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. 2017 Dec 1;159(2):579–596. doi: 10.1210/en.2017-03061

Figure 4.

Figure 4.

Inhibition of SHH signaling impairs zF regrowth. (A) Experimental design of GANT61 treatment. Three-week-old wild-type male mice were treated with dexamethasone for 14 days. Starting on the day of dexamethasone withdrawal, mice were injected with GANT61 or vehicle for 3 days followed by organ harvest. (B) Relative mRNA quantification of Gli1 and Gli2 after GANT61 treatment normalized to vehicle. (C) Adrenal weight of GANT61- and vehicle-treated mice. (D) mRNA expression of Sf1 following GANT61 treatment normalized to vehicle. (E) Hematoxylin and eosin staining of adrenals treated with GANT61 or vehicle (left) shows altered adrenal morphology. Immunofluorescence staining (right) of cortical SF1+ cells (red) and nuclei (DAPI, blue) displays fewer steroidogenic cells in the cortex, as confirmed by quantification of SF1+ cell (far right). (F) mRNA levels of Cyp11b1 in GANT61-treated mice normalized to vehicle. (G) Experimental design of LDE225 treatment: After tamoxifen pulse to activate cre-recombinase, Gli1CreERT2-R26REGFP mice were administered dexamethasone for 14 days, followed by LDE225 for 3 consecutive days, and were euthanized on the next day. (H) Adrenal weight of vehicle- and LDE225-treated mice. (I) mRNA expression of Sf1 following LDE225 treatment normalized to vehicle. (J) Relative mRNA quantification of Cyp11b1 after LDE225 treatment normalized to vehicle. (K) Immunofluorescent staining showing cortical SF1+ nuclei (in red) and Gli1-expressing cells and their descendant (green) in mice treated with LDE225 (bottom) or vehicle (top). Arrows point at cortical Gli1 descendants that coexpress SF1. A decreased delamination into the cortex of Gli1 descendants is appreciable in LDE225-treated mice compared with vehicle-treated animals. Quantification of EGFP+; SF1+ cells is shown in the histogram (right). (L) Relative mRNA quantification of Wnt4, Lef1, and Rspo3 of GANT61-treated mice compared with vehicle-treated mice. (M) Relative mRNA quantification of Wnt4, Lef1, and Rspo3 of LDE225-treated mice compared with vehicle-treated mice. For statistical analyses, n = 5 animals per treatment. Error bars represent the standard error of the mean. *P < 0.05; **P < 0.01; ***P < 0.001. Scale bar: 100 μm.