Figure 2. Activation of WNT signalling in SOX2⁺ pituitary stem cells (PSCs) and their descendants is necessary for long-term growth.

(A) Schematic of the experimental timeline used in panels A and B. Endogenous expression of tdTomato (magenta, Axin2 targeted cells) and EGFP (green, Sox2 expressing cells) in Axin2^CreERT2/+;Sox2^Egfp/+;ROSA26^tdTomato/+ pituitaries harvested at P24 sectioned in the frontal plane. Nuclei are counterstained with Hoechst in the merged panel. Scale bar: 50 μm. (B) A representative culture plate showing colonies derived from Tomato⁺, EGFP⁺, or Tomato⁺;EGFP⁺ cells that were isolated from Axin2^CreERT2/+;Sox2^Egfp/+;ROSA26^tdTomato/+ pituitaries by fluorescence-activated cell sorting (FACS) plated in stem cell promoting media at clonogenic densities and stained with crystal violet (left panel). The proportion of colony-forming cells in each subpopulation was quantified by counting the number of colonies per well (right panel). Each data point indicates individual wells, n = 5 separate pituitaries. p=0.0226, Mann–Whitney U-test (two-tailed). Scale bar: 10 mm. (C) Immunofluorescence staining against SOX2 (green) and Ki-67 (magenta) in Sox2^+/+Ctnnb1^LOF/LOF (control) and Sox2^CreERT2/+Ctnnb1^LOF/LOF (mutant) pituitaries from mice induced at P14 and analysed 22 weeks after induction (at P168) (bottom panel). Scale bar: 50 μm. (D) Dorsal view of whole mount pituitaries of Sox2^+/+;Ctnnb1^LOF/LOF (control) and Sox2^CreERT2/+;Ctnnb1^LOF/LOF (mutant), 22 weeks after induction (i.e. P168). Scale bars: 1 mm. (E) Model summarising the effect of Ctnnb1 deletion in SOX2⁺ PSCs. PL, posterior lobe; IL, intermediate lobe; AL, anterior lobe.

Figure 2—figure supplement 1. Activation of WNT signalling in SOX2⁺ pituitary stem cells (PSCs) and their descendants is necessary for long-term growth.

(A–E) Step-wise gating strategy to isolate WNT-responsive, SOX2-EGFP⁺ cells by flow sorting. (A and B) Single pituitary cells dissociated from Axin2^CreERT2/+;ROSA26^tdTomato/+;Sox2^eGFP/+ mice were gated to exclude debris (A) and gated for single cells according to SSC-A and SSC-W (B). (C) Dead cells were excluded according to incorporation of DAPI. (D) Three populations of fluorescent cells were identified and sorted according to the following profiles: GFP^-;tdTomato⁺, GFP⁺;tdTomato⁺, or GFP⁺;tdTomato^-. (E) Quantification of the number of GFP⁺ cells out of all gated cells (left, n = 5 biological repeats), the proportion of all GFP⁺ cells that were found to be tdTomato⁺ (right, n = 5 biological repeats), and a representation of the gating used for quantification (bottom).

Figure 2—figure supplement 2. Activation of WNT signalling in SOX2⁺ pituitary stem cells (PSCs) and their descendants is necessary for long-term growth.

(A) Confocal images of native GFP fluorescence in frontal sections from TCF/Lef:H2B-EGFP pituitaries at P21. Scale bar: 50 μm. (B) mRNA in situ hybridisation in TCF/Lef:H2B-EGFP pituitaries at P21, detecting Egfp transcripts (red). Double mRNA in situ hybridisation showing overlap between Sox2 (red) and Egfp (blue) transcripts in pituitaries at P21. White arrowheads indicate double-positive staining. Scale bars: 50 μm. (C) Immunofluorescence staining against SOX2 (magenta) and GFP (green) in TCF/Lef:H2B-EGFP pituitaries harvested from P21 mice. White arrows indicate double-positive cells. Graph of quantification of the in vitro colony forming potential of GFP cells isolated from P21 TCF/Lef:H2B-EGFP pituitaries by flow sorting. Each data point represents single well replicates. Error bars show SEM, p<0.001 (one-way ANOVA, n = 3 individual pituitaries). Scale bar: 50 μm. Representative scatter plot showing gating used for fluorescence-activated cell sorting and population percentages in each gate. (D) Immunofluorescence staining against PIT1, TPIT, and SF1 (magenta) in Sox2^CreERT2/+;Ctnnb1^LOF/+;ROSA26^mTmG/+ and Sox2^CreERT2/+;Ctnnb1^LOF/LOF;ROSA26^mTmG/+ pituitaries 22 weeks post-induction at P14 (age P24). Arrows indicate double-positive cells. Scale bar: 50 µm. (E) Immunofluorescence staining against β-catenin (magenta) and GFP (green) in Sox2^CreERT2/+; Ctnnb1^LOF/+;ROSA26^mTmG/+ and Sox2^CreERT2/+; Ctnnb1^LOF/LOF;ROSA26^mTmG/+ pituitaries 22 weeks post-induction. Arrowheads indicate double-positive cells, and arrows indicate GFP⁺ cells that have lost β-catenin expression in mutants. Scale bar: 50 µm. PL, posterior lobe; IL, intermediate lobe; AL, anterior lobe; Inf, infundibulum; RP, Rathke’s pouch; Sph, sphenoid bone.