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First published online 16 January 2008
doi: 10.1242/dev.012708

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Recombinase-mediated cassette exchange reveals the selective use of G_q/G₁₁-dependent and -independent endothelin 1/endothelin type A receptor signaling in pharyngeal arch development

¹ Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
² Tsukuba Safety Assessment Laboratories, Banyu Pharmaceutical Company Limited, 3 Okubo, Tsukuba, Ibaraki 300-2611, Japan.
³ Department of Developmental Medical Technology (Sankyo), Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
⁴ Institute of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, 69120, Heidelberg, Germany.

View larger version (32K): [in this window] [in a new window]   Fig. 1. RMCE in the mouse Ednra gene. (A) Strategy for targeting and knock-in of lacZ. Probes for genotyping are indicated as 5'- and 3'-probes. B, BamHI; Bl, BlnI; C, ClaI; P, PstI; Pm, PmeI. (B) Southern blot analysis of PstI- or BlnI/PmeI-digested genomic DNA of the offspring from an intercross of heterozygotes, probed with the 5'- or 3'-probes, respectively. (C) Genomic PCR analysis for sequential Cre-lox-mediated recombination with primers indicated in A.

View larger version (72K): [in this window] [in a new window]   Fig. 2. RMCE-mediated knock-in of lacZ into the Ednra locus. (A,B) Whole-mount staining of E8.25 (six-somite stage) (A) and E8.5 (nine-somite stage) (B) EdnralacZ2FRT/+ mouse embryos shows lacZ expression in the head mesenchyme at the hindbrain level (arrows). (C) An E8.5 transverse section at the rostral hindbrain level shows lacZ expression in mesenchyme throughout the head and pharyngeal arch regions, but not in the neuroepithelium, surface ectoderm and foregut endoderm. (D) High magnification of the boxed area in C demonstrates lacZ expression in migratory neural crest cells delaminating from the dorsal neuroepithelium (arrows). (E) At E9.0 (17-somite stage), lacZ is expressed in the head and pharyngeal arch regions, in the heart, and in the ventral half of the trunk. (F) An E9.0 sagittal section in the pharyngeal region demonstrates lacZ expression in neural crest-derived ectomesenchyme and in the head mesenchyme adjacent to the neuroepithelium, but not in the pharyngeal epithelium, vascular endothelium and many cells in the core mesenchyme (asterisks). (G) The E9.0 cardiac outflow region demonstrates lacZ expression in cardiac neural crest cells surrounding the second and third arch arteries and colonizing between the foregut and the aortic sac (arrow). (H-J) Whole-mount in situ hybridization of E9.0 wild-type embryos using Ednra (H), Crabp1 (I) and Snail1 (J) probes. (K-M) Comparison between Ednra-lacZ expression (K) and in situ hybridization patterns of Crabp1 (L) and Snail1 (M) in E9.0 transverse sections at the first pharyngeal arch level. White arrowheads, migratory neural crest cells; arrows, mesoderm-derived mesenchyme. Scale bars: 100 µm in C,F,G,K-M; 50 µm in D. aa2/3, second and third arch arteries; as, aortic sac; fb, forebrain; fg, foregut; hb, hindbrain; ht, heart; hv, head vein; mb, midbrain; ne, neuroepithelium; nt, neural tube; op, optic vesicle; ot, otic vesicle; pa1/2/3, first, second and third pharyngeal arches; pp2, second pharyngeal pouch.

View larger version (100K): [in this window] [in a new window]   Fig. 3. LacZ expression in the head mesenchyme and neural-crest derivatives in Ednra lacZ2FRT/+ mouse embryos. (A-C) Transverse sections at E10.0. LacZ expression is observed throughout the head mesenchyme adjacent to the neural tube and in the peripheral pharyngeal arch mesenchyme (A). Trigeminal ganglia are highly populated with lacZ-positive cells (B). LacZ expression is detectable within the lateral wall of the aortic sac and pharyngeal arch arteries (C). (D-H) Transverse sections at the levels of the central (D-F) and distal (G,H) regions of the lower jaw at E12.5. E,F,H are high magnification images of the boxed areas in D and G. LacZ expression is observed in mesenchyme underlying the oral epithelium in the lower and upper jaws (D,G). In the molar (E) and incisor (H) buds, lacZ expression is present in mesenchyme surrounding the dental lamina. LacZ expression is undetectable in the precartilage primordium contributing to the rod portion of Meckel's cartilage, whereas surrounding mesenchymal cells express lacZ (F). In the primordium of the rostral process of Meckel's cartilage, lacZ is highly expressed (H). Scale bars: 100 µm in A-C; 500 µm in D,G; 50 µm in E,F,H. aa2, second arch artery; as, aortic sac; da, dorsal aorta; dl, dental lamina; dm, dental mesenchyme; fg, foregut; gV, trigeminal ganglion; lj, lower jaw; nt, neural tube; pa1/2, first and second pharyngeal arches; pc, precartilage primordium of Meckel's cartilage; to, tongue; uj, upper jaw.

