rnascope probe m. musculus pou1f1  (Advanced Cell Diagnostics Inc)

Journal: eLife

Article Title: Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

doi: 10.7554/eLife.59142

Figure Lengend Snippet: ( 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.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Pou1f1 , Advanced Cell Diagnostics , 486441 , .

Techniques: Immunofluorescence, Staining, Flow Cytometry

Journal: eLife

Article Title: Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

doi: 10.7554/eLife.59142

Figure Lengend Snippet: ( 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.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Pou1f1 , Advanced Cell Diagnostics , 486441 , .

Techniques: Immunofluorescence, Staining, Clone Assay

Journal: eLife

Article Title: Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

doi: 10.7554/eLife.59142

Figure Lengend Snippet: ( 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.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Pou1f1 , Advanced Cell Diagnostics , 486441 , .

Techniques: Immunofluorescence, Staining, Control

Journal: eLife

Article Title: Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

doi: 10.7554/eLife.59142

Figure Lengend Snippet: ( 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.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Pou1f1 , Advanced Cell Diagnostics , 486441 , .

Techniques: Fluorescence, In Situ, Hybridization, Staining, Immunofluorescence, In Vitro, Isolation, FACS, Expressing

Journal: eLife

Article Title: Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

doi: 10.7554/eLife.59142

Figure Lengend Snippet: ( A ) Native EGFP protein expression in frontal cryosection of a P14 Sox2 Egfp/+ pituitary. Schematic of the workflow used for bulk RNA-sequencing analysis of Sox2 + and Sox2 − cells. Genome browser views of reads aligning to the Sox2 and Pit1 loci in the positive and negative fractions indicating good separation of the EGFP + population. Scale bar: 50 µm. ( B ) Sox2 + cells express a significant enrichment in markers associated with epithelial-to-mesenchymal transition (EMT), adherens, and tight junctions, consistent with their epithelial nature. Gene set enrichment analysis (GSEA) plots and immunofluorescence staining against E-Cadherin (adherens junction marker) and ZO1 (tight junction marker) in the marginal zone epithelium at P14. Scale bar: 50 µm. See for full GSEA gene lists. ( C ) Sox2 + cells express a significant enrichment in several signalling pathways, shown with respective GSEA plots. See for full GSEA gene lists. ( D ) Bar charts showing the FPKM values of components of the LGR/RNF43/ZNRF3/R-spondin module in the Sox2 + and Sox2 − fractions and the distribution of the Frizzled receptors. GSEA plot for components of the WNT pathway. Validation of sequencing: (i) mRNA in situ hybridisation with specific probes against Lgr4 (blue) and Sox2 (red) in P14 pituitaries showing co-expression. (ii) Double mRNA in situ hybridisation against Fzd4 (blue) and Sox2 (red) indicating co-expression in both the marginal zone epithelium and parenchymal Sox2 + cells. Boxed regions are magnified. Scale bars: 250 µm and 50 μm in boxed inserts. (iii) mRNA in situ hybridisation against Rspo1 , Rspo2 , Rspo3 , and Rspo4 in sagittal sections of wild-type pituitaries at P14. Boxed regions are magnified, only Rspo4 is detected. Scale bars: 250 µm and 100 μm in boxed inserts.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Pou1f1 , Advanced Cell Diagnostics , 486441 , .

Techniques: Expressing, RNA Sequencing, Immunofluorescence, Staining, Marker, Biomarker Discovery, Sequencing, In Situ, Hybridization

Journal: eLife

Article Title: Pituitary stem cells produce paracrine WNT signals to control the expansion of their descendant progenitor cells

doi: 10.7554/eLife.59142

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Pou1f1 , Advanced Cell Diagnostics , 486441 , .

Techniques: Isolation, Sequencing, RNAscope, Positive Control, Negative Control, Recombinant, Staining, Software