Time-course of Lrrn2 expression. Chick embryos from HH4–11 following in situ hybridisation for Lrrn2 and Hoxb1. All views are dorsal side up, anterior to the top. (A) HH4, (B) HH5, (C) HH7. Note asymmetric expression of Lrrn2 on the right side of the node (arrows in (A-C)). (D) HH8, (E) HH 9, (F) HH11. Expression of Lrrn2 starts to localise to the r4 region at HH8 and by HH9 is clearly distinguishable (red arrowheads in (D, E)). Strong staining is also seen in the prospective diencephalon-midbrain region (di/mb) (D). At HH11, r4 expression within the neuroepithelium can be seen (red arrowheads), along with expression in the adjacent mesoderm (black arrowheads in (F)). (G-K) Double in situ hybridisation with Lrrn2 (blue) and Hoxb1 (red). (G) HH6, (H) HH7; arrows indicate anterior boundary of Hoxb1 expression (future r3/4 boundary). (I) HH8-: the region between the double, black arrows highlights overlap in expression, corresponding to presumptive r4. (J) HH8+: high power view of hindbrain region with fluorescent image of Hoxb1 expression where the r3/4 boundary is clear, despite some quenching due to the NBT/BCIP staining of Lrrn2, and (K) brightfield image showing the same. The anterior boundary of the Lrrn2 stripe (bracket) coincides with the anterior boundary of Hoxb1, indicating that Lrrn2 is expressed in early r4. Scale bars: 100 μm.