Fig. 2. Determination of the En2-Cre expression domain using the R26R reporter and restricted ablation of Notch1 signaling. (A) Heterozygous Floxed Notch1 (
/wt) animals carrying an En2-Cre and R26R allele showed a restricted distribution of Cre-expressing cells (blue staining) and their progeny in the midbrain-hindbrain region of the developing brain at E10. The plane of the sections used in the subsequent analysis is depicted in red. (B) Homozygous Floxed Notch1 (/) littermates carrying an En2-Cre and R26R allele showed a similar distribution of cells expressing ß-galactosidase (blue) in the midbrain-hindbrain region of the brain to control embryos at E10. (C) In situ RNA hybridization on cross-sections through the neural tube, dorsal to the left and ventral to the right, of E10 mutant animals (/) showed normal expression of Notch1 mRNA in the ventral region of the hindbrain neural tube but loss of expression in the medial cerebellar primordium (arrow). (D) Control Floxed Notch1 (lox/lox) animals without an En2-Cre transgene showed normal distribution of Notch1 transcripts throughout the dorsal and ventral region of the hindbrain neural tube including the cerebellar primordium (arrow). (E) The downregulation of Notch1 expression in the medial portion of the cerebellar primordium of the mutants coincides with the region of recombination as indicated by expression of ß-galactosidase from the R26R allele on adjacent sections (F). (G) Hes5 expression was strongly reduced in the medial cerebellar primordium of mutant embryos (/) (arrow). By contrast, control embryos (lox/lox) showed homogeneous expression of Hes5 in the cerebellar primordium (H). Scale bars: in C, 100 µm for C,D,G,H; in E 100 µm for E,F.
