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First published online April 22, 2004
doi: 10.1242/10.1242/dev.01082

Development 131, 2125-2135 (2004)
Published by The Company of Biologists 2004
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Apaf1-dependent programmed cell death is required for inner ear morphogenesis and growth

Francesco Cecconi1, Kevin A. Roth2, Oleg Dolgov3, Eliana Munarriz1, Konstantin Anokhin3, Peter Gruss4 and Marjo Salminen5,*

1 Dulbecco Telethon Institute, Department of Biology, University of Rome `Tor Vergata', via della Ricerca Scientifica, 00133 Rome, Italy
2 Department of Pathology, University of Alabama at Birmingham, SC 961E, 1530 Third Avenue South, Birmingham, AL 35294, USA
3 P. K. Anokhin Institute of Normal Physiology RAMS, 6, Bol. Nikitskaya st, 103009 Moscow, Russia
4 Department of Molecular Cell Biology, Max-Planck Institute of Biophysical Chemistry, Am Fassberg 11, 37077 Goettingen, Germany
5 Institute of Biotechnology, University of Helsinki, Viikinkaari 9, 00710 Helsinki, Finland



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  		Fig. 1. Detection of Apaf1/lacZ and Bcl-XL in the developing inner ear at E12.5. Apaf1/lacZ expression could be detected in the otic epithelium and the surrounding mesenchyme at E9.5 (A), E10.5 (B) and E12.5 (D,E). No staining could be detected in sections from wild-type embryos (C). Bcl-XL protein could be detected in the otic epithelium but not in the periotic mesenchyme at E10.5 (G), E11.5 (H) and E12.5 (I). No staining in the otic region could be detected in control sections with only secondary antibody (F) or with TUJ1 antibody which specifically stains neurons (J). (K) Plane of the sections, (L) lacZ expression in a heterozygous Apaf1+/– embryo, (M) control staining in a wild-type embryo. (N) RT-PCR analysis of total RNA from E9.5 and E11.5 wild-type embryos. Only a 395 bp-long fragment corresponding to the Bcl-XL isoform was detected. Scale bar: 100 µm. a, anterior; c, cochlea; d, dorsal; ed, endolymphatic duct; ld, lateral semicircular duct; nt, neural tube; ov, otic vesicle; p, posterior; pd, posterior semicircular duct; sd, superior semicircular duct; us, utriculosaccular space; v, ventral.

 


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  		Fig. 2. Apoptosis during early otic vesicle development. (A) Plane of the sections. Dying cells were detected in cross-sections through the otic vesicle at E10.0 (B-D) and E10.5 (E-H) with the TUNEL method. (E) Negative control for TUNEL staining without terminal deoxynucleotidyl transferase. The genotypes of the embryos are indicated in each case. The arrowheads in F and H point to the dying cells observed between the otic vesicle and the surface ectoderm. A higher magnification of this area is shown in the left-hand corner in F and H. Scale bar: 50 µm.

 


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  		Fig. 3. Apoptosis in the inner ear at E12.5. TUNEL-positive cells in sections from wild-type (A-D,G), Apaf1 (E) and Bcl2l mutant (F,H) embryos. The arrowheads in A, C and G point to TUNEL-positive cells at the future fusion plate-forming epithelium and the adjacent periotic mesenchyme. The arrowheads in B point to dying cells at the areas where the ducts separating the utricle, saccule and cochlear duct will become thinner. The arrow in A points to the TUNEL-positive cells at the outer edge of the semicircular duct. Scale bar: 100 µm. hm, head mesenchyme; s, saccule; u, utricle.

 


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  		Fig. 4. Three-dimensional reconstructions of the inner ear epithelium at E13.5. Otic epithelium was reconstructed from serial sections through wild-type (A), Apaf1 (B) and Bcl2l (C) mutant inner ears. All three embryos came from the same double heterozygous mating. A lateral view is shown, dorsal is up, ventral down. The Apaf1 ear has been turned so that it corresponds to the two others. Endolymphatic duct is indicated with a blue color, the semicircular ducts with yellow, the utricle and saccule with red and the cochlea with an orange color. The white line shows the plane of the section through the posterior duct shown in D-F. The black dots indicate Netrin1 expression after RNA in situ hybridization in D-F. Scale bars: 60 µm in A-C; 100 µm in D-F.

 


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  		Fig. 5. Variability in Apaf1 and caspase 9 inner ear phenotype. Cross-sections through the inner ear at E16.5 (A-C) and E13.5 (D-F). The images in B and C show sections from the two ears from the same Apaf1 mutant embryo with no brain phenotype. The lack of superior semicircular duct is indicated with an asterisk in C. The section in E comes from a caspase 9 mutant embryo with a mild brain phenotype, and in F from an embryo with a severe brain phenotype. Scale bar: 100 µm. cc, crus commune.

 


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  		Fig. 6. Dorso-ventral determination of the otic epithelium. Radioactive in situ hybridization analysis on cross-sections through E11.5 (A,B,E,F) and E12.5 (C,D,G) embryos. The embryos were treated with Dlx5 (A,B), Pax2 (C,D) and Netrin1 (E,F) antisense probes and with Netrin1 sense probe (G). Scale bar: 100 µm.

 


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  		Fig. 7. Cell proliferation in Apaf1 and Bcl2l mutant otic epithelium. Proliferating cells were detected with an anti-BrdU antibody staining of the developing inner ear at E11.5 (A-C), at E10.5 (D-E) and E12.5 (F-H) in wild-type (A,D,F,H), Apaf1 (B,G) and Bcl2l (E) mutant otic epithelium. Control staining with secondary antibody only (C). Scale bar: 100 µm.

 


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  		Fig. 8. Analysis of the inner ear in Apaf1-/-/Bcl2l-/- double mutant embryos. Many TUNEL-positive cells could be observed in the otic epithelium of Bcl2l-/- embryos (A) at E11.5. No apoptosis could be observed in the Apaf1-/-/Bcl2l-/- double mutant otic epithelium (B). In the wild-type embryo, the superior semicircular duct has already formed (C), whereas more immature morphogenesis can be observed in the Apaf1-/-/Bcl2l-/- double mutant otic epithelium (D-E). The arrows in B and D point to the closure defect in the double mutant otic vesicles. In situ hybridization analysis shows Dlx5 (F) and Netrin1 (G) expression in the double mutant superior semicircular duct outpocketing. Scale bar: 100 µm.

 

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© The Company of Biologists Ltd 2004