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doi: 10.1242/10.1242/dev.00463

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The regional pattern of retinoic acid synthesis by RALDH2 is essential for the development of posterior pharyngeal arches and the enteric nervous system

¹ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP/Collège de France, BP 10142, 67404 Illkirch Cedex, CU de Strasbourg, France
² Departments of Medicine and Molecular and Cellular Biology, Center for Cardiovascular Development, Baylor College of Medicine, One Baylor Plaza, Houston, Texas 77030, USA

View larger version (96K): [in a new window]   Fig. 1. Thymus and parathyroid defects in RA-rescued Raldh2–/– mutants. Coronal histological sections (stained with tetrachrome) of an E18.5 wild-type fetus (WT; A,D) and two Raldh2–/– fetuses (B,E and C,F) that were rescued by maternal RA supplementation from E7.5 to E12.5. (A) A representative section of the wild-type thymus gland. (D) Sections of the left and right thyroid lobes and the adjacent parathyroid glands. (B) No thymus was seen in the mediastinum of the first mutant (note the presence of a persistent truncus arteriosus). (C) An ectopic cervical thymus rudiment was found in the second animal. (E-F) This rudiment was connected to the thyroid tissue via a pouch-like structure (F, inset). No parathyroid glands were seen in the mutants. At, atrium; PTA, persistent truncus arteriosus; Pth, parathyroid; Th, thymus; Tr, trachea; Ty, thyroid.

View larger version (90K): [in a new window]   Fig. 3. Altered gene expression in the posterior pharyngeal region of RA-rescued Raldh2–/– embryos. (A-D) Whole-mount in situ analysis of Hoxb1 transcripts in E9 (22 somites) wild-type (A,C) and Raldh2–/– (B,D) embryos. Profile views of the whole embryos (A,B) and enlargement of the posterior foregut region after medial bisection (C,D, inside views). (E,F) In situ hybridization analysis of Hoxa1 transcripts in the foregut area of E9 wild-type and Raldh2–/– embryos, respectively (viewed as in C,D). (G-J) In situ hybridization analysis of Fgf8 transcripts in E9.5 wild-type (G,I,) and Raldh2–/– (H,J) embryos. External profile views (G,H) show Fgf8 ectodermal labeling, whereas internal views after bisection (I,J) reveal the endodermal labeling. Insets in (I,J) are coronal views which make it possible to distinguish the ectodermal and endormal labeling. (K,L) In situ hybridization analysis of Pax9 expression in the foregut region of E9.5 (main panels) and E10.5 (insets: detail of the pharyngeal region caudally to the 2nd arch) wild-type and Raldh2–/– embryos, respectively. b1-b3, branchial arches; ec, ectoderm; en, endoderm; fg, foregut; me, mesoderm; p1-p3, pharyngeal pouches; r4, rhombomere 4; tb, tail bud.

View larger version (83K): [in a new window]   Fig. 2. (A-F) Defects in aortic arch formation in RA-rescued Raldh2–/– embryos. Embryos collected at E9.5 (A,B), E10.5 (C,D) or E11.5 (E,F) were incubated in phosphate-buffered saline and injected intracardially with china ink. Compare the aortic arch patterns in wild-type (A,C,E) and Raldh2–/– (B,D,F) embryos (see text for details). The inset in F shows a detail of a different Raldh2–/– embryo. Arrowheads point to the single embryonic vessel connecting the aortic sac and the dorsal aorta. (G-I) Analysis of Raldh2 expression pattern in the wild-type embryonic pharyngeal region. Whole-mount in situ hybridization of an antisense Raldh2 riboprobe was performed on E8.5 (10 somites, G) and E8.75 (16 somites, H,I) wild-type embryos. The arrowhead in G points to Raldh2 expression boundary in the posterior foregut region. (I) A higher magnification of the branchial arch region, viewed from the inside after bisection of the embryo. (J-L) Patterns of RA response in the branchial arch region of E8.5 wild-type (J) and Raldh2–/– (K,L) embryos carrying a RARE-hsp68-lacZ reporter transgene. Embryos are shown after whole-mount X-gal assay and medial bisection of their head and trunk region (inside views). a2-a6, aortic arches; as, aortic sac; b1-b3, branchial arches; da, dorsal aorta; en, endoderm; ey, eye; fg, foregut; me, mesoderm; nt, neural tube; so, somites.

