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. 2019 Mar 26;8:e43996. doi: 10.7554/eLife.43996

Figure 5. Postnatal expression of constitutively active YAP increases leads to an activation of SOX2+ pituitary stem cells.

(A) Schematic outlining the time course of doxycycline (DOX) treatment administered to Hesx1Cre/+;R26rtTA/+;Col1a1tetO-Yap/+ (YAP-TetO) and Hesx1+/+;R26rtTA/+;Col1a1tetO-Yap/+ controls to drive expression of YAP-S127A in mutant pituitaries. (B) At P42 (3 weeks of treatment), immunofluorescence staining on frontal anterior pituitary sections detects strong total YAP expression in YAP-TetO mutants compared to the control and no increase in pYAP(S127). Immunofluorescence for SOX2 and SOX9 reveals an expanded population of stem cells in YAP-TetO compared to control (quantification in F). (C) Graph showing the percentage of double Ki-67+;SOX2+ cells as a proportion of the total SOX2+ (p=0.027 (*)) or Ki-67+ (p=0.006 (**)) populations at P42 (n = 3 pituitaries per genotype). There is an increase in the numbers of cycling SOX2 cells in YAP-TetO mutant compared to controls. The image shows a representative example of double immunofluorescence staining against Ki-67 and SOX2 in a control and YAP-TetO section. (D) Schematic outlining the time course of doxycycline (DOX) treatment administered to Hesx1Cre/+;R26rtTA/+;Col1a1tetO-Yap/+ (YAP-TetO) and Hesx1+/+;R26rtTA/+;Col1a1tetO-Yap/+ controls to drive expression of YAP-S127A in mutant pituitaries for three weeks, followed by a three-week recovery period in the absence of DOX. (E) Immunofluorescence staining against YAP, SOX2 and SOX9 on control and YAP-TetO pituitaries treated as in D, shows comparable expression of YAP, SOX2 and SOX9 between genotypes. (F) Graph of quantification of SOX2+ cells as a percentage of total nuclei in control and YAP-TetO pituitaries at P42 p=0.0014 (**); P63 p=0.0044 (**); P105 p<0.0001(****) (n = 3 pituitaries per genotype). Following the Recovery treatment scheme in D, there is no significant difference in the numbers of SOX2+ cells between genotypes. (G) Schematic outlining the time course of tamoxifen induction and doxycycline (DOX) treatment administered to Sox2CreERT2/+;R26rtTA/mTmG;Col1a1tetO-Yap/+ (mutant) and Sox2CreERT2/+;R26mTmG/+;Col1a1+/+ (control) animals to drive expression of YAP-S127A in SOX2+ cells of mutants. (H) Lineage tracing of SOX2+ cells and immunofluorescence staining against SOX2 and GFP shows an expansion of GFP+ cells compared to controls at P63, where a proportion of cells are double-labelled. (I) Immunofluorescence staining against commitment markers PIT1, SF1 and terminal differentiation marker ACTH (TPIT lineage) together with antibodies against GFP detects double-labelled cells (arrows) across all three lineages in Sox2CreERT2/+;R26rtTA/mTmG;Col1a1tetO-Yap/+ pituitaries following the recovery period. Graph of quantification of GFP+;PIT1+, GFP+;SF1+ and GFP+;ACTH+ cells as a percentage of total GFP+ cells in Sox2CreERT2/+;R26rtTA/mTmG;Col1a1tetO-Yap/+ pituitaries at P63. Scale bars 100 µm. Data in C. and F. represented as mean ±SEM, analysed with Two-Way ANOVA with Sidak’s multiple comparisons. See also Figure 5—figure supplement 1.

Figure 5.

Figure 5—figure supplement 1. Postnatal expression of constitutively active YAP leads to an activation of SOX2+ pituitary stem cells.

Figure 5—figure supplement 1.

(A) Schematic outlining the time course of doxycycline (DOX) treatment administered to Hesx1Cre/+;R26rtTA/+;Col1a1tetO-Yap/+ (YAP-TetO) and Hesx1+/+;R26rtTA/+;Col1a1tetO-Yap/+ controls to drive expression of YAP-S127A in mutant pituitaries. Hematoxylin and eosin staining of control and YAP-TetO pituitaries at P42 (3 weeks treatment), P63 (6 weeks treatment) and P105 (12 weeks treatment). (B) RNAscope mRNA in situ hybridisation against YAP targets Cyr61 and Ctgf showing increased transcripts in YAP-TetO sections compared to controls at P42. (C) Analysis of YAP-TetO mutants at P105: double immunofluorescence staining against SOX2 and Ki-67 reveals regions of expanded SOX2+;Ki-67- cells compared to the normal expression pattern in the control. This region is SOX9+, does not accumulate TAZ or YAP and expresses pYAP as does normal anterior pituitary epithelium. Immunofluorescence against PIT1 shows the absence of commitment to this lineage, a pattern not seen in the control. Hematoxylin and eosin staining in consecutive sections identifies this region, which does not have neoplastic features. (D) Schematic outlining the time course of doxycycline (DOX) treatment administered to Hesx1Cre/+;R26rtTA/+;Col1a1tetO-Yap/+ (YAP-TetO) and Hesx1+/+;R26rtTA/+;Col1a1tetO-Yap/+ controls to drive expression of YAP-S127A in mutant pituitaries for three weeks, followed by a three-week recovery period in the absence of DOX. Hematoxylin and eosin staining of control and YAP-TetO pituitaries. RNAscope mRNA in situ hybridisation shows comparable levels of expression of targets Cyr61 and Ctgf. E) Graph comparing total fluorescence of Cyr61 and Ctgf by Fast Red RNAscope mRNA in situ hybridisation across sections from control, Hesx1Cre/+;R26rtTA/+;Col1a1tetO-Yap/+ (YAP-TetO) and Sox2CreERT2/+;Lats1fl/fl;Lats2fl/+ anterior pituitaries, normalised for total anterior pituitary area. There is a significant increase in the expression of both targets in Sox2CreERT2/+;Lats1fl/fl;Lats2fl/+ pituitaries compared to other genotypes (one-way ANOVA with Tukey’s post hoc test; Control v Sox2CreERT2/+;Lats1fl/fl;Lats2fl/+: p<0.0001 for Cyr61 (****), p=0.001 for Ctgf (***); YAP-TetO v Sox2CreERT2/+;Lats1fl/fl;Lats2fl/+: p<0.0001 for Cyr61 (****), p=0.0049 for Ctgf (**)). Scale bars 250 µm in A, 100 µm in B-D.