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First published online 18 February 2004
doi: 10.1242/dev.01017

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Bapx1 regulates patterning in the middle ear: altered regulatory role in the transition from the proximal jaw during vertebrate evolution

Abigail S. Tucker^1,*, Robert P. Watson^2,*, Laura A. Lettice², Gen Yamada³ and Robert E. Hill^2,

¹ Department of Craniofacial Development and Orthodontics, Kings College London, Floor 28, Guy's Tower, Guy's Hospital, London SE1 9RT, UK
² MRC Human Genetics Unit, Western General Hospital, Crewe Rd, Edinburgh EH4 2XU, UK
³ Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan

View larger version (88K): [in a new window]   Fig. 1. Analysis of the middle ear ossicles in E18.5 Bapx1-/- mice. Alizarin Red and Alcian Blue staining of dissected middle ears from E18.5 wild-type (A) and Bapx1-/- (B) embryos. Development of the middle ear bones appears to be largely unaffected by loss of Bapx1 expression. The stapes and incus develop normally, as do the joints between the three middle ear ossicles (inserts in A and B, and data not shown). Although elements of the malleus, such as the processus brevis and the manubrium appear normal, the body of this ossicle is significantly narrower in mice lacking Bapx1 (B,C). The length of the malleus is unaffected in Bapx1-/- mice (B,D). Measurements of malleal width and length were taken between the T-bars indicated in A. I, incus; M, malleus; MM, manubrium of the malleus; PB, processus brevis.

View larger version (90K): [in a new window]   Fig. 2. Analysis of the structural elements of the middle ear in E18.5 Bapx1-/- mice. Alizarin Red and Alcian Blue staining of dissected middle ears from E18.5 wild-type (A,B), heterozygous (C,D) and homozygous mutant (E,F) embryos. (A,C,E) Lateral view. (B,D,F) Frontal view. At E18.5, the tympanic ring and gonium are evident as discrete ossified elements located below Meckel's cartilage and adjacent to the developing middle ear ossicles (A,B). Embryos heterozygous for the Bapx1 mutation have no overt defects associated with the tympanic ring but do display variable hypoplasia of the gonium (C,D). All homozygous-null embryos lack a gonium and display hypoplasia of the anterior part of the tympanic ring (E,F). Arrowheads in B-D indicate the extremities of the gonium. G, gonium; M, malleus; Me, Meckel's cartilage; Ty, tympanic ring.

View larger version (128K): [in a new window]   Fig. 3. Analysis of malleal/gonial bone fusion in Bapx1+/- neonatal mice. Alizarin Red staining of dissected middle ears from P2 wild-type (A,B) and Bapx1 heterozygous (C,D) mice. In Bapx1+/- neonatal mice, hypoplasia of the gonium resulting from the loss of one copy of the Bapx1 gene is still evident (black arrowheads). Despite this, these mice show signs of fusion between the gonium and the malleus (white arrowheads in inserts indicate extremities of fused region). In all adult heterozygous mice examined to date, the malleus has been found to be fused to the tympanic bone (data not shown). M, malleus; Ty, tympanic ring.

View larger version (131K): [in a new window]   Fig. 4. Expression of Bapx1 in the mouse middle ear. (A) Expression of Bapx1 during middle ear development can first be detected at E10.5, with expression of the gene in a discreet domain in the first branchial arch (blue arrowhead). (B) By E12.5, Bapx1 expression can be detected in the region of the condensing middle ear ossicles. (C,D) Closer inspection of this expression pattern reveals Bapx1 expression around Meckel's cartilage (defined in C by expression of -collagen) and in condensations that correspond to the condensing tympanic ring (yellow arrowhead) and gonium (black arrowhead). (E-P) Analysis of Bapx1 expression during late embryogenesis through sagittal sections stained with either Alcian Blue and chlorantine Fast Red (E,G,I,K,M,O) or by 35S radioactive in situ hybridisation (F,H,J,L,N,P). (F,H,J,L) Dark-field photographs, Bapx1 expression shown by white silver grains. (N,P) Bright-field photos, Bapx1 expression shown by black grains. (E-J) E15.5, (K,L) E14.5, (M-P) E17.5. (E,F) Expression of Bapx1 both within and between (arrow) the malleus and incus. (G,H) Expression surrounding the malleus and incus in the incudo-malleal joint region. (I,J) Expression in the developing gonium (arrow) and around Meckel's cartilage, but not within the differentiating tympanic ring. (K,L) Expression under the EAM (arrow). (M,N) Expression around the developing gonium (arrow) and tympanic. (O,P) Expression in the incudo-malleal joint (arrow). Me, Meckel's cartilage; M, malleus; I, incus; S, stapes; Ty, tympanic ring; G, gonium; EAM, external auditory (acoustic) meatus.

