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Embryonic retinoic acid synthesis is required for forelimb growth and anteroposterior patterning in the mouse

Karen Niederreither*,{dagger}, Julien Vermot{dagger}, Brigitte Schuhbaur, Pierre Chambon and Pascal Dollé{ddagger}

Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France
* Present address: Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA
{dagger} These authors contributed equally to this work



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  		Fig. 1. Morphology of the Raldh2–/– forelimbs. (A-C) Scanning electron micrographs of wild-type (A) and Raldh2–/– (B,C) embryos collected at E13.5 after RA supplementation by oral gavage from E6.75 to E9.25. (C) A close-up of the left mutant forelimb. (D) Raldh2–/– fetus collected at E18.5 after RA supplementation via the maternal food supply (100 µg/g food) from E7.5 to 8.5. An enlargement of the forelimb rudiment is shown (inset). (E) Raldh2–/– fetus collected at E18.5 after RA supplementation via maternal food (100 µg/g food from E7.5 to 8.5 and 250 µg/g food from E8.5 to E14.5). The left forelimb shows mirror-image digit duplications and the right forelimb has an abnormal digital cleft (arrowhead). (F,G) Abnormal digit patterning in two Raldh2–/– fetuses collected at E18.5 after the same conditions of supplementation. The arrowhead in G points to the enlarged first digit. hl, hindlimb; fl, forelimb.

 


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  		Fig. 2. Skeletal defects of the Raldh2–/– forelimbs. (A) comparative view of the right forelimbs of control (left) and Raldh2–/– embryos after four different conditions of RA supplementation (experimental group numbers as in Table 1). Note the progressive improvement of limb size and skeletal patterning according to the experimental group. (B) Skeletal pattern of a wild-type forelimb. (C-G) Examples of the Raldh2–/– abnormal forelimb patterns. The embryos shown were from experimental groups 1 (C), 2 (D), 4 (E) and 5 (F,G). See Text for a full description of the skeletal patterns. In F and G, an arrowhead points the abnormally long and triphalangeal first digit (compare with B). h, humerus; r, radius; s, scapula; u, ulna; 1 and 5: digit number.

 


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  		Fig. 3. (A-L) Analysis of the patterns of embryonic RA response during maternal RA supplementation. Embryos carrying the RARE-hsp68-lacZ RA reporter transgene, collected from RA-supplemented or untreated mothers, were concomitantly X-gal stained. (A-D) Profile views of E9.5 embryos. (E-I) Dorsal views of the forelimb bud region of E9.5 embryos. Brackets indicate the outgrowing forelimb buds. (J,K) Dorsal views of the forelimbs of E10.5 embryos. (L) Transverse section of an E10.5 embryo at the level of the hindlimb buds. WT, wild-type embryos; –/–, Raldh2–/– embryos; –RA, untreated embryos. The embryos in B-D and F-I were RA-treated from E7.5 to E8.5 with 100 µg/g food and from E8.5-E9.5 with 100 µg/g food or 250 µg/g food, as indicated in the panels (100 and 250, respectively). The embryo in K was RA-treated until E10.5, and the one in L until E9.5 (250 µg/g food). (M-P) Detection of RARß transcripts in E9.5 wild-type (M,N) and Raldh2–/– (O,P) embryos that were untreated (M,P) or RA-treated from E7.5 to E9.5 (250 µg/g food) (N,O). Arrowheads point to the similar rostral expression boundaries in the hindbrain of untreated and RA-treated wild-type embryos. Profile views. (Q,R) RARß transcript distribution in the forelimbs of E10.5 wild-type (Q) and Raldh2–/– (R) embryos that received RA from E7.5 to E10.5 (250 µg/g food). Dorsal views. fl, forelimb buds; hl, hindlimb buds; mn, mesonephros; sc, spinal cord; so, somite.

