Establishment of rhombomere 1: Isthmic gene expression is refractory to Hoxa2 overexpression (A) At stage [st.] 6, Otx2 and Hoxa2 expression (both in blue) are separated by a Gbx2-positve domain (red) that defines rhombomere 1 (r1). The domain of Gbx2 expression encompasses r1–3. (B) At st.9-, the boundaries of the Otx2 and Hoxa2 expression domains define the segmental boundaries of r1 as determined by fate-map experiments [2, 17]. (C) Viral overexpression of RCASBP(B)Hoxa2 (red) at embryonic day (E) 0 fails to suppress the activation of either FGF8 (blue) or (D) en1 at E3 (e3). Scale bar (A,B) = 200 μm. mhb – midbrain/hindbrain isthmus.