Fig. 8. Role of shifting RA boundaries during posterior hindbrain segmentation. A model for RA action during mouse hindbrain development is shown. Our findings demonstrate that RA generated by Raldh2 in the paraxial mesoderm travels anteriorly to presumptive r3 and r4 during establishment of Hoxb1 expression, but that this is very transient. Initially, RA forms an early anterior boundary at r2/r3 (next to the r2 border of Cyp26a1 expression) followed soon after by a late anterior boundary at r4/r5 (next to the r4 border of Cyp26c1 expression). RA is therefore present in r3/r4/r5 to directly regulate Hoxb1 induction and repression through previously described 3' and 5' RAREs (Marshall et al., 1994; Studer et al., 1994). We also demonstrate that vHnf1 requires RA for posterior hindbrain expression, and that vHnf1 is needed to limit the posterior extent of Hoxb1 expression to help establish the r4/r5 expression boundary for Hoxb1. Thus, RA acts directly to induce Hoxb1 expression and then RA acts both directly and indirectly (through induction of vHnf1) to restrict Hoxb1 expression to r4. Also shown is a mutual repression between Irx3 and vHnf1 at the r4/r5 boundary, which has been demonstrated in zebrafish (Lecaudey et al., 2004) and is supported by our findings in mouse.