Figure 3

PDX1 and NKX6.1 expression in somatostatin- and glucagon-positive cells following alloxan plus caerulein. (a) Cells coexpressing PDX1 (white) and glucagon (red) were found transiently following alloxan plus caerulein, peaking at day 13 (quantified in e). Coexpression of glucagon and NKX6.1 (green) was not found. (b) β-catenin (green), which localizes primarily to the cell membrane, was used to outline cells to confirm coexpression of PDX1 (white) and glucagon (red) in the same cell. PDX1-glucagon coexpression frequently occurred in the absence of insulin expression (Supplementary Figure 4), suggesting that PDX1 induction was an early event in the process of α- to β-cell transdifferentiation. (c) Cells coexpressing NKX6.1 (green) and somatostatin (red) were found transiently following alloxan plus caerulein, peaking at day 20, but not after alloxan alone (quantified in g; see also Supplementary Figure 3B). Note that in many of the cells coexpressing NKX6.1 and somatostatin at day 20, NKX6.1 was present in the cytoplasm (additional examples in Supplementary Figure 5). (d) β-catenin (white) was used to outline cells to confirm the coexpression of NKX6.1 (green) and somatostatin (red). No such cells were found in islets from mice injected with alloxan alone (Supplementary Figure 3B). Further examination of the cells coexpressing NKX6.1 and somatostatin revealed that in cells expressing somatostatin and NKX6.1 but not insulin, the NKX6.1 was invariably cytoplasmic (area outlined in red in Supplementary Figure 5E).³⁹ In cells that coexpressed NKX6.1, somatostatin and insulin, NKX6.1 was usually nuclear (Supplementary Figures 5C–F), but in rare cells was cytoplasmic. No cells coexpressing insulin and somatostatin but not NKX6.1 were found. Cytoplasmic expression of NKX6.1 has been described, with the suggestion that it is controlled by the redox state of the cell and that its localization may be important in Type II diabetes.³⁹ PDX1 also exhibited a transient increase in expression in cells expressing somatostatin, peaking at day 20 (quantified in f), which did not occur following alloxan alone (Supplementary Figure 3) In alloxan-injected mice, expression of PDX1 in δ-cells remained constant over time at 9.5% (Supplementary Figure 3B, quantified in Figure 3f), but rose to more than 40% at day 20 in alloxan plus caerulein treated mice (Figure 3c, quantified in f). Scale bar=75 μm