rnascope probe sox2  (Advanced Cell Diagnostics Inc)

Journal: Nature Communications

Article Title: Uncovering minimal pathways in melanoma initiation

doi: 10.1038/s41467-025-60742-0

Figure Lengend Snippet: a Tumors derived from Braf CA/+ and Braf CA/+ ; Pten Δ/+ mouse models were passaged by transplantation onto NSG (immune deficient) mice. Created in BioRender. Shiu, J. (2025) https://BioRender.com/m4kraw3 . b Single-cell gene expression profiles were used to subcluster NC-derived cell clusters (32,611 cells), among which principal tumor, melanocyte and LNM clusters were identified. UMAP visualizations of cell-types and sample distribution are shown. rd = Round of transplantation. Tx = transplant. c Gene expression profiles of the NC-derived clusters identified in ( b ). Gene signatures shared among LNM1 and LNM2 are labeled in black. Each column in the heatmap represents a cell from the corresponding group. d 182,950 cells from Braf CA/+ and Braf CA/+ ; Pten Δ/+ primary tumors ( n = 9) and transplanted NSG tumors ( n = 6) were subjected to single-cell RNA-seq. UMAP visualization of cell-types is shown. e Ridge plots of Aqp1 and Sox2 expression. Note the high expression of Aqp1 and Sox2 in both LNM1 and LNM2, compared to other cell types present in the whole skin. Percentages on the side denote the fraction of cells expressing Aqp1 and Sox2 in each cell type, respectively. Exp.: Expression. f Conditional Sox2 deletion in melanocytic/NC-derived cells inhibits tumor development. Tumor incidence in mice of the indicated genotypes is shown, and each dot represents an individual biological replicate; error bars represent the group mean ± SD. Tumor numbers were compared using an unpaired two-tailed t -test. Asterisks denote statistical significance: p < 0.05 (*), p < 0.01 (**); ns: not significant. Source data are provided as a Source Data file.

Article Snippet: RNAscope probe , Sox2 , ACD Bio , 401041-C3 , Used for RNAscope FISH.

Techniques: Derivative Assay, Transplantation Assay, Gene Expression, Labeling, RNA Sequencing, Expressing, Two Tailed Test

Journal: Nature Communications

Article Title: Uncovering minimal pathways in melanoma initiation

doi: 10.1038/s41467-025-60742-0

Figure Lengend Snippet: Skin contains both pigmented melanocytes and rare, poorly-pigmented LNM cells. Upon Braf activation, both populations expand, but melanocytes arrest as nevi, unless loss-of-function mutation in tumor suppressor gene Pten allows them to develop rapidly into pigmented melanomas (Pathway 1). Otherwise, Braf -activated LNM cells can undergo rare, stochastic transitions to produce scantly pigmented melanomas (Pathway 2). The probability of such transitions depends on host pigmentation status, Sox2 expression, and whether both alleles of Pten have become inactivated. LNM cells persist in both types of tumor. Created in BioRender. Shiu, J. (2025) https://BioRender.com/pf330qi .

Article Snippet: RNAscope probe , Sox2 , ACD Bio , 401041-C3 , Used for RNAscope FISH.

Techniques: Activation Assay, Mutagenesis, Expressing

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: Movie of z -stack through a neonatal AP organoid immunofluorescently stained for SOX2 (red) and E-cadherin (green). Hoechst33342 was used as nuclear stain (blue).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques:

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: 3D reconstruction of a neonatal AP organoid immunofluorescently stained for SOX2 (red) and E-cadherin (green). Hoechst33342 was used as nuclear stain (blue).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques:

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Violin plots showing distribution of number of genes detected per cell (nFeature), total counts per cell (nCount), and percentage of mitochondrial content (percent.mito) per sequenced neonatal anterior pituitary (AP) sample (CTRL, control; DMG, damaged; two biological replicates each). Dashed lines indicate cutoff values for filtering low-quality/dead cells and doublet exclusion, that is removing cells with nFeature below 750 and above 8000 (representing potential doublets) and with percent.mito above 17.5%. ( B ) Dot plot displaying percentage of cells (dot size) expressing indicated marker genes with average expression levels (color intensity; see scales on top) in the collective AP samples (i.e., adult and neonatal AP). ( C ) Ridge plot displaying mRNA expression level of indicated genes in SC1 and SC2 of adult and neonatal AP. ( D ) Left : Immunofluorescence staining of SOX2 (red) and Ki67 (green) in adult and neonatal pituitary (separate channels and merge). Nuclei are stained with Hoechst33342 (blue). Boxed area is magnified (scale bar, 100 μm). Right : Bar graphs showing proportion of SOX2 + cells in adult and neonatal AP, or of SOX2 + Ki67 + cells in SOX2 + cell population (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 3 for Adult, n = 6 for Neonatal; unpaired t -test).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Control, Expressing, Marker, Immunofluorescence, Staining

