Fig. 8. Selective mesodermal changes consistent with disturbed AER function in T^-/- limb buds. Potential changes in limb mesoderm due to altered AER function were evaluated in T^-/- embryos (right sides) compared to wild type (wt, left sides). Proliferation was assessed by immunostaining multiple transverse sections with the mitosis-specific anti-phospho-histone H3 (representative sections shown). Similar numbers of mitotic cells were present in wild-type and T^-/- limb buds at both E9.5 (not shown) and at E9.75 (A). Apoptosis was evaluated by TUNEL assay at E9.5 (not shown) and E9.75 (B) using fluorescein-UTP (green) and propidium iodide nuclear counterstaining (red) and also by whole-mount Nile Blue sulfate staining at E10 (not shown). At all stages, levels of apoptosis in T^-/- limb buds were minimal and similar to wild type. Lmx1b and Shh were checked to evaluate overall limb `maturity' and level of AER signaling. Lmx1b is expressed uniformly at early stages and dorsally later. Shh activation depends on AER signals. While Lmx1b expression was preserved and showed nornal dorsal restriction in T^-/- limb buds at E10 (C), Shh was not expressed at all in T^-/- forelimb bud by E10 (D), but was detected at levels similar to wild type in hindgut (data not shown). d, dorsal; v, ventral; a, anterior; p, posterior.