View larger version (100K): [in a new window]   Fig. 4. Post-otic neural crest cell alterations in RA-rescued Raldh2–/– embryos. (A-D) Whole-mount in situ analysis of EphA4 (A,B) and EphA2 (C,D) transcripts in E9.5 wild-type (A,C) and Raldh2–/– (B,D) embryos. In (D), EphA2-labeled cells are confined dorsally to the foregut pocket (arrowhead). E-I, Distribution of Crabp1 transcripts in E9.5 wild-type (E,H) and Raldh2–/– (F,G,I) embryos. (E-G) Profile and (H,I) dorsal views. Brackets in F,G delineate the mutant post-otic NCC populations (which normally would colonize the 3rd, 4th and 6th branchial arches). An asterisk in G indicates cells that are abnormally confined along the dorsal foregut wall, and may correspond in part to pre-otic (r4-derived) NCC. Mutant embryos also exhibit abnormal patterns of connectivity between the post-otic hindbrain and NCCs (arrowheads and bracket in H and I, respectively). b1-b6, branchial arches; fg, foregut; fl, forelimb bud; ot, otocyst; p1-p3, pharyngeal pouches; r3-r6, rhombomeres.

View larger version (75K): [in a new window]   Fig. 5. Growth and patterning defects of posterior cranial nerves in RA-rescued Raldh2–/– mutants. Whole-mount anti-neurofilament stainings of E11.5 embryos were examined after removal of the surface ectoderm to expose the nerve tracts. (A) wild-type embryo. (B-D) Raldh2–/– embryos with increasingly severe phenotypic defects. Cranial nerves are indicated in Arabic numerals (5-12). g9-g10, distal sensory ganglia of the 9th and 10th nerves, respectively. The arrowhead in D points to an aborted 9th-11th common axonal tract.

View larger version (77K): [in a new window]   Fig. 6. Rhombomere and cranial ganglion patterning in RA-rescued Raldh2–/– embryos. (A-D) Hoxa2 and kreisler rhombomeric transcript patterns were analyzed in E8.5 wild-type (A,C) and Raldh2–/– (B,D) embryos (dorsal views). (E,F) Analysis of Ret transcripts, which mark the prospective cranial ganglia NCCs in E9.5 wild-type embryos (E), reveals a selective deficiency of the 9th and 10th ganglion cells in mutant embryos (F, a bracket indicates the few post-otic Ret-labeled cells). ot, otocyst; r2-r6, rhombomeres; 7-8, 9, 10, prospective cranial nerve ganglia.

View larger version (102K): [in a new window]   Fig. 7. Lack of enteric nervous system development in RA-rescued Raldh2–/– mutants. In situ hybridization was performed with a 35S-labeled Ret probe on cryosections of wild-type (A,C,E,G,I) and Raldh2–/– (B,D,F,H,J) embryos. Each panel shows bright-field (left) and dark-field (right) views of the same section to show, respectively, the histology and the in situ hybridization signal (white dots). At E10.5, mutant embryos only show scattered Ret-labeled prospective ganglioblasts (arrows in B), which failed to colonize the midgut levels (compare A and B). E12.5 mutant embryos have rudimentary vagal nerve tracts (compare C and D, arrows) and their stomach and gut wall are devoid of Ret-positive ganglion cells (compare E,G and F,H). Other domains of Ret expression (e.g. in the trigeminal ganglia: compare I and J) are unaffected in mutants. fg, foregut; g5, trigeminal ganglion; mg, midgut; sc, spinal cord; st, stomach; ur, ureteric bud.