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Left : UMAP plot of neonatal and adult anterior pituitary (AP) combined. Middle : UMAP plot of the annotated cell clusters in the integrated AP samples (i.e., collective single-cell transcriptome datasets from neonatal and adult AP). Somato, somatotropes; Lacto, lactotropes; (Prolif) Cortico, (proliferating) corticotropes; Gonado, gonadotropes; Thyro, thyrotropes; Melano, melanotropes; SC1 and SC2, stem cell clusters 1 and 2; EC, endothelial cells; IC, immune cells; MC, mesenchymal cells; PL, posterior lobe cells; Gonado Prog, gonadotrope progenitor cells; Gonado Prec, gonadotrope precursor cells. Right : Bar plots showing proportions of each cell cluster at both ages. ( B ) Dot plot displaying percentage of cells (dot size) expressing indicated marker genes with average expression levels (color intensity; see scales on top) in the collective AP samples (i.e., adult and neonatal AP). ( C ) Top : Heatmap displaying scaled expression of selected epithelial and mesenchymal marker genes in the stem cell clusters SC1, SC2, and Prolif SC of neonatal and adult AP. Bottom : Immunofluorescence staining of CK8/18 (green), VIM (red/green), and SOX2 (red) in neonatal and adult AP. Nuclei are stained with Hoechst33342 (blue). Boxed area is magnified (scale bar, 100 µm).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Expressing, Marker, Immunofluorescence, Staining

