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First published online February 2, 2004
doi: 10.1242/10.1242/dev.00978

Development 131, 923-931 (2004)
Published by The Company of Biologists 2004
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A direct role for Fgf but not Wnt in otic placode induction

Bryan T. Phillips, Elly M. Storch, Arne C. Lekven and Bruce B. Riley*

Biology Department, Texas A&M University, College Station, TX 77843-3258, USA



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  		Fig. 1. Effects of disrupting Wnt8 function. (A-F) Lateral views of live embryos at 30 hpf. wnt8 morphant (A), moderately affected dkk1-injected wild-type embryo (B) and severely affected dkk1-injected wild-type embryo lacking hindbrain and otic tissue (C). Enlarged view otic vesicles of wnt8 morphant (D), moderately affected dkk1-injected wild-type embryo (E) and wild-type embryo (F). (G,H) Lateral views of pax8 expression at tailbud stage in a wild-type embryo (G) and wnt8 morphant lacking the otic domain (H). (I-K) Dorsal views of pax8 expression at six-somite stage in a wild-type embryo (I), severely affected dkk1-injected embryo (J) and Dfw8 homozygote (K). Arrowheads indicate the preotic region. Abbreviation: mhb, midbrain-hindbrain border. (A-F) Anterior is to the left and dorsal upward. (G,H) Lateral views with anterior upward. (I-K) Dorsal views with anterior upward. Scale bars: A-C, 150 µm; D-F, 30 µm; G-K, 200 µm.

 


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  		Fig. 2. Cross-regulation of Wnt8 and Fgf. (A-D) fgf3 expression in the hindbrain of tailbud stage wild-type embryo (A), tailbud stage wnt8 morphant (B), tailbud stage Dfw8 homozygote (C) and sixsomite stage Dfw8 homozygote (D). (E-H) fgf8 expression in the hindbrain of tailbud stage wild-type embryo (E), tailbud stage wnt8 morphant (F), tailbud stage Dfw8 homozygote (G) and six-somite stage Dfw8 homozygote (H). (I-K) wnt8 ORF2 expression in the hindbrains of a wild-type embryo at 90% epiboly (I), wild-type embryo injected with fgf3- and fgf8-MOs at 90% epiboly (J), wild-type embryo at tailbud stage (K) and tailbud stage wild-type embryo injected with fgf3- and fgf8-MOs (L). Arrowheads indicate hindbrain domain. p, prechordal plate. Dorsal views with anterior upward. Scale bar: 200 µm.

 


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  		Fig. 3. Effects of Fgf misexpression. (A,B) Two-color in situ hybridization of three-somite stage. Wild-type (A) and fgf3 (B) plasmid-injected embryos showing pax8 expression (blue) and krox-20 expression (red). The left preotic domain is enlarged significantly and a region with ectopic upregulation of pax8 (arrowhead) is evident. (C,D) Two-color in situ hybridization of three-somite stage embryo injected with fgf3 plasmid showing fgf3 expression (blue) and pax8 expression (red). (C) Brightfield and (D) fluorescent images showing relationship between ectopic fgf3-expressing cells (arrow) and ectopic pax8-expressing cells (arrowhead). The boxed area in C is enlarged in D. The endogenous preotic domain on the left is enlarged in the vicinity of misexpressed fgf3. The r4 domain of fgf3 is faintly visible in C. (E,F) foxi1 expression at three-somite stage in wild-type (E) and fgf8 plasmid-injected embryo (F). Ectopic expression in anterior region of the injected embryo is indicated (arrowhead). (G) Ectopic pax2.1 expression (arrowhead) adjacent to the midbrain of a four-somite stage embryo injected with fgf3-plasmid. (H) Low magnification view of a 30 hpf wild-type embryo injected with fgf8-plasmid showing that overall axial development is essentially normal, although development of anterior sensory structures is perturbed. (I) Higher magnification of the embryo shown in H. Ectopic otic vesicles are indicated (arrowheads). Development of adjacent eye tissue is perturbed, but general features of brain development, such as the epiphysis and midbrain-hindbrain boundary are produced. (J) Frontal/lateral view of an embryo injected with fgf3-plasmid. An ectopic otic vesicle is indicated (arrowhead). Development of adjacent nasal and eye tissue is severely perturbed. (K) Lateral view of a wnt8 morphant injected with fgf8-plasmid. Ectopic otic vesicles (arrowheads) are seen next to the midbrain-hindbrain boundary. Abbreviations: e, epiphysis; mhb, midbrain-hindbrain border; op, endogenous otic placode; r3, rhombomere 3. (A-E,G) Dorsal view with anterior upward. (F) Dorsal-anterior view. (H,I,K) Lateral views with anterior to the left. (J) Frontal-lateral view with anterior to the left. Scale bars: A-C,E-G, 150 µm; D, 60 µm; H, 175 µm; I-K, 75 µm.

 


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  		Fig. 4. Effects of Wnt8 misexpression. (A-C) Three-somite stage embryos globally expressing wnt8 ORF1 showing expression of fgf8 (A), erm (B) and pax8 (C). The r4 domain of fgf8 is indicated. The weaker anterior fgf8 expression corresponds to midbrain-hindbrain boundary. Asterisks mark the anterior limit of the neural plate. (D) pax8 expression in an embryo globally expressing ORF1 and coinjected with fgf3-MO and fgf8-MO. Preotic expression is nearly ablated. (E) Ectopic otic vesicles (arrowheads) in a live embryo at 30 hpf globally expressing ORF1. Note that anterior sensory structures and morphological landmarks in the brain such as the midbrain-hindbrain boundary and epiphysis are not produced. (F) Loss of otic tissue in a 30 hpf embryo globally expressing ORF1 and coinjected with fgf3-MO and fgf8-MO. (A-D) Dorsal views with anterior upward. (E,F) Lateral views with anterior to the left. Scale bars: A-D, 200 µm; E-F, 75 µm.

 


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  		Fig. 5. Expression of Fgf- and Wnt-inducible reporter genes. All images show gene expression patterns in wild-type embryos at tailbud stage. (A) Expression of erm. (B) Costaining of erm and fgf8 (darker staining). (C) Two-color staining showing expression of erm (red) and fgf8 (blue). (D) TOPdGFP expression. (E) Costaining of TOPdGFP and fgf8. (F,G) Parasagittal sections at the locations indicated in E showing TOPdGFP expression in a domain lateral and posterior to the hindbrain (F) and fgf8 expression in the hindbrain (G). Preotic domains are indicated by asterisks. Abbreviations: p, prechordal plate; hb, hindbrain. (A,B,E) Dorsal views with anterior upward. (C) Dorsolateral view with anterior upward. (D) Lateral view with anterior upward and dorsal to the right. (F and G) Dorsal is to the right and anterior is upward. Scale bars: A,B,E, 150µm; C, 40 µm; D, 175 µm; F,G, 25 µm.

 

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© The Company of Biologists Ltd 2004