Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality

Notch family genes encode transmembrane proteins involved in cell-fate dete rmination. Using gene targeting procedures, we disrupted the mouse Notch2 g ene by replacing all but one of the ankyrin repeat sequences in the cytopla smic domain with the E, coli beta-galactosidase gene. The mutant Notch2 gen e encodes a 380 kDa Notch2-beta-gal fusion protein with beta-galactosidase activity, Notch2 homozygous mutant mice die prior to embryonic day 11.5, wh ereas heterozygotes show no apparent abnormalities and are fully viable. An alysis of Notch2 expression patterns, revealed by X-gal staining, demonstra ted that the Notch2 gene is expressed in a wide variety of tissues includin g neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arc hes, but not heart. Histological studies, including in situ nick end labeli ng procedures, showed earlier onset and higher incidence of apoptosis in ho mozygous mutant mice than in heterozygotes or wild type mice. Dying cells w ere particularly evident in neural tissues, where they were seen as early a s embryonic day 9.5 in Notch2-deficient mice, Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch 2-beta-gal fusion protein is not toxic per se. In contrast to Notch1-defici ent mice, Notch2 mutant mice did not show disorganized somitogenesis, nor d id they fail to properly regulate the expression of neurogenic genes such a s Hes-5 or Mash1, In situ hybridization studies show no indication of alter ed Notch1 expression patterns in Notch2 mutant mice. The results indicate t hat Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function.