(a) Hh signaling components are enriched in α- vs β-cells and their expression is unchanged 30 days upon DT-induced β-cell ablation, Pdx1OE or both. Log2FC are taken from the RNA-Seq presented in Fig. 3. (b) Indian Hedgehog ligand expression in islets of control mice. Immunofluorescence was repeated two times with similar results. (c) Non-quantitative RT-PCR of Hedgehog pathway’s components in sorted α-, δ- and β-cells. Liver, duodenum and uterus were used as controls. Experiments were repeated three times with similar results. Unprocessed scans of the blots are provided in Supplementary Fig. 7. (d) Transgenes required for simultaneous α-cell lineage tracing, Smo co-receptor downregulation and DT-induced β-cell ablation. (e) Experimental design. (f) Smo and (g) α-cell genes Arx and Glucagon are downregulated in α-cells with reduced Smo activity upon DOX treatment. n = 3 mice. qPCR was performed once. Two-tailed unpaired t-test; SMO: P = 0.0012, GCG: P = 0.0325 fl/+ vs +/+ P = 0.0141 fl/fl vs +/+ and P = 0.0177 fl/fl vs fl/+, ARX: P = 0.0214 fl/+ vs +/+, 0.0245 fl/fl vs +/+). Center indicates the mean. (h) Smo inactivation in α-cells in absence of β-cell loss or insulin signaling impairment does not lead to insulin production in intact islets. Immunofluorescence was repeated once, on 2 slides of 3 different mice. (i) Transgenes required to lineage trace δ-cells, experimental design and characterization of labeling specificity in the Sst-rtTA line. Only δ-cells express YFP. n = 5, 4, 4 for δ-, β- and α-cells, respectively. (j) Transgenes required for lineage tracing and Smo coreceptor inactivation in δ-cells, and for DT-induced β-cell ablation and experimental design. Smo is downregulated in purified δ-cells after DOX treatment. n = 3 mice. qPCRs were performed once. Two-tailed unpaired t-test, P = 0.0048. Downregulation of Smo in δ-cells does not alter the expression of the δ-cell genes Somatostatin and Hhex. n = 3 mice. qPCR was performed once. Center indicates the mean. Insulin protein is not expressed in δ-cells upon Smo inactivation in intact islets. Immunofluorescence was repeated once, on 3 slides of 3 different mice. (k) Experimental design for δ-cell lineage tracing and DT-induced β-cell loss in δ-Smo-KO mice. A fraction of δ-cells express insulin after β-cell ablation in δ-Smo-KO mice, but their percentage is not increased when compared to mice with intact Smo expression. n = 3, 3, 3 mice in no DT wt/wt, wt/fl, fl/fl respectively and n = 4, 4, 3 mice in DT wt/wt, wt/fl, fl/fl respectively. Immunofluorescence was repeated once, on 3 to 5 slides per mouse. Center in the graph indicates the mean. Two-tailed unpaired t-test. (l) Transgenes required for α-cell tracing and DT-induced β-cell ablation, experimental design and immunofluorescence staining of mouse islets 1 month after DT+GANT61 treatment. Immunofluorescence was repeated once, on 3 to 5 slides of n = 8 mice treated with DT+GANT61. A representative islet is shown. (m) The % of α-cells expressing insulin (n = 5 and 4 mice for DT+DMSO and DT+GANT61, respectively), the number of insulin-expressing cells per islet section (n = 8 mice for DT+DMSO and DT+GANT61), and the % of islets containing insulin+ cells (n = 8 mice and n = 9 mice for DT+DMSO and DT+GANT61, respectively) are increased in mice treated with GANT61 after DT-induced β-cell loss. Two-tailed unpaired t-test (P = 0.0236 panel left, P = 0.0085 middle, P = 0.0213 right graph. (n) Transgenes required for simultaneous β- and δ-cell ablation, experimental design and percentage of cells coexpressing insulin and glucagon after β- and δ-cell loss. n = 4, 5, 3 mice for β-, β+δ and δ-cell ablation, respectively. Center indicates the mean. Two tailed unpaired t-test, P = 0.0494. Scale bars: 10 μm. See Supplementary Table 1m,n,q as source data.