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First published online January 23, 2009
doi: 10.1242/10.1242/dev.016204

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Early mouse caudal development relies on crosstalk between retinoic acid, Shh and Fgf signalling pathways

Vanessa Ribes^*,, Isabelle Le Roux^*,, Muriel Rhinn^*, Brigitte Schuhbaur and Pascal Dollé

¹IGBMC (Institut de Génétique et de Biologie Moléculaire et Cellulaire), BP 10142, Illkirch, F-67400 France. ²Inserm, U 964, Illkirch, F-67400 France. ³CNRS, UMR 7104, Illkirch, F-67400 France. ⁴Université Louis Pasteur, Faculté de Médecine, Strasbourg, F-67000 France.

View larger version (112K): [in this window] [in a new window]   Fig. 1. Dynamic RA signalling in primitive streak, node and caudal neural plate. Whole-mount detection of Raldh2 (A-B',E,E') and Cyp26a1 (G,G') transcripts in wild-type embryos, and lacZ mRNA (C-D',F,F') in RARE-lacZ embryos, at E7.5 (A-D') and E7.75 (E-G'). A,C,E',F',G', caudal views; E,F,G, lateral views; A',C', insets in E',F', details of the node region (broken lines or asterisks); B,D, transverse sections of the embryos shown in A,C, at the level of the medial region of the primitive streak (ps); B',D', high-power magnifications of the primitive streak (boxed in B,D); inset in G', transverse section of the embryo through the mid-primitive streak. al, allantois; ed, endoderm; ep, epiblast; hf, headfold; ms, mesoderm; ps, primitive streak.

View larger version (113K): [in this window] [in a new window]   Fig. 2. RA is required to restrict the expression of primitive streak markers. Whole-mount in situ hybridization of follistatin (A-D''), brachyury (E-H'') and Wnt3a (I-J'') in control and Raldh2-/- embryos (genotypes indicated above) at E7.75 (A-B'',E-F''I,J, insets) and three- to four-somite stages (C-D'',G-J''). Main panels, posterior views; insets, lateral views. Transverse sections at the level of the node ('') and caudal primitive streak region (') are also shown (approximate section levels indicated in main panels). Brackets show lateral expansion of follistatin (C,D) and brachyury (G,H) in mutants. Arrowheads and arrows (E'-F'') indicate the lateral border of brachyury expression in epiblast and mesoderm, respectively. hb, hindbrain; nc, notochord; no (or asterisks), node; ps, primitive streak; so, somites.

View larger version (119K): [in this window] [in a new window]   Fig. 3. RA deficiency affects expression of caudal neural plate markers. (A-H) Whole-mount in situ hybridization of Sox2 (A,B), Wnt8a (C,D), Cdx1 (E,F), Hoxb9 (G,H) and Hoxb8 (G,H, insets) in control and Raldh2-/- embryos (genotypes indicated above) at the three- to four-(A-F) and five- to six-(G,H) somite stages. Asterisks indicate the node. Brackets (C-F) indicate a lack of Wnt8a and Cdx1 expression in lateral regions of mutants. Owing to overall shortening of the trunk, the Hoxb8 domain appears shorter in the mutant neural tube (nt).

View larger version (94K): [in this window] [in a new window]   Fig. 4. Impaired neurogenesis in Raldh2-/- embryos. (A-P) Whole-mount in situ hybridization of Wnt3a (A,B), Msx1 (C,D), Sox10 (E,F, main panels), Sox9 (E,F, insets), Delta1 (G-J), Hes5 (K-N) and Ngn2 (O,P). (Q,R) Anti-neurofilament immunolabelling on transverse brachial spinal cord sections. Genotypes are as indicated. Developmental stages are four- to five-somite stage (G,H,K,L), 13-15 somites (C-F,I,J,M,N, insets in O,P) and E9.5 (A,B,O,P,Q,R). drg, dorsal root ganglion; mn, motoneurons.

View larger version (91K): [in this window] [in a new window]   Fig. 5. Decreased Shh signalling in Raldh2-/- embryos. Immunodetection of Shh on whole-mount embryos (A-B') and on transverse cryosections of the brachial spinal cord (C-F). Whole-mount in situ hybridization of Shh (G-J) and Gli1 (K-N''). G-J,M',M'',N',N'' are vibratome transverse sections of the brachial spinal cord (G-J,M'',N'') and hindbrain (M',N'). Genotypes are indicated above. Developmental stages are E7.75 (A-B',K-L'), 6 somites (M,N), 11-12 somites (M'-N''), 14-15 somites (C,E,G,I) and E9.5 (D,F,H,J). Brackets indicate Gli1 expression in newly formed mesoderm (K,L) and lateral epiblast (K',L'). Asterisks indicate the node. nc, notochord; fp, floor plate.

View larger version (147K): [in this window] [in a new window]   Fig. 6. Exogenous Shh-N does not rescue Gli1 expression in Raldh2-/- embryos, even at supra-physiological concentrations. (A-P) Whole-mount in situ hybridization of Gli1 on E7.5 embryos cultured for 14 hours in medium containing no drug (A-B'), and 5 nM (C-D'), 50 nM (E-F') or 200 nM (G-H') Shh-N, and embryos cultured for 14 hours in medium containing 0.001% ethanol (vehicle, I,J, insets in M,N), and 200 nM AT-RA (K,L), 10 nM AT-RA (M,N), or 10 nM AT-RA and 50 nM Shh-N (O,P). Genotypes are indicated above.

View larger version (89K): [in this window] [in a new window]   Fig. 7. Altered Fgf signalling in Raldh2-/- embryos. (A-J) Whole-mount in situ hybridization of sprouty 2 (A-F) and Mkp3 (G-J) in wild-type and Raldh2-/- embryos (genotypes indicated above) at E7.75 (A,B), 4-5 somites (C,D,G,H) and E9.5 (E,F,I,J). Asterisks indicate the node. Brackets in C,D indicate expression in condensing somites as a landmark, and in E,F,I,J indicate regions with abnormally low expression. lb, limb bud; s, somites. (K-R) Whole-mount Gli1 in situ hybridization on embryos (genotypes indicated above) collected at E7.5 and cultured for 14 h (K-N) or 6 h (O-R) in medium containing 0.1% DMSO (vehicle, K,L,O,P) and 50 µM SU5402 (M,N,Q,R).

View larger version (111K): [in this window] [in a new window]   Fig. 8. Retinoic acid negatively regulates Gli2 expression, independently of Fgf signalling. (A-D) Whole-mount in situ hybridization of Gli2 in control and Raldh2-/- embryos at E7.75 (A,B, main panels, anterior views; insets, posterior views) and 13- to 14-somite stages (C,D). (E-L) Gli2 in situ hybridization on embryos cultured for 6 hours (E-J) or 14 hours (K,L) in medium containing 0.001% ethanol (E,G), 0.1% DMSO (I,K), 200 nM AT-RA (F,H) or 50 µM SU5402 (J,L). Genotypes indicated above.

View larger version (18K): [in this window] [in a new window]   Fig. 9. Summary of retinoic acid-dependent events during body axis extension and caudal development. Schematic overview of the abnormal caudal phenotype of early somite-stage Raldh2-/- embryos (A) and of the main regulatory interactions underlying these defects (B). This summarizes regulatory loops occurring at pre- and early somite stages, characterized by previous work on avian models (see Diez del Corral and Storey, 2004; Olivera-Martinez and Storey, 2007) and supported (or uncovered) in our mouse model (broken lines).

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