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Left : Volcano plot displaying differentially expressed genes (DEGs) in neonatal anterior pituitary (AP). Colored dots represent significantly up- (blue) and down- (gray) regulated genes in Prolif SC versus SC1 and SC2. A selection of cell cycle-associated genes is indicated. Right : DEG-associated Gene Ontology (GO) terms linked with cell cycle processes enriched in Prolif SC versus SC1+SC2 of neonatal AP. ( B ) Left : Volcano plot displaying DEGs in neonatal AP. Colored dots represent significantly up- (purple, pink) and down- (orange, yellow) regulated genes in Prolif Pou1f1 + cells versus Somato and Prolif Cortico versus Cortico, respectively. A selection of cell cycle-associated genes is indicated. Middle: DEG-associated GO terms linked with cell cycle processes enriched in Prolif Pou1f1 + cells versus Somato or Prolif Cortico versus Cortico, respectively, of neonatal AP. Right : Immunofluorescence staining of Ki67 (red) and PIT1 or ACTH (both green) in neonatal pituitary. Nuclei are labeled with Hoechst33342 (blue). Boxed areas are magnified (scale bar, 100 μm). ( C ) Left : Volcano plot displaying DEGs in neonatal AP. Colored dots represent significantly up- (blue) and down- (gray) regulated genes in Gonado Prog versus SC1, SC2, and Prolif SC. A selection of endocrine/secretory-associated genes is indicated. Middle : DEG-associated GO terms linked with endocrine/secretory processes enriched in the Gonado Prog versus SC1+SC2 +Prolif SC of neonatal AP. Right : Immunofluorescence staining of SOX2 (red) and αGSU (green) in neonatal pituitary. Nuclei are labeled with Hoechst33342 (blue). Boxed area is magnified (scale bar, 100 μm). Figure 1—figure supplement 2—source data 1. Differentially expressed gene (DEG) and Gene Ontology (GO) analysis in Prolif SC versus SC1+SC2 of neonatal anterior pituitary (AP). (A) DEG analysis in Prolif SC versus SC1+SC2 of neonatal AP. The columns represent: gene names (Gene), p values (p_val), average log fold change expression (avg_logFC; positive values indicate upregulation and negative values downregulation in Prolif SC), percentage of cells expressing the indicated gene in Prolif SC (pct.1) and in SC1+SC2 (pct.2), and the FDR adjusted p value (p_val_adj). (B) Gene Ontology (GO) analysis of genes upregulated in Prolif SC versus SC1+SC2 in neonatal AP. Analyses were performed using Reactome overrepresentation analysis. Pathway name, number of mapped identifiers that match the pathway (#Entities found), total number of identifiers in the pathway (#Entities total), total entities in the pathway/total number of entities for the entire species (Entities ratio), p value, FDR, and −log10(FDR) are shown in the columns. Associated with . Figure 1—figure supplement 2—source data 2. Differentially expressed gene (DEG) and Gene Ontology (GO) analysis in Prolif Pou1f1 + and Prolif Cortico cells versus Somato and Cortico, respectively, of neonatal anterior pituitary (AP). (A) Differentially expressed gene (DEG) analysis in Prolif Pou1f1 + cells versus Somato of neonatal anterior pituitary (AP). (B) DEG analysis in Prolif Cortico versus Cortico of neonatal AP. The columns represent: gene names (Gene), p values (p_val), average log fold change expression (avg_logFC; positive values indicate upregulation and negative values downregulation in first mentioned cluster), percentage of cells expressing the indicated gene in the first mentioned cluster (pct.1) and in the second mentioned cluster (pct.2), and the FDR adjusted p value (p_val_adj). (C) Gene Ontology (GO) analysis of genes upregulated in Prolif Pou1f1 + cells versus Somato in neonatal AP. (D) GO analysis of genes upregulated in Prolif Cortico versus Cortico in neonatal AP. Analyses were performed using Reactome overrepresentation analysis. Pathway name, number of mapped identifiers that match the pathway (#Entities found), total number of identifiers in the pathway (#Entities total), total entities in the pathway/total number of entities for the entire species (Entities ratio), p value, FDR, and −log10(FDR) are shown in the columns. Associated with . Figure 1—figure supplement 2—source data 3. Differentially expressed gene (DEG) and Gene Ontology (GO) analysis in Gonado Prog versus SC1+SC2+Prolif SC of neonatal anterior pituitary (AP). (A) DEG analysis in Gonado Prog versus SC1+SC2+Prolif SC of neonatal AP. The columns represent: gene names (Gene), p values (p_val), average log fold change expression (avg_logFC; positive values indicate upregulation and negative values downregulation in Gonado Prog), percentage of cells expressing the indicated gene in Gonado Prog (pct.1) and in SC1+SC2+Prolif SC (pct.2), and the FDR adjusted p value (p_val_adj). (B) Gene Ontology (GO) analysis of genes upregulated in Gonado Prog versus SC1+SC2+Prolif SC in neonatal AP. Analysis was performed using Gorilla. Pathway name, p value, FDR q -value, enrichment score [( b / n )/( B / N )], and −log10(FDRq) are shown in the columns. N , total number of genes; B , total number of genes associated with a specific GO term; n , number of genes in the top of the user’s input list or in the target set when appropriate; b , number of genes in the intersection. Associated with .

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Selection, Immunofluorescence, Staining, Labeling, Expressing

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Experimental schematic for organoid culturing. PD, postnatal day; wks, weeks; MZ, marginal zone; PP, posterior pituitary. Mouse icons obtained from BioRender. ( B ) Organoid formation efficiency (passage 0, P0) from adult and neonatal anterior pituitary (AP) cultured in PitOM or RSpECT medium. Left : Representative brightfield pictures of organoid cultures (scale bar, 500 μm). Right : Bar graph indicating number of organoids developed per well (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 4 for Adult, n = 3 for Neonatal; two-way analysis of variance (ANOVA) with Tukey’s multiple comparison test). ( C ) Immunofluorescence staining of SOX2, E-cadherin (E-cad), TACSTD2 (all red), and CK8/18 (green) in neonatal pituitary and derived organoids. Nuclei are labeled with Hoechst33342 (blue) (scale bar, 100 μm). ( D ) Organoid formation efficiency (P0) starting from AP cells of adult ROSA26 mT/mG (tdTomato + (tdT)) mice, or from a 1:1 mixture of adult ROSA26 mT/mG and neonatal wild type (WT) AP cells (P0). Top left : Light microscopic (LM) and epifluorescence (tdT) pictures. Arrowheads indicate tdT + organoids (scale bar, 500 µm). Top right : Bar graph indicating number of adult (tdT + ) organoids developing per 5000 cells in indicated cultures (mean ± SEM). Data points represent biological replicates ( n = 4, paired t -test). Bottom : Immunofluorescence staining of Ki67 (green) and tdT (red) in AP organoids derived from adult ROSA26 mT/mG , neonatal WT or a 1:1 mixture of adult ROSA26 mT/mG and neonatal WT AP cells. Nuclei are labeled with Hoechst33342 (blue) (scale bar, 100 μm). Bar graphs showing percentage of Ki67 + cells in organoids as indicated (mean ± SEM). Data points represent individual organoids ( n = 3). ( E ) Organoid formation efficiency from AP of neonatal Il6 +/+ and Il6 −/− mice in RSpECT with or without interleukin-6 (IL-6) (P0). Left : Representative brightfield pictures of organoid cultures (scale bar, 500 μm). Right : Bar plot showing number of organoids developed per well in conditions as indicated (mean ± SEM). Data points represent biological replicates ( n = 5 for Il6 +/+ , n = 6 for Il6 −/− ; two-way ANOVA with Tukey’s multiple comparison test). ( F ) Neonatal AP organoid passageability with or without IL-6. Left : Representative brightfield images at indicated passage (scale bar, 500 µm). Right : Bars depicting the passage number reached at the end of this study.

