A, B) and NeuroD (C, D) after Eya1 injections two different phenotypes are depicted. Abbreviations as in Fig. 2. gPrV: profundal-trigeminal ganglionic complex; L: lens placode; PN: pronephros.
	Fig. 5. Effects of overexpression of GR–Eya1 or GR–Eya1+GR–Six1 on various neurogenesis markers at tail bud stages (stage 26). Injected (A1–N1) and control (A2–N2) sides of tail bud stage embryos after unilateral injection of GR–Eya1 (A–H) or GR–Eya1+GR–Six1 (I–N) and DEX activation at stage 16–18. In all cases, where myc-GFP was co-injected as lineage tracer (A–F, I–M), embryos are shown superimposed with green fluorescent channel. Black asterisks indicate reductions, while black or white arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm. Yellow asterisks identify expression in pharyngeal pouches. For NeuroD (C, D) and p27Xic1 (G, H) after GR–Eya1 injection two different phenotypes are depicted. Abbreviations as in Fig. 2 and Fig. 4.
	Fig. 7. Dose-dependent effects of Eya1 on neurogenesis markers as revealed in tissue sections. Distribution of N-Tubulin (A), NeuroD (B), Ngnr1 (C) and Delta1 (D) in stage 26 embryos after injection of myc-Eya1 or co-injection of Eya1 with myc-GFP mRNA. Embryos are shown as wholemounts (injected side: A1–D1; control side: A2–D2) and in transverse sections (at level indicated by black line) shown in bright field (A3–D3), superimposition of green (myc-immunostaining) and blue (DAPI) fluorescence (A4–D4, no DAPI image available in panel D), and as a merged image (A5–D5). Boxed areas are magnified in inserts. The DAPI channel is not shown in the inserts of panel C to make Ngnr1 staining more clearly visible. Hatched boxed area in panels C3–C5 shows Ngnr1 positive epibranchial placode on control side in an adjacent section. Asterisks indicate reductions, while arrows mark ectopic marker gene expression in the placodal or non-neural ectoderm. Double arrows identify areas of residual expression. Arrowheads mark displaced otic vesicle on injected side. Bar in panel D5: 100 μm (for A3–5 – D3–5).
	Fig. 9. Eya1 and Six1 promote proliferative SoxB1-positive neuronal progenitors. (A–C) Transverse sections through neural plate (A) or tail bud stage (B, C) embryos that received unilateral myc-Eya1 mRNA injection at the two cell stage. (A, B) Panels show Sox3 expression in bright field (A1–B1), distribution of injected myc-Eya1 protein (A2–B2), PCNA together with DAPI staining (A3–B3) and a merged image (A4–B4). (C) Panels show distribution of injected myc-Eya1 protein (C1), PCNA (C2) and a merged image (C3) together with DAPI staining. Ectoderm that received high levels of Eya1 expresses Sox3 (A1–B1, arrows), is drastically thickened (A1–C1, red lines) compared to control side (black or white lines) and consists of multiple layers of PCNA positive cells (A3, B3, C2). The otic vesicle has failed to form on the injected side of embryos in panels B and C. Arrowheads identify cells coexpressing myc-Eya1 and PCNA. Double arrows in panel C identify the basal lamina of the ectoderm on the injected side. Abbreviations: nt: neural tube. vOt: otic vesicle. All bars: 100 μm. (D–G) Effects of Eya1 and Six1 gain and loss of function on phosphohistone H3 (PH3). (D–F) PH3 immunopositive nuclei in placodal ectoderm of neural plate stage embryos after injection of lacZ (D) Eya1+lacZ (E) or Eya1+Six1+ lacZ mRNAs (F). (G) Relative mitotic index after injections of Six1 or Eya1 mRNAs or morpholinos. Standard deviations and significant increases are indicated (t-test; *: p < 0.05, **: p < 0.001).
	Fig. 10. Ectopic neurons in embryos overexpressing Eya1 or Eya1+Six1 originate from Sox3 immunopositive neuronal progenitors. Panels show distribution of NeuroD expressing cells in relation to Sox3-immunopositive cells in tail bud stage embryos after injection of myc-Eya1 mRNA alone (A) or together with Six1 mRNA (B). Embryos are shown as wholemounts (injected side: A1–B1; control side: A2–B2) and in transverse sections (at level indicated by black line). Panels show bright field images (A3–B3), myc-immunostaining together with DAPI (A4–B4), Sox3-immunostaining together with DAPI (A5–B5), and merged images (A6–B6). Asterisks indicate reductions, while arrows mark ectopic NeuroD expression in the non-neural ectoderm. Ectopic NeuroD expression is found next to Sox3 immunopositive cells on the fringes of ectodermal regions that received high levels of myc-Eya1. Sox3 cells reside in ectoderm that received high levels of myc-Eya1 (arrowheads) as well as in areas of lower myc-Eya1 levels immediately abutting NeuroD expressing cells (double arrows). Boxed areas are shown at higher magnification in inserts. All bars: 100 μm
	Fig. 11. Role of SoxB1 genes in placodal neurogenesis. (A) Immunoblot showing that Sox2MO and Sox3MO specifically and efficiently block protein synthesis from co-injected utr-Sox2 and utr-Sox3 mRNA, respectively. (B–E, F–I) Effects of unilateral injection (lower half) of Sox2+Sox3MO (B–E), or Sox3 mRNA (F–I) on various neurogenic markers at neural plate stage. Embryos are shown superimposed with green fluorescent channel to reveal distribution of myc-GFP co-injected as lineage tracer. Asterisks indicate reductions, while arrows mark increased or ectopic marker gene expression in the placodal or non-neural ectoderm. (J–L) Effects of unilateral injection of GR–Sox3 and DEX activation at stage 16–18 on neurogenic markers at tail bud stages (J1–L1: injected side; J2–L2: control side). Asterisks indicate reductions in the placodal or non-neural ectoderm. (M, N) Ectopic neuronal differentiation (arrows) after injection of GR–Eya1+GR–Six1 mRNAs (M1: inj. side; M2: control side) and DEX activation at stage 16–18 is significantly reduced after co-injection of Sox2/Sox3 MO (M3: inj. side; M4: control side; N: quantitation, χ2 test; : p < 0.001). (O–R) Ectopic expression (arrows) of Sox3 (O1, O3: inj. side; O2, O4: control side) and Sox2 (P1, P3: inj. side; P2, P4: control side) but not of NeuroD (Q1, Q3: inj. side; Q2, Q4: control side) and p27Xic1 after injection of GR–Eya1+GR–Six1 mRNAs and DEX activation at stage 16–18 persists and is more extensive with CHX treatment (R: quantitation; χ2 test; : p < 0.05, : p < 0.001).

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