View larger version (117K): [in a new window]   Fig. 5. Analysis of the relationship between Bapx1 and Gsc in murine middle ear development. (A-C) Whole-mount in situ hybridisation analysis of Bapx1 (A), Gsc (B) and Bapx1/Gsc (C) expression in E10.5 branchial arches. At E10.5, Bapx1 is expressed in two discreet domains within the mandibular component of the first branchial arch, including both the neural crest-derived mesenchyme (white arrowhead) and a small region of the branchial arch epithelium (yellow arrowhead). Gsc expression is restricted to the neural crest derived mesenchyme in the caudal half of the first branchial arch. (D) Schematic representation of the two expression domains highlighting the area of overlap in the caudoproximal region of the mandibular component of the first branchial arch. (E-N) In situ hybridisation analysis of Bapx1 expression in wild-type and Gsc-/- E15.5 embryos. (E-G,J-L) Sagittal sections through the middle ear region of an E15.5 mouse embryo. Stained with Alcian Blue and chlorantine Fast Red (E,J), and by 35S radioactive in situ for Bapx1 (F,K) and Gsc (G,L). Bapx1 is expressed around and within the developing malleus (depending on the level of section within this ossicle) and coincides with Gsc expression in the anterior part of the tympanic ring (yellow arrows) (F,G). Bapx1 expression overlaps Gsc in the caudal mesenchyme above the developing EAM (red arrows) (K,L). High levels of Bapx1 are observed within and around the gonial (F), where only very weak expression of Gsc is observed (G). Unlike Gsc, which is expressed throughout the tympanic ring (yellow arrows in G,L), Bapx1 is expressed only around the anterior part of the tympanic (F,K). (H,I,M,N) Expression in Gsc-/-. The expression pattern of Bapx1 is maintained around and in the gonium and where the tympanic ring would normally form (H,I). Expression is also maintained under the malleus (red arrow), despite failure of the EAM to form correctly (M,N). (O-R) Results of the reciprocal experiment in which Gsc expression was analysed in E10.5 (O,P) and E15.5 (Q,R) Bapx1-/- embryos. Expression of Gsc is unaffected by the loss of Bapx1 expression within the first branchial arch. At E15.5 expression of Gsc is seen around Meckel's cartilage (Me) and in the pharyngeal cleft between the first and second branchial arches (arrowhead). EAM, external auditory meatus; G, gonium; M, malleus; Me, Meckel's cartilage; Ty, tympanic ring.

View larger version (53K): [in a new window]   Fig. 6. Comparison of membranous bone ossification in the middle ear and jaw joint. Alcian Blue and Alizarin Red stained skeletal preparations. (A-C) Development of the cartilages and bones of the murine middle ear, side view. (D-F).Development of the cartilages and bones of the chick jaw joint, dorsal view. (A) Formation of the malleus and incus at E14. The malleus develops at the proximal end of Meckel's cartilage. There is no bone ossification at this stage. (B) Ossification of the tympanic ring at the base of the malleus at E16. (C) Ossification of the gonium in between the malleus and tympanic ring at E18.5. (D) Formation of the jaw joint between the articular and the quadrate at E7. The articular lies at the proximal end of Meckel's cartilage. (E) Ossification of the angular under the articular and Meckel's at E9. (F) Ossification of the prearticular next to the angular at E13. I, incus; M, malleus; Me, Meckel's cartilage; Ty, tympanic ring; G, gonium; A, articular; Q, quadrate; An, angular; P, prearticular.

View larger version (90K): [in a new window]   Fig. 7. Expression of Bapx1 in the chick jaw joint. (A) Section stained with Haematoxylin/Eosin. (C,E,H) Sections stained with Alcian Blue and chlorantine Fast Red. (B,D,F,I) 35S radioactive in situ hybridisation for Bapx1. (G) 35S radioactive in situ hybridisation for Runx2. (A,B,E-I) Frontal sections. (C,D) Sagittal sections. (A,B) E7. (C-G) E9. (H,I) E12. (A,B) Expression of Bapx1 in the quadrate and articular cartilages of the jaw and in between in the developing jaw joint (arrow). (C,D) By E9, expression of Bapx1 is downregulated in the cartilages of the jaw but remains high in the jaw joint itself (arrow). (E) Frontal view showing the developing membranous bones associated with the articular. The presumptive prearticular is marked by an asterisk. (F) Expression of Bapx1 coinciding with the region of the presumptive prearticular and around the angular and surangular bones surrounding the jaw joint. (G) Expression of the bone marker Runx2 within the developing membranous bones (angular, surangular and prearticular). (H,I) Expression of Bapx1 around the membranous bones surrounding the articular at E12 but not within these bones. A, articular; Q, quadrate; An, angular; S, surangular, P, prearticular.

View larger version (131K): [in a new window]   Fig. 8. Analysis of malleal/incal joint formation in the murine middle ear. (A-C) 35S radioactive in situ for Nkx3.1. (D) Section stained with Alcian Blue and chlorantine Fast Red. (E,I) 35S radioactive in situ for Bapx1. (F) 35S radioactive in situ for Eya1. (G,J) 35S radioactive in situ for Gdf5. (H,K) 35S radioactive in situ for Gdf6. (A-C,G-K) Transverse sections E14.5. (D-F) Sagittal sections E14.5. (A-C,J,K) Bapx1-/- embryos. (A-C) Contrary to expectations, Bapx1-/- mice display no malleal/incal joint phenotype raising the possibility of functional compensation by a related gene. 35S radioactive in situ for Nkx3.1 reveals that expression is unaffected in the Bapx1 mutant head (A), with no upregulation seen in the middle ear (C). Expression of Eya1, a gene that, when inactivated, gives rise to malleal/incal joint defects, is expressed around the developing malleus and incus (F) but, in contrast to Bapx1 (E), is excluded from the region between the two ossicles. (G-I) Expression of Gdf5 and Gdf6 overlap with that of Bapx1 in the incudo-malleal joint but expression of both genes is unaffected in the Bapx1 mutant (J,K).