Fig. 1. The anterior region of the Six3-null mouse brain progressively caudalizes during development. (A-G') At the 15- to 17-somite stage, (A) Irx1 is expressed in the mesencephalon, pretectum and thalamus of the wild-type brain, (B) Lhx5 in the prethalamus and the telencephalon, (C) Arx in the prethalamus and dorsal telencephalon, (D) Fezf1 and (E) Fezf2 in the ventral prethalamus and telencephalon, (F) Nkx2.1 in prethalamus and hypothalamus, and Six6 (G) in the Rathke's pouch (arrow) and anterior hypothalamus. (H-K') At the 15- to 22-somite stages, Irx3 is normally expressed in the mesencephalon, pretectum and thalamus (H,I). At the 19- to 22-somite stages, Tcf4 is normally expressed in the pretectum, thalamus (bracket) and ventral prethalamus (arrow) (J,K). At these somitic stages, the anterior-most part of the Six3-null brain has not fully posteriorized, as indicated by the anteriorly expanded Irx1 expression (A', arrowhead) and lack thereof in the dorsal brain (arrow), the presence of Lhx5 (B') and the absence of Irx3 and Tcf4 expression (H',J', brackets). Reduced Nkx2.1 expression (F') and the absence of Six6 (G') confirmed the absence or reduction of the hypothalamus in the Six3-null brain. Residual Six6 expression in surface ectoderm at the 15-somite stage suggested the presence of the Rathke's pouch region (arrow); however, no such structure is present at later stages. Later, the alar plate of the Six3-null brain gradually caudalizes, and at the 22- to 23-somite stage, the expression of Irx3 and Tcf4 (I',K', brackets) is anteriorly expanded. (L-N') At the 17-somite stage, an abnormally large number of TUNEL⁺ cells were seen in the anterior region of the Six3^-/- brain (L'), as compared with wild type (L). Similar results were observed when the TUNEL assay was performed on sections of wild-type (M) and Six3^-/- (M') brains. The plane of the tissue sections shown in M,M' is indicated by a line in L,L'. A drastic reduction in the number of apoptotic cells was observed in the anterior region of the Six3-null brain between the 17- and 21-somite stages (M',N'). (N) Bar chart showing the number of TUNEL⁺ cells per 100 DAPI⁺ cells at the 17-somite stage; white bar, wild-type; black bar, Six3-null. Arrows in B,C,D indicate the dorsal limit of expression. Anterior is to the right.