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Development, Vol 126, Issue 15 3415-3424, Copyright © 1999 by Company of Biologists
JOURNAL ARTICLES |
Y Hamada, Y Kadokawa, M Okabe, M Ikawa, JR Coleman and Y Tsujimoto
National Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan. hamada@nibb.ac.jp
Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as 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 Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that 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.
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