Notch3 activation retains cells in a nonproliferative state. (A) Schematic diagram of the N3IC targeting vector used for the generation of R26-N3IC^SAT knockin mice. The intracellular domain of the Notch3 receptor (N3IC) was knocked in to the ROSA26 locus followed by an internal ribosome entry site and a YFP gene. A lox-STOP-lox cassette blocks the expression of the transgene in the absence of Cre recombinase. (B) Quantification of clonal expansion upon constitutive Notch3 activation. Mean (±SD) percentage of clones (single cells, two to four cell clones, and clones bigger than five cells) over the total number of clones counted from Notch3-CreERT2^SAT/R26^mTmG/R26-N3IC^SAT mice (4wk8wk N3IC) compared with Notch3-CreERT2^SAT/R26^mTmG mice (4wk8wk). Mice were induced with a single dose of 4-OHT at 4 wk of age and analyzed at 8 wk of age. *, P ≤ 0.05. The p-values were calculated using the Student’s t test. (C) Genomic DNA PCR showing the unfloxed N3IC transgene (top) and the floxed N3IC allele (bottom, indicated by a white arrow). The floxed allele is amplified only upon 4-OHT induction. Lanes: (1) 1-kb molecular weight marker; (2) N3IC mouse1 no 4-OHT; (3) N3IC mouse1 + 4-OHT; (4) N3IC mouse2 no 4-OHT; (5) N3IC mouse2 + 4-OHT; and (6) nontransgenic control mouse. (D) qRT-PCR with specific oligos recognizing the extracellular (N3EC) or intracellular (N3IC) domain of Notch3 indicates sustained N3IC expression in GFP⁺ cells sorted from Notch3-CreERT2^SAT/R26^mTmG/R26-N3IC^SAT mice 4 wk after induction. Note that the levels of endogenous Notch3 (detected with the N3EC oligos) are unchanged between GFP⁺ and GFP⁻ cells because the analysis has been performed on the progeny of Notch3-expressing cells 4 wk after induction, containing both Notch3⁺ and Notch3⁻ cells. (E) qRT-PCR analysis of sorted GFP⁻ and GFP⁺ cells from Notch3-CreERT2^SAT/R26^mTmG/R26N3IC^SAT mice 4 wk after induction shows elevated expression levels of Arf and p21 in N3IC-expressing GFP⁺ cells. (D and E) Error bars represent the SD of at least three independent experiments. (F) Proposed model for the observed behavior of GFP-labeled cells. A luminal cell expressing high levels of Notch3 receptor (N3^high) is kept in a resting nonproliferative state until it down-regulates Notch3 expression (N3^low/−), allowing it to enter the cell cycle and give rise to a luminal lineage. The assumption is that only cells expressing high levels of Notch3 (N3^high) will be marked by Cre recombination, whereas proliferative N3^low/− cells will not be labeled by GFP in this system.