Fig. 1. High levels of Hes1 expression in boundaries. (A) Whole-mount in situ hybridization at E10.5. Hes1 mRNA is expressed at high levels in the ZLI (left arrow) and the isthmus (right arrow). Left and right lines indicate sections shown in (H-O) and (B,C), respectively. (B) Immunohistochemistry for Hes1. Hes1 protein is expressed at high levels in the isthmus (boxed) and the floor plate (asterisk). (C) A higher magnification of a boxed region in B. Inset in C shows Hes1 protein expression in the isthmus at E9.5. (D-G) Serial parasagittal sections of the isthmic region at E11.5 (boxed in A). Wnt1-expressing cells (D, arrow) also express Hes1 (E, arrow) at high levels in the isthmus. These cells do not efficiently take up BrdU (F,G, arrows), compared with the surrounding region. (H-K) Hes1 protein is expressed at high levels in the ZLI (H, asterisk). Cells expressing Hes1 (I, bracket) also express Shh (J,K, brackets) in the ZLI. Inset in J shows a lower magnification of in situ hybridization for Shh. (L-O) Hes1 protein (L) and Hes1 mRNA (M) are highly expressed in the ZLI (asterisks). Cells in the ZLI display lower BrdU uptake (N,O, asterisks). (P) Flat-mount in situ hybridization of Hes1 at E9.5. The midbrain-hindbrain region was cut along the dorsal midline. Hes1 mRNA is expressed in the interrhombomeric boundaries. Arrowheads indicate the isthmus. Top is rostral. (Q-V) Frontal section of the hindbrain at E9.5. Cells at interrhombomeric boundaries express Hes1 protein at high levels. They are mostly negative for phosphorylated histone 3 (pH3) (U,V, arrows), indicating that interrhombomeric boundary cells do not actively proliferate. The boxed region in Q is enlarged in the insets in Q-S. PI, propidium iodide. Scale bars: 500 µm in O; 200 µm in V; 100 µm in C,G; 50 µm in K.