Fig. 2. Pbx1 expression and skeletal structures in wild-type and mutant embryos. (A-D) Immunohistochemical analysis of Pbx1b localization visualized with DAB (brown staining) on tissues counter-stained with Hematoxylin. Diffuse nuclear Pbx1b expression is present in the epidermis and mesenchymal tissues of the embryo at E13.5 (A), most intensely in mesenchyme that is condensing to form rib (black arrowheads) and vertebral (red arrowheads) cartilage (B). Abundant Pbx1b expression in proliferating rib chondrocytes at E14.5 (C), which is nuclear (C, inset), is reduced to undetectable levels in hypertrophic chondrocytes at E16 when ossification is imminent (D). BA2, second branchial arch; vert, vertebrae. (E,F) Differential bone and cartilage staining of skeletal elements. E16 embryos were stained with Alcian Blue and Alizarin Red to visualize cartilage and bone, respectively, in the forming skeletal structures. Lateral views demonstrate agenesis of the ventral ribs, hypoplasia of the sternum and clavicles, together with rib malformations and fusions (red arrowhead) in the Pbx1-/- embryo (E, right). There is also thinning, compressions (black arrows) and fusions (red-tipped arrows) of the vertebral bodies and neural arches in the Pbx1-/- embryo (F, bottom). The styloid process and hyoid lesser horns (green arrows) are malformed, as are the structures of the occipital arch. Costal cartilage, Cc; clavicle, cv; exoccipital, EO; otic capsule, OC; ossified rib, orb; supraoccipital arch, SOA; sternum, stn. Asterisks indicate malformed structures.
