Figure 8. Ectopic lenses cannot induce Wnt2b or collagen IX gene expression in optic cup tissue. A: A whole mount posterior view of Hamburger-Hamilton (HH) stage 23 eye at the initiation of the experiment. Note that the endogenous lens is out of view. Lenses were placed under the retinal pigment epithelium (RPE) in the posterior of the eye. L1 indicates the position of a HH stage 15 implanted lens, L2 indicates the position of a HH stage 24 implanted lens and an arrow indicates the optic nerve. B: A section through the eye in A after 48 h in culture, and after in situ hybridization for collagen IX gene expression. The arrows indicate endogenous expression around the lens in the anterior of the eye and implanted lens are demarcated with dashes and labeled as in A. No collagen IX gene expression is seen around implanted lenses. The RPE tissue appears black and is broken at ectopic lens insertion points. C-E: Immuno-histochemistry for δ-crystallin and collagen IX expression on sections adjacent to those in B. Implanted lenses are demarcated with dots, and are positive for δ-crystallin (green signal). C: CollagenIX staining (red signal) is found in the endogenous anterior optic cup lip (arrowhead, indicates same position as arrowhead in D) and no signal is found associated with the ectopic lens, L2. D: CollagenIX expression (red signal, single and double arrowheads) is found in the anterior optic cup, in association with the native lens. E: CollagenIX protein (donor derived) is found associated with the HH stage 15 lens (asterisk, red signal), but is not found in host optic cup tissue. F: Wnt2b gene expression is shown in sections adjacent to those in B. The implanted lens L2 and the native lens have slight expression in lens ectoderm (double arrowheads). A strong Wnt2b signal is seen in the optic cup lip (arrow) but is not found in associated with implanted lens L2. Thickened areas of RPE are artifacts of growth in culture. G: Slight Wnt2b expression is found in the lens ectoderm of implanted lens L1 (asterisk), but is not found in the host optic tissue. Scale bars in B, F, and G are equal to 200 µm, and in C, D, and E are equal to 100 µm.