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Cell Culture, Comparison, Immunofluorescence, Staining, Derivative Assay, Labeling

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Organoid development from neonatal anterior pituitary (AP) cells in PitOM lacking the indicated factors (P0). Left : Representative brightfield pictures of organoid cultures (scale bar, 500 μm). Right : Bar plot showing percentage organoids developed per well in indicated conditions (relative to PitOM, set as 100% [dashed line]). ( B ) Left : Violin plots displaying mRNA expression level of indicated genes in adult and neonatal SC clusters (SC1+SC2+Prolif SC) as exposed by single-cell RNA-sequencing (scRNA-seq) analysis. Right : Bar plots depicting relative expression level of indicated genes in neonatal AP organoids (relative to adult AP organoids, set as 1 [dashed line]) (mean ± standard error of the mean [SEM]), grown in RSpECT (P0). Data points represent biological replicates ( n = 3). ( C ) Organoid formation from AP cells of neonatal Sox2 eGFP/+ or wildtype (WT) mice, or from a 1:1 mixture (Mix) of neonatal Sox2 eGFP/+ and WT AP cells (P0). Light microscopic (LM) and epifluorescence (enhanced green fluorescent protein [eGFP]) images are shown. Boxed areas are magnified in the bottom row (scale bars, 500 µm). ( D ) Representative, still brightfield images of live time-lapse recordings of neonatal AP organoid formation (P0) using IncuCyte S3 at days indicated. Arrow points to a single structure at consecutive days of culture, starting from an individual cell. ( E ) Immunofluorescence staining of αGSU, ACTH, PRL (all green), and GH (red) in neonatal pituitary and derived organoids. Nuclei are labeled with Hoechst33342 (blue) (scale bar, 100 μm). ( F ) Bar graph showing percentage of organoid-initiating SOX2 + cells per well of 10,000 seeded adult or neonatal AP cells (mean ± SEM). Data points represent biological replicates ( n = 3 for Adult, n = 4 for Neonatal; unpaired t -test).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Expressing, RNA Sequencing, Immunofluorescence, Staining, Derivative Assay, Labeling

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Left : Bar plot depicting relative Il6 gene expression level as determined by RT-qPCR (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 2). Middle : Violin plot displaying mRNA expression level of Il6 in adult and neonatal anterior pituitary (AP) as exposed by single-cell RNA-sequencing (scRNA-seq) analysis. Right : Stat3 regulon activity projected on UMAP plot of adult and neonatal AP, with indication of cell clusters. ( B ) Left : Bar graph displaying interleukin-6 (IL-6) protein levels in supernatant (SN) medium from neonatal AP organoid cultures at indicated passages (mean ± SEM). Data points represent technical replicates ( n = 2; one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test). Right : Bar plot depicting relative gene expression level of Il6 in neonatal AP organoids at indicated passages (mean ± SEM). Data points represent biological replicates ( n = 6). ND, not detectable. ( C ) Left : Bar graph showing relative gene expression levels of indicated genes in early (P2) and late (P8–15) passage organoids from neonatal AP (mean ± SEM). Data points represent biological replicates ( n = 2). Right : Immunofluorescence staining of SOX2 and E-cad (red) and ACTH, αGSU, PRL, and GH (all green) in IL-6-expanded, late passage organoids from neonatal AP. Nuclei are labeled with Hoechst33342 (blue) (scale bar, 100 μm).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, RNA Sequencing, Activity Assay, Immunofluorescence, Staining, Labeling