 


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  		Fig. 4. Abnormal patterns of Fgf expression in the Raldh2–/– forelimbs. (A) Comparative view of E9.5 wild-type (left) and Raldh2–/– embryos treated with RA from E7.5 to E8.5. Note comparable expression levels in various expression domains, except in forelimb ectoderm. br, branchial arches; flb, forelimb buds; mhb, mid-hindbrain region; tb, tail bud. (B,C) Details of Fgf8 expression in the forelimb ectoderm of E9.5 wild-type (B) and Raldh2–/– (C) embryos after RA supplementation from E7.5-8.5. (D) Fgf8 expression pattern in an E10.5 wild-type forelimb AER. (E-H) Altered patterns of Fgf8 expression and AER morphology in E10.5 Raldh2–/– forelimbs, after RA treatment from E7.5 to E9.5 (250 µg/g). (I-K) Fgf10 expression in the distal mesoderm of E10.5 wild-type (I) and Raldh2–/– (J,K) forelimbs. Whereas Fgf10 is expressed throughout the distal mesoderm in (I), a preferential anterior distribution is seen in (J) and an abnormal anterior tissue outgrowth with specific Fgf10 expression is found in (K). (L-N) Fgf4 expression in wild-type (L) and Raldh2–/– (M,N) E10.5 forelimb buds. Fgf4 is expressed in the posterior two-thirds of the AER in wild type. No expression is detectable in the bud in M, whereas the one in N shows an ectopic patch of expression in the anterior part of the AER.

 


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  		Fig. 5. Altered expression patterns of Shh, Bmp2 and Gli3 in Raldh2–/– forelimbs. (A-C) Shh transcripts in E9.5 wild-type (A) and Raldh2–/– (B,C) forelimb buds after short-term (E7.5 to E8.5) RA supplementation. (D-F) Shh transcripts in E10 wild-type (D) and Raldh2–/– (E,F) forelimb buds after long-term (E7.5 to E9.5) RA supplementation. (G-I) Bmp2 transcripts in E10.5 wild-type (G) and Raldh2–/– (H,I) forelimb buds after short-term (H) and long-term (I) RA supplementation, respectively. (J-M) Gli3 transcripts in E9.5 wild-type (J) and Raldh2–/– (K-M) forelimb buds analyzed at E9.5 after short-term (J-L) and long-term (M) RA supplementation, respectively.

 


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  		Fig. 6. Expression of dHand in Raldh2–/– forelimbs. (A-C) Wild-type (A) and Raldh2–/– (B,C) forelimb buds analyzed at E10.5 after short-term and long-term RA supplementation, respectively. Note the lack of posterior asymmetry of dHand expression in mutant forelimbs. (D-G) Early expression pattern of dHand in wild-type (D) and Raldh2–/– (E-G) forelimb buds analyzed at E9-9.5 after short-term (D,E,G) and long-term (F) RA supplementation. Although dHand transcripts are posteriorly restricted during early forelimb outgrowth in mutants (brackets), only the anteroproximal forelimb margin remains free of dHand transcripts during further limb growth (arrowheads).

 


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  		Fig. 7. Meis2, Sprouty4, CRABP I, Wnt7a and En1 transcript distributions in wild-type (A,C,E,H,M) and Raldh2–/– (B,D,F,G,I,J,K,L,N) limb buds. All embryos are at E10-E10.5, except in M,N. (A-D) Dorsal views of the forelimb buds of Meis2-hybridized embryos. (E-G) Ventral views of the forelimb buds of E10.5 Sprouty4-hybridized embryos. (H-J) Profile views of CRABP I-hybridized embryos. (K,L) Ventral views of Wnt7a-hybridized embryos, where brackets indicate patches of ectopically expressing ventral cells. (M,N) Profile views of E9.5 En1-hybridized embryos, with insets showing En1 expression in E10.5 forelimb buds. dm, dermomyotome.

 


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  		Fig. 8. Altered distributions of Hoxd11 (A-E) and Hoxd12 (F-J) transcripts in the Raldh2–/– forelimbs. In mutants, deregulated expression along the forelimb AP axis is seen prior to the formation of the autopod (A,B,F,G: E9.5). Later on (C-E,H-J: E11.5), both the autopodal (arrowheads) and non-autopodal (arrows) expression domains extend ectopically along the anterior margin of the mutant limbs.

 




© The Company of Biologists Ltd 2002