View larger version (60K): [in this window] [in a new window]   Fig. 4. Craniofacial defects of Ednra-null mice. Facial appearances (A,B) and skeletal structures (C-O) of E18.5 wild-type (A,C,E,I,J,L,N) and Ednraneo/neo (B,D,F-H,K,M,O) mice are shown. (A,B) The mutant lower jaw (arrow in B), in contrast to the normal one (A), appears a mirror image of the upper jaw with duplicated arrays of vibrissae. (C,D) Lateral and caudal views demonstrate defected and transformed mandibular (ventral) structures and malformed hyoid fused to the basisphenoid in mutants (D). (E,F) Mandibular elements including the dentary, goniale and ectotympanic (E) are mostly lost and replaced by ectopic structures resembling the maxilla, jugal and palatine, whereas the lower incisor with surrounding alveolar bone was formed in mutants (F). The zygomatic process of squamosal is often lost in mutants (arrowhead in F). (G-I) The ectopic bone in the mutant lower jaw (G) appears a duplication of the ipsilateral maxilla (H), which is almost identical to the wild-type one (I), except for the distal incisive alveolus. Broken lines in H and I indicate the margin articulating to the premaxilla. (J,K) A set of the palatine, pterygoid, ala temporalis and lamina obturans, the upper jaw elements in normal mice (J), are duplicated and connected to the ectopic maxilla (mx*) through palatomaxillary-like articulation in mutants (K). (L,M) In mutants, the malleus and incus are malformed, giving an ectopic structure extending to the squamosal (arrowhead in M). The stapes has nearly normal appearance, but is connected to the hyoid and displaced out of the fenestra ovalis (M). The styloid process was truncated in mutants (M). (N,O) Unlike the normal hyoid (N), the mutant hyoid is fused bilaterally to the basisphenoid (arrowhead in O) and connected to the stapes through an aberrant cartilageous strut (O). amx, alveolus of maxilla; at, ala temporalis; bh, body of hyoid; bs, basisphenoid; btp, basitrabecular process; dnt, dentary; etm, ectotympanic; fmx, frontal process of maxilla; fovl, fenestra ovalis; ghh, greater horn of hyoid; gn, goniale; hy, hyoid; in, incus; ina, incisive alveolus of dentary; iof, infraorbital foramen; jg, jugal; lhh, lesser horn of hyoid; LI, lower incisor; lo, lamina obturans; ma, malleus; mx, maxilla; pl, palatine; pmx, premaxilla, ppmx, palatal process of maxilla; ptg, pterygoid; sp, styloid process; sq, squamosal; st, stapes; zpmx, zygomatic process of maxilla; *, ectopic structure.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Phenotypes of Ednra and Ednrb knock-in mice. (A) RMCE-mediated knock-in of Ednra and Ednrb cDNA into the Ednra locus. Partial structures of the wild-type and knocked-in Ednra allele and RT-PCR analysis for the expression of knocked-in genes in mandibular arches of E9.5 Ednra- and Ednrb-knock-in heterozygous embryos are shown. PCR primers were represented by arrowheads in the left panel. Note that 753- and 659-bp bands correspond to knocked-in and endogenous Ednra transcripts, respectively. (B-H) Facial appearances (B,C) and skeletal structures (D-H) of E18.5 EdnralacZ/A (Ednra-knock-in) (B,D) and EdnralacZ/B (Ednrb-knock-in) (C,E-H) mice. Ednra knock-in restored normal facial appearance (B) and skeletal structures (D). Ednrb knock-in mice still exhibit craniofacial defects resembling the Ednra-null phenotype (C,E), except for some differences described below. White arrowhead, ectopic cartilage connecting the incus and extended basitrabecular process. (F) Comparison of the morphology of incisive alveolar bones (boxed with dotted line) among the Ednra-null (Ednraneo/neo), Ednrb-knock-in (EdnralacZ/B) and wild-type (Ednra+/+) mandible. Incisors were removed. The EdnralacZ/B incisive alveolus is well developed, comparable with the wild-type one. (G) A representative of the EdnraB/B hyoid, which is, unlike the Ednra-null hyoid, not fused to the basisphenoid. The body has an extended ossification center and is connected to the stapes though a transformed lesser horn. The great horn is fused with the superior horn of the thyroid (arrow). (H) Ectopic cartilage (arrowhead) between the malleal/incal region and ala temporalis in EdnralacZ/B mice. at, ala temporalis; bh, body of hyoid; bs, basisphenoid; dnt, dentary; fovl, fenestra ovalis; ghh, greater horn of hyoid; hy, hyoid; in, incus; jg, jugal; lhh, lesser horn of hyoid; LI, lower incisor; ma, malleus; mx, maxilla; pmx, premaxilla, sp, styloid process; st, stapes; zpmx, zygomatic process of maxilla; *, ectopic structure.

