Fig. 1. Boundaries of Cyp26a1, vHnf1 and Irx3 expression in the early mouse hindbrain. Anterior is oriented towards the top in all panels, and all embryos are wild type (WT). (A-E) Cyp26a1 mRNA at E7.75-E8.0 is localized anteriorly with its posterior extent to presumptive rhombomere 2 (r2) of the hindbrain, as determined by whole-mount in situ hybridization. Shown is an E7.75 embryo stained for expression of Cyp26a1 (A), and an E8.0 embryo double-stained for expression of Cyp26a1 and Krox20, a known marker for r3 (B). Also shown is an E7.75 embryo double-stained for expression of Cyp26a1 and Hoxb1, a known marker for r4 (C); neither gene is expressed in r3. Double-staining for Cyp26a1 and Epha2 (a marker for presumptive r4 and the adjacent mesoderm) at E7.75 (D) and E7.9 (E) demonstrates a space between the two expression domains where presumptive r3 lies; expression of Epha2 in the mesoderm adjacent to r4 overlaps the lateral regions of presumptive r3 from this viewpoint. (F,G) Expression of vHnf1 is localized posteriorly with its anterior border at the r4/r5 boundary Shown are embryos double-stained for expression of vHnf1 and Krox20 at E8.0 when Krox20 is expressed only in r3 (F) and at E8.25 when Krox20 is expressed strongly in r3 and weakly in r5 (G). There is a gap between Krox20 and vHnf1 expression at both stages, indicating that vHnf1 expression extends to r5 but not into r4. (H) Double staining for Epha2 and vHnf1 shows that vHnf1 is expressed in r5 up to the r4/r5 boundary. (I) Expression of Irx3 at E8.25 is localized anteriorly with a posterior border at the r4/r5 boundary when compared with the expression pattern of vHnf1 (G). (J) Double staining for Irx3 and vHnf1 expression at E8.0 provides evidence that these two expression domains meet at r4/r5.