rnascope probe m. musculus sox2  (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 Sox2 , Advanced Cell Diagnostics , 401041 , .

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 Sox2 , Advanced Cell Diagnostics , 401041 , .

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 Sox2 , Advanced Cell Diagnostics , 401041 , .

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 ) 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.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Sox2 , Advanced Cell Diagnostics , 401041 , .

Techniques: Expressing, Derivative Assay, Isolation, Fluorescence, FACS, Staining, MANN-WHITNEY, Two Tailed Test, Immunofluorescence, Control, Mutagenesis

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–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).

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Sox2 , Advanced Cell Diagnostics , 401041 , .

Techniques:

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 Sox2 , Advanced Cell Diagnostics , 401041 , .

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 ) Immunofluorescence staining against GFP (green) and SOX2 (magenta) in Axin2 CreERT2/+ ; ROSA26 mTmG/+ pituitaries 48 hr post-induction. Graph representing a quantification of the proximity of individual GFP + cells to the nearest SOX2 + cell as quantified by the number of nuclei separating them. Plotted data represents the proportion of GFP+ cells that fall into each category of the total GFP+ cells, taken from n = 3 separate pituitaries. Scale bars: 50 μm. ( B ) Experimental paradigm for RNA Seq analysis of Sox2 positive and negative cells. ( C ) Graphs representing the FPKM values of Wls and Porcupine in Sox2 positive and negative cells (black and grey bars, respectively). mRNA in situ hybridisation for Sox2 and for Wls on wild-type sagittal pituitaries at P14, demonstrating strong Wls expression in the marginal zone epithelium. Scale bars: 250 μm. ( D ) Bar chart showing the FPKM values of Wnt genes in the Sox2 + and Sox2 − fractions. Double mRNA in situ hybridisation against Wnt2 , Wnt5a, and Wnt9a (blue) together with Sox2 (red) validating expression in the Sox2 + population. Boxed regions through the marginal zone epithelium are magnified. Scale bars: 100 μm and 50 μm in boxed inserts.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Sox2 , Advanced Cell Diagnostics , 401041 , .

Techniques: Immunofluorescence, Staining, RNA Sequencing, In Situ, Hybridization, 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 Sox2 , Advanced Cell Diagnostics , 401041 , .

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: ( A ) Schematic of time points for induction by tamoxifen induction and tissue harvesting of control Sox2 +/+ ;Wls fl/fl and mutant Sox2 CreERT2/+ ;Wls fl/fl pituitaries. ( B ) Whole mount, dorsal views of control Sox2 +/+ ;Wls fl/fl (top panel) and mutant Sox2 CreERT2/+ ;Wls fl/fl (bottom panel) pituitaries at P21, representative of n = 4 controls and n = 5 mutants. Scale bars: 500 μm.

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Sox2 , Advanced Cell Diagnostics , 401041 , .

Techniques: Control, Mutagenesis

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 SOX2 (green) and Ki-67 (magenta) in Sox2 +/+ ;Wls fl/fl (control) and Sox2 CreERT2/+ ;Wls fl/fl (mutant) pituitaries induced from P14 and analysed after 1 week. Nuclei were counterstained with Hoechst. (i and ii) represent magnified fields of view of regions indicated by white boxes in top panels. Scale bars: 50 μm. Graph of quantification of cycling cells marked by Ki-67 among cells negative for SOX2. Values represent mean ± SEM, p=0.0008, unpaired t -test. Graph of quantification of cycling cells marked by Ki-67 among SOX2-positive cells. Values represent mean ± SEM, p=0.0121, unpaired t -test. Each data point shows the mean of one biological replicate, n = 4 pituitaries from controls and five pituitaries from mutants. ( B ) Double mRNA in situ hybridisation using specific probes against Lef1 (blue) and Sox2 (red) in control and mutant pituitaries following tamoxifen induction from P14 and tracing for 7 days. Scale bars: 250 μm and 50 μm in boxed regions. ( C ) Model summarising paracrine WNT secretion from SOX2 + PSCs to lineage-committed progenitors and the effects of abolishing WNT secretion from SOX2 + PSCs through the deletion of Wls .

Article Snippet: Sequence-based reagent , RNAscope probe M. musculus Sox2 , Advanced Cell Diagnostics , 401041 , .

Techniques: Immunofluorescence, Staining, Control, Mutagenesis, 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 Sox2 , Advanced Cell Diagnostics , 401041 , .

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