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. 2015 Jan 2;14(1):9–17. doi: 10.4161/15384101.2014.988027

Figure 2.

Figure 2

(See previous page). Notch inhibition confers clonal dominance. (A) Side views of 3-dimensional reconstructions of confocal z stacks showing clonal areas of wholemounts of esophageal epithelium immunostained for DN-Maml1 (green) at one month and 1 year post induction. Dapi is blue, scale bars 500 μm. (B) Effect of clonal DN-Maml1 expression on progenitor cell dynamics. At early time points DN-Maml1 expression (green) increases the rate of progenitor cell division and decreases the rate of differentiating cell stratification. In addition, divisions resulting in 2 differentiating cells are absent, blocking clone loss by differentiation. In combination these changes result in exponential clonal expansion in a background of wild type cells (blue). (C) Notch inhibition induces differentiation of adjacent wild type cells. Side view of 3-dimensional reconstructions showing typical appearances of DN-Maml1 induced and uninduced age-matched control epithelial wholemounts. Progenitor cells were labeled with a pulse of Ethinyl deoxy Uridine (EdU, red), taken up by progenitors that were in S phase 48 hours before staining. At the boundary of a DN-Maml1 clone (green), an increased proportion of non-mutant suprabasal EdU+ cells (arrowed) is seen compared with controls, indicative of an increased rate of progenitor differentiation. Dotted line indicates basement membrane, scale bars 10 μm. (D) XZ cross sections of a wholemount confocal z-stack from DN-Maml1 induced treated with EdU as described in (C). Accelerated stratification at the wild type edge shows typical markers of esophageal differentiation. GFP green, EdU is red and differentiation marker Keratin 4 white, dotted line indicates basement membrane, arrows suprabasal EdU positive cells. Scale bars 10 μm. (E) Model of wild type cell elimination through competition with Notch mutant cells. Notch signaling is activated preferentially in wild type cells at the clonal edges due to inhibition of Notch pathway in mutant cells. This prompts stratification and differentiation of wild type progenitors. Clone expansion is accelerated by the active expulsion of wild type cells through differentiation.