Figure 1. Axin2 expressing cells contribute to pituitary growth and expansion of all lineages.

(A) Immunofluorescence staining against GFP (green) with markers of pituitary stem cells (PSCs) or lineage commitment (magenta) in Axin2^CreERT2/+; ROSA26^mTmG/+ pituitaries harvested from mice induced at P14 and lineage traced for 2 days (top panel) and 14 days (bottom panel). Scale bar: 10 μm. (B) Quantification of lineage expansion between 2 and 14 days following induction at P14. Graph shows that the proportion of lineage committed cells (either PIT1⁺, TPIT⁺, or SF1⁺) and PSCs (SOX2⁺), that is, that are transcription factor (TF)⁺ cells that are GFP⁺ increases between 2 days (black bars) and 14 days (grey bars) post-induction. PIT1 p=0.000004, TPIT p=0.008 multiple t-tests. n = 4 animals per time point. (C) Immunofluorescence staining against GFP (green) in pituitaries harvested from Axin2^CreERT2/+;ROSA26^mTmG/+ mice induced at P14 and lineage traced for 2 days, 2 weeks, and 8 weeks. Bottom panel shows magnified fields of view of regions of interest indicated by white boxes in panels above. Scale bars: 50 μm. (D) Top panel showing the quantification of the proportion of all cells in Axin2^CreERT2/+;ROSA26^mTmG/+ pituitaries that are GFP⁺ at 2, 7, 14, 28, and 56 days post-induction as analysed by flow cytometry. Days 2–7 p<0.0001 unpaired t-test. Data points show individual measurements from biological replicates, n = 4–8 pituitaries per time point. (Bottom) Graph of the absolute number of GFP+ cells (green) and as a proportion of total cells (blue) at the time points indicated. (E) X-gal staining in Axin2^CreERT2/+;ROSA26^LacZ/+ pituitaries harvested from mice induced at P14 and lineage traced for 8 weeks (left) and 1 year (right). Scale bars: 500 μm. (F) Model summarising the major contribution of WNT-responsive progenitors of all lineages to pituitary growth, in addition to that of SOX2⁺ PSCs.

Figure 1—figure supplement 1. Axin2 expressing cells contribute to pituitary growth and expansion of all lineages.

(A) Schematic of the combined experimental timeline used in panels A–C. Immunofluorescence staining against GFP (green) and markers of hormone-secreting endocrine cells (GH [somatotrophs], ACTH [corticotrophs], PRL [lactotrophs], TSH [thyrotrophs], FSH/LH [gonadotrophs]) in Axin2^CreERT2/+;Rosa26^mTmG/+ pituitaries induced at P14 and lineage traced for 48 hr. Scale bar: 10 µm. (B) Graph of quantification of expansion of the WNT-responsive SF1⁺ population in Axin2^CreERT2/+;ROSA26^mTmG/+ pituitaries induced at P14 and lineage traced for 2 or 28 days. There is a significant increase of GFP⁺;SF1⁺ cells as a proportion of the total SF1⁺ cells at P28. p=0.0048, unpaired t-test (n = 2 at 2 days, 3 at 28 days). (C) Immunofluorescence staining against GFP (green) and markers of hormone-secreting endocrine cells of the PIT1 lineage (GH [somatotrophs], PRL [lactotrophs], TSH [thyrotrophs]) in Axin2^CreERT2/+;ROSA26^mTmG/+ pituitaries induced at P14 and lineage traced for 14 days. Scale bars: 50 µm. Graph showing expansion of each of the Hormone⁺ cell types (Hormone⁺;GFP⁺) as a percentage of the total Hormone⁺ population between 2 and 14 days post-induction. There is a significant increase in GH⁺ somatotrophs (p=0.000548) and TSH⁺ thyrotrophs (p=0.0016), whilst there is no significance (ns) between PRL⁺ lactotroph populations between the two time points. Multiple t-test (n = 3 at 48 hr, n = 4 at 14 days post-induction). (D) Clonal analysis of individual cells targeted in Sox2^CreERT2/+;ROSA26^Confetti/+ (left panel) and Axin2^CreERT2/+;ROSA26^Confetti/+ pituitaries (right panel), induced at P14 and harvested after 4 weeks (P42). Arrows point to individual clones, numbered for the number of cells in the clone. Scale bar: 100 µm.

Figure 1—figure supplement 2. Axin2 expressing cells contribute to pituitary growth and expansion of all lineages.

(A) Dorsal wholemount view of Axin2^CreERT2/+; Ctnnb1^LOF/+;ROSA26^mTmG/+ and Axin2^CreERT2/+; Ctnnb1^LOF/LOF;ROSA26^mTmG/+ pituitaries induced at P14 and lineage traced for 5 days. Scale bars: 500 μm. Immunofluorescence staining against GFP (green) and pH-H3 (magenta) in Axin2^CreERT2/+; Ctnnb1^LOF/+;ROSA26^mTmG/+ and Axin2^CreERT2/+; Ctnnb1^LOF/LOF;ROSA26^mTmG/+ pituitaries. Scale bar: 50 µm. Quantification of the contribution of lineage traced cells in control and mutants. Each data point represents the mean from one individual. p=0.0313, unpaired t-test (n = 3). (B) Immunofluorescence staining against GFP (green) and PIT1, SF1, and ACTH (magenta) in Axin2^CreERT2/+; Ctnnb1^LOF/+;ROSA26^mTmG/+ and Axin2^CreERT2/+;Ctnnb1^LOF/LOF;ROSA26^mTmG/+ pituitaries induced at P14 and lineage traced for 5 days. Quantification of the percentage of GFP⁺ cells, double-positive for each of the lineage markers, showing no significant changes for each lineage between controls and mutants (unpaired t-test, PIT1 p=0.1729, SF1 p=0.9488, ACTH p=0.6186. n = 4 controls, two mutants). Scale bars: 50 µm. (C) Immunofluorescence against GFP (green) and SOX2 (magenta) in Axin2^CreERT2/+; Ctnnb1^LOF/+;ROSA26^mTmG/+ and Axin2^CreERT2/+;Ctnnb1^LOF/LOF;ROSA26^mTmG/+ induced at P14 and lineage traced for 5 days (n = 4 controls, two mutants). Scale bars: 50 µm. (D) Immunofluorescence against GFP (green) and Cleaved Caspase-3 (magenta) in Axin2^CreERT2/+; Ctnnb1^LOF/+;ROSA26^mTmG/+ and Axin2^CreERT2/+;Ctnnb1^LOF/LOF;ROSA26^mTmG/+ induced at P14 and lineage traced for 5 days (n = 4 controls, two mutants). Scale bars: 50 μm.