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Left : Immunofluorescence staining of SOX2 (red) and Ki67 (green) in Il6 +/+ and Il6 −/− neonatal pituitary. Nuclei are labeled with Hoechst33342 (blue). Boxed areas are magnified. Arrowheads indicate SOX2 + Ki67 + cells (scale bar, 100 μm). Right : Bar graphs showing proportion of SOX2 + cells in Il6 −/− neonatal anterior pituitary (AP) and of SOX2 + Ki67 + cells in SOX2 + cell population (relative to Il6 +/+ AP, set as 100% [dashed line]) (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 4–5 for Il6 +/+ , n = 3 for Il6 −/− ). ( B ) Bar graph showing relative gene expression levels of indicated genes in Il6 +/+ and Il6 −/− neonatal AP (mean ± SEM). Data points represent biological replicates ( n = 4). ( C ) Bar graph showing relative gene expression levels of indicated genes in Il6 +/+ and Il6 −/− neonatal AP (mean ± SEM). Data points represent biological replicates ( n = 4; unpaired t -test). ( D ) Organoid development and expansion from WT ( Il6 +/+ ) neonatal AP cells, cultured and exposed to cytokines as indicated. Left : Representative brightfield pictures of organoid cultures (scale bar, 500 μm). Right : Bar graphs showing number of organoids developed per well under the conditions as indicated (mean ± SEM). Data points represent biological replicates ( n = 10 for RSpECT, n = 8 for +LIF, n = 5 for +IL-11, n = 3 for +TNFα and +IL-1β). ( E ) Organoid development and expansion from Il6 −/− neonatal AP cells, cultured and exposed to cytokines as indicated. Left : Representative brightfield pictures of organoid cultures (scale bar, 500 μm). Right : Bar graphs showing number of organoids formed per well under the conditions as indicated (mean ± SEM). Data points represent biological replicates ( n = 8 for RSpECT, n = 6 for +TNFα and +IL-1β, n = 3 for +IL-11 and +LIF; one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test). ( F ) Left : Bar graph depicting relative gene expression level of Il6 (relative to expression in RSpECT, set as 1 [dashed line]) (mean ± SEM). Data points represent biological replicates ( n = 3; two-way ANOVA with Dunnett’s multiple comparisons test). Right : IL-6 protein level in SN medium (collected 3 days after seeding/passaging) of neonatal AP organoids in indicated passages and culture conditions (mean ± SEM). Data point represents mean of two technical replicates ( n = 1). ( G ) Representative brightfield pictures of organoid cultures from neonatal AP treated with STATTIC, or not (RSpECT), in indicated passages (scale bar, 500 μm).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Immunofluorescence, Staining, Labeling, Gene Expression, Cell Culture, Expressing, Passaging

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Heatmap displaying scaled expression of selected WNT-associated differentially expressed genes (DEGs) in the stem cell clusters SC1, SC2, and Prolif SC between adult and neonatal anterior pituitary (AP). ( B ) DEG-associated Gene Ontology (GO) terms linked with WNT signaling enriched in SC1, SC2, and Prolif SC of neonatal versus adult AP. ( C ) Left : Tcf7l1 and Tcf7l2 regulon activity projected on UMAP plot of neonatal AP, with indication of cell clusters. Right : Violin plots displaying regulon activity of Tcf7l1 and Tcf7l2 in the indicated clusters of adult and neonatal AP. ( D ) Dot plot displaying selected WNT-associated ligand–receptor interactions revealed by CellPhoneDB in neonatal SC1, SC2, Prolif SC, and MC clusters. p values are indicated by dot size, means of average expression of interacting molecule 1 in cluster 1 and interacting molecule 2 in cluster 2 are specified by color intensity (see scales on top). ( E ) Organoid development from neonatal AP cells, cultured and exposed to compounds as indicated (P0). Left : Representative brightfield pictures of organoid cultures (scale bar, 500 μm). Right : Bar graphs showing number of organoids formed per well under conditions as indicated (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 6; one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test). ( F ) Top : Schematic of in vivo treatment schedule and analysis. IF, immunofluorescence. Mouse icon obtained from BioRender. Middle : Bar plots depicting relative expression level of indicated genes in neonatal AP of mice treated as indicated (relative to vehicle [VEH], set as 1 [dashed line]) (mean ± SEM) ( n = 3; unpaired t -test). Bottom : Bar graph showing percentage of SOX2 + Ki67 + cells in SOX2 + cell population in neonatal AP of mice treated as indicated (relative to VEH, set as 100% [dashed line]) (mean ± SEM). Data points represent biological replicates ( n = 4; paired t -test). ( G ) UMAP plot of the annotated cell clusters in human fetal pituitary and projection of selected WNT-associated genes’ expression. PL, posterior lobe (pituicyte) cells; CC, cell cycle cells; RBC, red blood cells; Pro.PIT1, progenitor cells of PIT1 lineage; Pre.Gonado, precursor cells of gonadotropes. Figure 3—source data 1. Differentially expressed gene (DEG) and Gene Ontology (GO) analysis in SC1+SC2+Prolif SC of neonatal versus adult anterior pituitary (AP). (A) DEG analysis in SC1+SC2+Prolif SC of neonatal versus adult AP. The columns represent: gene names (Gene), p values (p_val), average log fold change expression (avg_logFC; positive values indicate upregulation and negative values downregulation in neonatal), percentage of cells expressing the indicated gene in neonatal (pct.1) and in adult (pct.2), and the FDR adjusted p value (p_val_adj). (B) Gene Ontology (GO) analysis of genes upregulated in neonatal versus adult SC1+SC2+Prolif SC. Analyses were performed using Reactome overrepresentation analysis. Pathway name, number of mapped identifiers that match the pathway (#Entities found), total number of identifiers in the pathway (#Entities total), total entities in the pathway/total number of entities for the entire species (Entities ratio), p value, FDR, and −log10(FDR) are shown in the columns. Associated with .

