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Fig. 1 Notch1 activity in the gastrulating mouse embryo. A: Amino acid sequence comparison of various Notch1 homologues spanning the γ-secretase cleavage site. The blue arrowhead indicates the S3 cleavage site preceding V1744, the amino terminal amino acid of N1ICD. This S3 cleavage site is conserved in mouse, human, and chick Notch1; zebrafish Notch1a; and C. elegans glp1, but not in Drosophila Notch and zebrafish Notch1b, where it is shifted one amino acid (green arrowhead). Boxes mark conserved amino acid stretches; identical amino acids appear in red. TM, transmembrane region; NICD, Notch intracellular domain. B: Western blot of embryonic day (E) 9.5 and E13.5 mouse embryos probed with anti-N1ICD (V1744) antibody, showing a 110-kDa N1ICD band in different tissues (top). Western blot from several mammary tumor cell lines (bottom). Note that only the highly invasive MDA-MB231 line expressed N1ICD. SMC-3 is a 140-kDa cohesin used as loading control. C: Planes of sections shown in D-K. D,E: Nuclear N1ICD staining (red) in the intraembryonic mesoderm region (iem, arrow) of the E7.0 wild-type (wt) embryo. Note that headfold region (hf) and embryonic ectoderm (ee) are negative. F: Detail of allantois with developing vessel expressing N1ICD (arrowhead). G: At E7.5, N1ICD is expressed in primitive endocardium (ec), cells of the cephalic mesenchyme (cm), amnion (am), and allantois (al). H: Detail of headfold region showing N1ICD staining in primitive endocardium (ec). ne, neuroectoderm; cm, cephalic mesenchyme. I: Detail showing N1ICD expression in blood island (bli) precursors. J: At E7.7, N1ICD is expressed in endocardium, cephalic mesenchyme and amnion (am). K: Detail of J, showing N1ICD expression in endocardium and primordium of dorsal aorta (da). Nuclei are counterstained with 4′,6-diamidine-2-phenylidole-dihydrochloride (DAPI, blue). Scale bars = 150 μm in D,G,J, 60 μm in E,F,H,I,K.