Fig. 4. (A) At stage 20, invagination of the ectoderm was obtained (red arrowhead) in the DD2-misexpressed limb bud. (B) Expression of Fgf8 was observed in the invaginated ectoderm (red arrowhead). (C) Cyclin D1 was expressed normally in the underlying mesenchyme. A red arrowhead indicates the invaginated epithelium. (D) In the normal limb bud, ßIII-tubulin proteins were detected by the TuJ1 antibody in both the ventral ectoderm and the motoneurons entering the limb. (E) In the DD2-misexpressed limb bud, ßIII-tubulin proteins were detected in the invaginated epithelium, suggesting that the invaginated ectoderm was derived from the ventral half. (F,G) The boxed shown in E was magnified. Clear TuJ1 staining was observed in the invaginated part (white arrowheads). In its distal-most end, thickening of the ectoderm was evident (blue arrowheads). EGFP proteins were visualized in green. (H) Fgf8 was expressed in this thick ectoderm (blue arrowhead). (I) In a ventral view, expression of Fgf8 was broad and flat. (J,K) At stage 24, two protrusions were formed with strong expression of Shh (J) and Gremlin (K) in the dorsal and the ventral protrusions, respectively. (L,M) When developed further, polydactylous limbs arose. Digits of such limbs were arranged in two rows, with digits a, b and c in one line, and digits a' and b' in another. A distal view was shown in M. (N,O) Skeletal patterns obtained by Alcian Blue staining. Lateral and posterior views are shown in N and O, respectively. (P) Schematic representation of the digit pattern in a dorsal view. Approximately 22% of the DD2-misexpressed limb buds displayed this phenotype (n=27).