DEV02733 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Generation of N1::cre mice. (A) Schematic of the expected genomic fragments after homologous replacement of the Notch1 locus with the N1::cre targeting vector. Indicated is a Neo selection cassette flanked by FRT sites, the Cre ORF with nuclear localization signal (nls) and with the addition of six Myc epitopes and SV40 polyadenylation signal (not shown). The FRT-flanked Neo was deleted in the germ line by crossing N1::cre/+ mice with CMV-Flp mice. All experiments reported here were performed with neo-deleted mice. (B) Southern blot analysis of HindIII-digested DNA from G418-resistant ES cells probed with external 5′-probe to identify wild-type N1 (∼9.3 kb) and N1::cre (∼8.6 kb). The internal Cre-ORFf probe identifies single integration of N1::cre clone #28 (and #160, not shown). RC is control R26-Cre-ER^T DNA (Vooijs et al., 2001). (C) RNA expression analysis of E13.5 N1::cre/+ (# 4,5) or wild type (# 3) with mN1-b probe (left panel, identifying the N1 extracellular domain) or Cre probe (right panel) confirms reduced expression of N1::cre receptor relative to the wild-type N1 receptor. The presence of the N1::cre allele does not impact upon the expression level of wild-type N1. Equal loading of samples is confirmed by Ethidium-Bromide staining (bottom panels). B, body; H, head.

• Supplemental Figure 2 -

  Fig. S2. N1::cre proteolysis is presenilin-dependent. Transfection assay in mammalian HEK293 cells with N1ΔE::cre-6Myc fusion protein demonstrates presenilin-dependent cleavage at Val1744. 16 hours in the presence of γ-secretase-inhibitor DAPT (GSI) abolishes cleavage. Transfection of N1ΔE::cre-6Myc fusion protein in presenilin-null (PS1/2dKO) MEFs results in no proteolysis; co-transfection with wild-type presenilin1 (PS1) restores cleavage. Upper panels: total protein expression detected by anti-Myc immunoblotting. Middle panels: Cre-immunoblot. Lower panel: α-VLLS (Val1744) to detect cleaved Notch1.

• Supplemental Figure 3 -

  Fig. S3. Proximal to distal gradient of N1 activity in the adult intestine. (A-F) Whole-mount X-Gal staining of N1::cre intestines showing abundant activity in duodenum; activity is reduced in the jejunum and ileum. Only a few labeled crypts are found in the colon. Controls: R26R colon without cre and reporter activity in the colon of CMV-cre;R26R double transgenic mice showing many lacZ-labeled crypts.

• Supplemental Figure 4 -

  Fig. S4. N1::cre activity in peripheral blood. (A) PCR-based assay on peripheral blood confirms that recombination at the R26R locus has occurred (lower panel). DNA was isolated from peripheral blood or proximal intestine from N1::cre;R26R (+) mice (n=4) or N1::cre (−) R26R (+) mice (n=2). PCR primers are according to Akagi et al. (Akagi et al., 1997). All animals inheriting N1::cre are positive for the ΔR26R band absent in N1::cre or R26R-only lanes. (B,C) Absence of follicular (B-cell) staining in adult X-Gal-stained N1::cre;R26R spleens. Original magnification: B, 2×; C, 40×.

• Supplemental Figure 5 -

  Fig. S5. N1::cre activity in tissues of ectodermal, mesodermal and endodermal origin. (A) α-VLLS staining of E14.5 thymus identifies cleaved Notch1 in the nuclei (DAB stain, inset 60×). (B) N1::cre-labeled thymocytes in E14.5 embryos (inset 60×). (C) Infrequent labeling of clones in E14.5 somite derivatives (note labeled intrasomitic artery, arrow; see Fig. 4C). (D,E) Infrequent labeling by N1::cre in pancreatic primordium (D, E14.5) and adult pancreas (E) where acinar and β-cell islet are labeled (note endothelial staining in vessels). (F) Robust R26R activity in adult pancreas from germline-deleted R26R mice. (G,H) Infrequent labeling of epithelial cells at E14.5. In adult kidney only a few proximal tubules are labeled, in contrast to heavy labeling of endothelial cells throughout the kidney and in glomeruli (inset 40×). (I) Ubiquitous R26R activity throughout adult kidney germline-deleted R26R mice. (J) Active Notch signaling in goblet cells shown by α-VLLS staining (arrow) of adult proximal intestine. (K) Expression of Notch1 confined to the proliferative compartment in crypt epithelial cells of the small intestine. Also note expression in endothelium below (arrowhead). (L) Low magnification of N1::cre labeling throughout the adult cerebellum. CPE, Choroid Plexus Epithelium; ML, Molecular layer; GL, Granular layer; PL, Purkinje layer. Note in addition to neuronal staining, extensive labeling in ML and GL but not in the PL. Counterstains: HE (A) or Neutral Red (B-J,L). Original magnification: A-I,K (inset 60×) 20×; J, 40×; L, 2.5×.