Fig. 3. RA generated by Raldh2 is required for Cyp26c1 expression in r4. Anterior is oriented towards the right in all panels except B, where anterior is towards the top. (A,B) Lateral (A) and dorsal (B) views of E7.75 Raldh2^–/– (–/–) and wild-type (WT) embryos double stained for expression of RARE-lacZ (ß-galactosidase activity) observed posteriorly followed by staining for Cyp26c1 mRNA (whole-mount in situ hybridization) observed anteriorly. The Raldh2^–/– embryo lacks RARE-lacZ expression, but Cyp26c1 expression at this stage (head mesoderm) is not affected. (C-E) Cyp26c1 expression in dorsal view of wild-type embryos at E7.75 (C), E7.9 (D) and E8.0 (E) showing its initial expression in r4, with the remaining expression occurring in head mesoderm. (F-I) Dorsal view of double staining for expression of Raldh2 (posterior) and Cyp26c1 (anterior) in Raldh2^–/– and wild-type embryos at E8.25 (F-G) and E8.5 (H-I). Raldh2^–/– embryos (marked by a large reduction in Raldh2 mRNA) lack the r4 domain of Cyp26c1 expression while retaining the r2 expression domain normally observed by E8.5. Raldh2^–/– embryos also exhibit an abnormal posterior extension of Cyp26c1 expression in head mesoderm at E8.5 marked by an asterisk (I). The anterior extent of Raldh2 mRNA in wild-type embryonic mesoderm is somite 1 (s1), which is adjacent to rhombomere 8 (r8) at E8.25 (F).