Fig. 4. Requirement of RA for induction of Hoxb1 and vHnf1, and for repression of Irx3. Anterior is towards the right (A-D,G-J,N-O) or towards the top (E-F,K-M). (A-C) Hoxb1 expression normally exhibits an anterior extension into the hindbrain, but this extension is eliminated in Raldh2^–/– embryos. Arrowheads indicate the anterior extent of Hoxb1 expression, which is at the level of the node in a wild-type embryo at E7.5 (A), and anterior to the node in the hindbrain at E7.75 (B) and E8.0 (C). The anterior expansion of Hoxb1 expression into the hindbrain is eliminated in an Raldh2^–/– embryo (C). (D) Double in situ hybridization at E8.0, showing a lateral view of vHnf1 expression posteriorly in the hindbrain and Cyp26a1 expression anteriorly. The Raldh2^–/– embryo lacks vHnf1 expression, but Cyp26a1 is not affected by a loss of RA. (E,F) A dorsal view of vHnf1 expression at E8.25, showing a complete loss of vHnf1 mRNA in an Raldh2^–/– embryo. (G,H) vHnf1 expression at E8.5 is normally present in the anterior spinal cord, as well as the posterior hindbrain up to the otic vesicle (ov), but an Raldh2^–/– embryo lacks vHnf1 mRNA in these neural tissues. (I,J) Neural expression of Hoxb1 at E8.5 is normally limited to r4 in the hindbrain and to the posterior spinal cord, but an Raldh2^–/– embryo lacks the r4 expression domain and instead exhibits a weak expression domain shifted to the most posterior region of the hindbrain (asterisk). (K,L) Double-staining for expression of Cyp26c1 (anterior) and Hoxb1 (posterior) at E8.25 demonstrates that both overlap in r4 of wild-type embryo, but that the Raldh2^–/– embryo lacks expression of both in the region where r4 should have developed (arrowhead). The brackets indicate a region where the Raldh2^–/– embryo retains expression of Hoxb1 in the most posterior region of the hindbrain (asterisk), whereas this domain of Hoxb1 expression is normally lost by E8.25. (M) Double staining for expression of Irx3 and Raldh2 in E7.9 wild-type and Raldh2^–/– embryos demonstrates the normal posterior border of Irx3 expression at r4 separated from the Raldh2 domain, whereas the mutant exhibits a loss of Raldh2 expression and an expansion of Irx3 expression into the posterior hindbrain (bracket and asterisk). (N,O) Irx3 expression at E8.25 is normally present in the anterior hindbrain with a posterior limit at r4 (arrowhead), but an Raldh2^–/– embryo exhibits a posterior expansion of Irx3 expression to the hindbrain/spinal cord junction (bracket and asterisk).