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First published online December 20, 2005
doi: 10.1242/10.1242/dev.02204

Development 133, 351-361 (2006)
Published by The Company of Biologists 2006
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Retinaldehyde dehydrogenase 2 (RALDH2)-mediated retinoic acid synthesis regulates early mouse embryonic forebrain development by controlling FGF and sonic hedgehog signaling

Vanessa Ribes1,*, Zengxin Wang2,*, Pascal Dollé1,{dagger} and Karen Niederreither2,{dagger}

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS, INSERM, Université Louis Pasteur, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France.
2 Departments of Medicine and Molecular and Cellular Biology, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA.



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  		Fig. 1. RALDH2 mediates RA signaling at early stages of embryonic craniofacial development. (A-L) RALDH2 protein distribution (A,E,I), ß-galactosidase activity of the RARE-hsp68-lacZ transgene (B,C,F,G,J,K) and Raldh3 mRNA (D,H,L) were analyzed at successive stages (as indicated on the left) in wild-type and Raldh2-/- embryos (as indicated above). (A'-K') Transverse sections at levels indicated (black lines) in the main panels. Insets show stage-matched embryos of the alternate genotype. anr, anterior neural ridge; m, mesenchyme; ov, optic vesicle; se, surface ectoderm; te, telencephalon.

 


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  		Fig. 2. Defective forebrain and optic development in Raldh2-/- embryos. (A,A') Serial frontal sections through the forebrain neuroepithelium of a headfold (10 somite)-stage wild-type embryo at the level of the optic evagination (ov) and prospective telencephalon (te). (B,B') Comparative sections of a stage-matched Raldh2-/- embryo. Brackets indicate regionally disorganized neuroepithelium. There is an excess of overlying mesenchymal cells (ms). (C,D) Comparative frontal sections at an early stage of optic vesicle (ov) development (14- to 15-somite stages). Arrowheads indicate the presence of cell debris in the Raldh2-/- forebrain lumen. (E,F) Sections at the anterior forebrain level, showing defective telencephalic vesicle outgrowth and excess mesenchymal cells (in the mutant, F). (G-J) Serial transverse sections through the telencephalic (te), diencephalic (di) and optic (ov) vesicles of E9.5 wild-type (G,I) and Raldh2-/- (H,J) embryos. Insets show details of the mesencephalic (me) neuroepithelium (G,H) and of the distal tip of the optic vesicle and surface ectoderm (se) (I,J). (K-M) Details of the infundibulum (in) and Rathke's pouch (rp) in a control (K) and two Raldh2-/- (L,M) embryos. pp, prechordal plate. Hematoxylin and Eosin staining.

 


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  		Fig. 3. Region-specific apoptosis and defective cell proliferation in the RA-deficient forebrain. (A,B) Immunofluorescent TUNEL detection of apoptotic cells on transverse forebrain sections of 14- to 15-somite stage wild-type (A) and Raldh2-/- (B) embryos. Overlays of the DAPI nuclear staining (blue) and TUNEL signal (red) are shown in false colors. (C,D) Whole-mount TUNEL analysis of wild type (C) and mutant (D) E9.5 embryos. Frontal views of the head. (E,F) Immunofluorescent detection of phospho-histone H3 on transverse forebrain sections of 14- to 15-somite stage embryos (false color overlays as in A,B). (G) Numbers of phospho-histone H3-positive cells as percentages of total cell numbers along the whole ventral forebrain neuroepithelium (fb) and three subregions, the rostroventral diencephalon (di), optic vesicles (ov) and ventral telencephalon (te) of 14-somite stage wild-type and Raldh2-/- embryos. Four embryos of each genotype were sectioned transversely, and all sections encompassing the aforementioned regions were counted (fb: wild type, 8%±1.3; mut, 5.1%±1.1; mean±s.e.m.; t-test P=0.0147; di: wild type, 6.5%±1.7; mut, 3.3%±0.9; P=0.0158; ov: wild type, 8.9%±1.6; mut, 5.4%±1.3; P=0.0131; te: wild type, 8.0%±2.0; mut, 6.3%±1.5; P=0.219). (H-M) Whole-mount in situ hybridization of p21 (H), cyclin D2 (J,K) and cyclin D3 (L,M) in wild-type and Raldh2-/- embryos. H,I: ventral views of E9.5 heads; J-M: profile views of 13- to 14-somite stage embryos. Insets show ventral (J,K) and frontal (L,M) views of the forebrain. ma, maxillary process; me, mesencephalon.