View larger version (67K): [in this window] [in a new window]   Fig. 6. Apoptosis analysis. TUNEL staining of E10.5 transverse sections through the mandibular arch reveals that TUNEL-positive cells (arrows) are confined to the proximal region in wild-type mouse embryos (A), but extend widely into the distal arch region in Ednraneo/neo (B) and Ednraneo/B (C) embryos. Scale bars: 100 µm. pa1, first pharyngeal (mandibular) arch.

View larger version (112K): [in this window] [in a new window]   Fig. 7. Gene expression analysis of the pharyngeal arches in Ednrb-knock-in embryos. (A,B) Whole-mount lacZ staining of E10.5 EdnralacZ/+ (A) and EdnralacZ/B (B) mouse embryos. (C-L) Whole-mount in situ hybridization for Dlx3 (C,D), Dlx5 (E,F), Dlx6 (G,H), Hand2 (I,J) and Goosecoid (K,L) in wild-type (C,E,G,I,K) and EdnraB/B (D,F,H,J,L) embryos at E9.5 (C-H), E10.0 (K,L) or E10.5 (I,J). The expression of Dlx3, Dlx5, Dlx6, Hand2 and Goosecoid was downregulated in EdnraB/B arches.

View larger version (75K): [in this window] [in a new window]   Fig. 8. Craniofacial defects of neural-crest-specific Gq/G11-deficient mice. (A-E) Skeletal structures of E18.5 P0-Cre-/-;G flox/floxq;G11 -/- (control) (A,C), P0-Cre-/+;G flox/floxq;G -/-11 (Gq/G11-deficient) (B,D) and EdnralacZ/B (E) mice. Gq/G11-deficient mice have a duplicated set of the maxilla, jugal, palatine, pterygoid and lamina obturans, and relatively well-developed incisive alveolus in the distal mandibular region (arrowheads), as EdnralacZ/B mice. (F-H) Transformed mandibular components (ectopic maxilla, jugal and palatine bones) of Gq/G11-deficient (F), EdnralacZ/B (G) and Ednraneo/neo (H) mice. (I,J) Hyoid and thyroid cartilages of control (I) and Gq/G11-deficient (J) mice. Unlike Ednra-null hyoid, the Gq/G11-deficient hyoid is not fused to the basisphenoid. Instead, the body has an extended ossification center and is fused with the lesser horn of the hyoid and the superior horn of the thyroid (arrow). bh, body of hyoid; bs, basisphenoid; btp, basitrabecular process; dnt, dentary; etm, ectotympanic; ghh, greater horn of hyoid; hy, hyoid; ina, incisive alveolus of dentary; jg, jugal; lhh, lesser horn of hyoid; lo, lamina obturans; mx, maxilla; pl, palatine; pmx, premaxilla; ptg, pterygoid; sht, superior horn of the thyroid; sq, squamosal; *, ectopic structure.

View larger version (27K): [in this window] [in a new window]   Fig. 9. Scheme illustrating the possible role of Gq/G11-dependent and -independent Edn1/Ednra signaling in pharyngeal arch development.