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First published online November 17, 2003
doi: 10.1242/10.1242/dev.00881

Development 130, 6329-6338 (2003)
Published by The Company of Biologists 2003
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Requirements for FGF3 and FGF10 during inner ear formation

Yolanda Alvarez1,*, Maria Teresa Alonso1,2,*, Victor Vendrell1,*, Laura Cecilia Zelarayan1, Pablo Chamero1,2, Thomas Theil3, Michael R. Bösl1, Shigeaki Kato4, Mark Maconochie5, Dieter Riethmacher1 and Thomas Schimmang1,{dagger}

1 Center for Molecular Neurobiology Hamburg, University of Hamburg, Falkenried 94, D-20251 Hamburg, Germany
2 Instituto de Biología y Genética Molecular, Universidad de Valladolid y Consejo Superior de Investigaciones Cientificas, Departamento de Bioquímica, Biología Molecular y Fisiología, Facultad de Medicina, E-47005 Valladolid, Spain
3 Institute for Animal Developmental and Molecular Biology, Heinrich-Heine-University, D-40225 Düsseldorf, Germany
4 Institute of Molecular and Cellular Biosciences, University of Tokyo, Bunkyo-Ku, Tokyo 113, Japan
5 School of Life Sciences, University of Sussex, Falmer, Brighton BN1 9QG, UK



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  		Fig. 1. Deletion of the Fgf3-coding region in mice. (A) The genomic locus with the exons and coding regions of the Fgf3 gene indicated. The coding region was replaced by a FGF3 cDNA and a neor gene flanked by loxP sites by homologous recombination. The introduced sequences were removed by Cre-mediated excision between the external loxP sites. (B) Southern blot analysis was carried out using the probes indicated in A to detect the correct targeting event in ES cells and subsequent deletion of the introduced sequences. (C) PCR analysis using primer pairs indicated by pink arrows in A demonstrating the presence and absence of specific products amplified from the targeted locus and after generation of the knock out allele. (D) Whole-mount RNA hybridisation analysis of Fgf3 expression in the hindbrain of an E8.5 Fgf3–/– mutant embryo and a wild-type littermate. Scale bar: 100 µm.

 


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  		Fig. 3. Ectopic expression of FGFs and formation of ectopic otic vesicles. (A) Construct for the generation of transgenic mice. An Epha4 r3/r5 enhancer in combination with a ß-globin minimal promoter is used to drive expression of FGFs and an IRES-lacZ reporter gene in the hindbrain. (B) Formation of an ectopic vesicle (arrow) anterior to the otic vesicle (OV) in an FGF10 transgenic embryo. (C) Section through the hindbrain of an FGF10 transgenic embryo stained for lacZ. An ectopic vesicle (arrow) has formed next to rhombomere (r) 4. (D,E) Expression of Dlx5 and Lmx1 in ectopic vesicles (arrows) of FGF10 transgenic mice. Scale bars: in C, 80 µm for C and 200 µm for B,D; in E, 200 µm for E.

 


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  		Fig. 2. Tail and inner ear phenotype of Fgf3–/– mutants. (A) Fgf3–/– adult mutants show a shortened, thickened and kinked tail. (B,C) Sections through the otic vesicle of a wild-type and a Fgf3–/– mutant littermate at E10.75. The endolymphatic duct is indicated by an asterisk. Note the reduced size of the otic vesicle in the mutant embryo compared with the wild-type control animal. (D,E) Transverse sections through the inner ear at E13.5. The cochleovestibular ganglion (cvg), endolymphatic duct (ed) and posterior semicircular canal are indicated (pc). (F,G) Sections through the cochlea of wild-type and Fgf3–/– adult mutant littermates. The cochlear ganglion is indicated by arrows. Scale bars: in C, 200 µm for B,C,F,G; in D, 200 µm for D,E.

 


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  		Fig. 4. Expression of Fgf3 and Fgf10 during inner ear formation. (A-C) Expression of Fgf3 was detected at the stages indicated by whole-mount in situ hybridization. The position of the prospective rhombomeres and rhombomeres are indicated. The arrow in B indicates the level of the transversal section shown in C, where expression in the hindbrain and otic placode can be observed. (D-N) Expression of Fgf10 was detected by whole-mount RNA in situ hybridisation at the stages of development indicated. (D-F) Expression of Fgf10 is observed in the anterior mesenchyme (m). The arrow in D indicates the level of the transversal section shown in E. A transverse section at this level in a four-somite stage embryo shows that Fgf10 expression is localized to ventral anterior mesenchyme (F). (G-N) Expression of Fgf10 in the hindbrain. To facilitate the localisation of Fgf10 expression relative to the position of rhombomeres 5 and 6, a probe for the Mafb gene was used in G-K. (L-N) Transverse sections at the levels indicated by arrows in H,I,K, respectively, show expression of Fgf10 restricted to the ventral part of the hindbrain. The broken line in I indicates the position the otic placode. In K and N, Fgf10 expression is also detected in the anterior part of the otic cup. Scale bars: in A, 200 µm for A; in B, 200 µm for B; in D, 50 µm for D; in G, 200 µm for G; in H, 200 µm for H; in I, 200 µm for I; in J, 200 µm for J; in K, 200 µm for K.

 


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  		Fig. 5. Defects in inner ear formation in Fgf3 and Fgf10 double mutant mice. (A-D) Sections at the level of the otic placode at E8 (eight somites) and the invaginating placode (E8.75) which have been hybridized with the indicated probes. Note the absence of Dlx5 staining in B. At E8.75, the otic placode in the mutant embryo has only initiated its invagination and shows very weak Pax2 expression (D), whereas strong expression is detected in the otic cup formed in the wild-type embryo (C). (E,F) Toluidin Blue stained sections through the otic vesicle of a wild-type and a Fgf3–/–/Fgf10–/– mutant littermate at E10.75. Note the absence of the cochlear ganglion in the mutant (indicated by an arrow in the wild-type animal) and a more ventralized position of the vesicle relative to the border of the neural tube (marked by asterisks). (G-P) Expression of the indicated otic marker genes by whole-mount RNA in situ hybridisation in wild-type embryos (G,I,K) and Fgf3–/–/Fgf10–/– mutant littermates at E9.5 (H,J,L,O,P). (M,N) Sections corresponding to the embryos shown in K,L. The punctated circle in J,L,N indicates the circumference of the residual otic tissue formed in the mutants. Note the complete absence of Dlx5 staining in the vesicle of the mutant animal in L,N. Scale bars: in A, 40 µm for A-D; in I, 100 µm for E-P.

 

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© The Company of Biologists Ltd 2003