 


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  		Fig. 4. Abnormal distribution of facial neural crest cells in Raldh2-/- embryos. Whole-mount in situ hybridization of Crabp1 (A,B), Ap2a (C,D), Sox10 (E,F) and Sox9 (G,H). Profile views. Embryos are at 12-14 somites (A-F) or E9.5 (G,H, and insets in C,D). Genotypes are indicated above. Brackets (A,B) indicate a deficit of labeled cells in the post-optic area, and arrows (D,F) indicate an excess of labeled cells in the preoptic (frontonasal) region of mutant embryos. ov, optic vesicle. Insets in A,B show transverse sections of 13- to 14-somite stage embryos.

 


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  		Fig. 5. Molecular analysis of forebrain development in Raldh2-/- embryos. Whole-mount in situ hybridization of Foxg1 (A,B: 13-14 somites; C,D: E9.5), Emx2 (E,F:11-12 somites; G,H: E9.5), Nkx2.1 (I,J: 11-12 somites; K,L: E9.5), Delta1 (M,N: 13-14 somites) and Hes5 (O,P: 15-16 somites). Embryo genotypes are indicated above. Main panels are profile views, except in C,G,H (frontal views) and D,K,L (ventral views of the forebrain). Insets show profile views of the same embryos (C,D,G,H,K,L), or additional embryos at the 14- to 15-somite stage (E,F,I,J) or E9.5 (M,N). di, diencephalon; hb, hindbrain; mb, midbrain; te, telencephalon.

 


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  		Fig. 6. Molecular analysis of the optic vesicle and infundibulum in Raldh2-/- embryos. (A,B) Whole-mount immunodetection of PAX6 protein in 10- to 11-somite stage (main panels) and 20- to 22-somite stage (insets) embryos. Profile views. Brackets indicate PAX6 expression in the dorsal telencephalon and diencephalon. (C-D') PAX6 immunohistochemistry on frontal sections through the optic vesicle (C,D) and telencephalon (C',D') of wild-type and Raldh2-/- embryos (13-14 somites). (E-L) Whole-mount in situ hybridization of Six3 (E,F), Pax2 (G,H), Hesx1 (I,J) and Bmp4 (K,L) in 12- to 14-somite stage embryos. Genotypes are indicated above. Profile views. Insets in E,F show frontal views of Six6-hybridized embryos (brackets indicate infundibulum). Other insets show details of the optic vesicle of E9.5 embryos (G,H) or ventral views of the same embryos (I-L). ov, optic vesicle; te, telencephalon.

 


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  		Fig. 7. Molecular analysis of Fgf gene expression and signaling in Raldh2-/- embryos. (A-D) Whole-mount Fgf8 in situ hybridization on 13- to 14-somite stage (A,B) and E9.5 (C,D) embryos. Main panels: frontal views of the ANR. Insets show profile views (A,B) or transverse sections (C,D) of the same embryos. (E,F) Whole-mount p-ERK1/2 immunodetection (main panels, E9.5) and Mkp3 in situ hybridization (insets, 12- to 14-somite stage). Profile views. (G,H) Whole-mount Spry1 in situ hybridization on 12- to 14-somite stage embryos (main panels, profile views; insets, frontal views). Genotypes are indicated above. anr, anterior neural ridge; se, surface ectoderm.

 


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  		Fig. 8. Molecular analysis of SHH signaling in Raldh2-/- embryos. Whole-mount in situ hybridization with Shh (A-D), Ptch1 (E,F), Gli1 (G,H), Gli3 (I,J) and Olig2 (K,L) probes. Embryos are at E7.75 (A,B), E9.5 (C,D) and 12-15 somites (E-L). Profile views, except in A,B (ventral views). Insets show transverse sections of the infundibulum (red brackets, C,D), ventral views of the same embryos (G,H) or details of the Olig2-positive region in older (E9.5) embryos (K,L). Genotypes are indicated above. ov, optic vesicle; pp, prechordal plate; te, telencephalon.

 

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© The Company of Biologists Ltd 2006