Figure 3

CG-1A is localized to developing chicken leg bud precartilage condensations. (a) Staining by in situ hybridization for CG-1A mRNA in the core of a 5-day leg bud with the distal-most cells underlying the AER unstained (red arrowhead). Inset shows control 5-day leg bud with probe omitted. In a 6-day leg (b), digits stain for CG-1A mRNA (black arrowheads) but interdigits are unstained (red arrowhead) (c) Indirect immunostaining for CG-1A protein in the 3½-day leg bud stylopod is predominantly extracellular (high-magnification view of the field enclosed by the blue box). Flank cells are sparsely stained (yellow box). (Scale bar: 50 μm). (d) In a 5-day leg section there is staining for CG-1A in the stylopod perichondrium (white arrowhead) but not its core (red arrowhead). In the zeugopod, the two intensely stained primordia (blue box) are separated by a region of unstained cells (yellow box). The developing autopodium is unstained except for a crescent representing the fourth digit condensation (blue arrowhead). Inset shows leg section with primary antibody omitted. (e) The peristylopod mesenchyme in a 6-day leg section stains for CG-1A, but not the core (red arrowhead). Fusiform cells in the intensely stained mid-leg were confirmed in companion sections to be myoblasts. Strong CG-1A staining is seen in the perichondria and leading edges of the third and fourth digits (yellow box), and in the ultimately regressing "fifth" digit condensation (violet box). Interdigit cells as well as those subjacent to the ectoderm stain sparsely (blue box). The fourth and fifth digits are marked as d4 and d5. (f) 5-day wing stylopod, showing intense CG-1A staining in the humeral crescent. Inset shows a comparable region (at lower magnification) of a control wing with primary antibody omitted. (Scale bar (e): 1 mm). Pull-out images were photographed using a 63× objective (Scale bar: 50 μm).