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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.

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