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Expressing, Activity Assay, Cell Culture, In Vivo, Immunofluorescence

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Differentially expressed gene (DEG)-based GSEA plots of indicated WNT-related hallmarks in neonatal versus adult stem cell clusters (SC1, SC2, and Prolif SC). Normalized enrichment score (NES), and p and FDR values are listed. ( B ) Heatmaps displaying scaled expression of several WNT ligand and receptor genes in adult and neonatal anterior pituitary (AP). ( C ) Left : Violin plots displaying mRNA expression level of indicated genes in SC1, SC2, MC, and Prolif SC of adult and neonatal AP. Right : Bar graphs displaying relative gene expression of indicated genes in neonatal AP, as determined by RT-qPCR (relative to expression in adult AP, set as 1 [dashed line]) (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 3; unpaired t -test). ( D ) Left : Projection on UMAP plot of selected WNT-associated genes’ expression in neonatal stem cell (SC1, SC2, and Prolif SC) and MC clusters, with indication of cell clusters. Right : RNAscope in situ hybridization analysis of neonatal pituitary for Sox2 (red), Lgr4 , Lgr6 , and Rspo3 (all green). Nuclei are stained with DAPI (blue). Arrowheads indicate the marginal zone (MZ) (scale bar, 100 μm).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, RNAscope, In Situ Hybridization, Staining

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Schematic of in vivo treatment schedule and analysis. DT, diphtheria toxin; m, months. Mouse icons obtained from BioRender. ( B ) Ablation of somatotropes (GH + cells) in neonatal mouse anterior pituitary (AP). Left : Immunofluorescence staining of GH (green) in control and damaged pituitary following DT injection (PD7). Nuclei are labeled with Hoechst33342 (blue) (scale bar, 100 μm). Right : Bar graph showing proportion of GH + cells in AP as indicated (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 5; paired t -test). ( C ) SOX2 + stem cell reaction to damage. Left : Immunofluorescence staining of SOX2 (red) and Ki67 (green) in control and damaged pituitary following DT injection (PD7). Nuclei are labeled with Hoechst33342 (blue) (scale bar, 100 μm). Right : Bar graphs showing proportion of SOX2 + cells in AP as indicated, or of SOX2 + Ki67 + cells in SOX2 + cell population (mean ± SEM). Data points represent biological replicates ( n = 5 for % SOX2 + cells, n = 3 for % SOX2 + Ki67 + cells). ( D ) Regeneration of somatotropes in neonatal mouse AP after their ablation. Left : Immunofluorescence staining of GH (green) in control and damaged pituitary 2 months after DT-induced ablation. Nuclei are labeled with Hoechst33342 (blue) (scale bar, 100 μm). Right : Bar graph showing proportion of GH + cells in AP as indicated (mean ± SEM). Data points represent biological replicates ( n = 4 for Control, n = 5 for Damaged). ( E ) Left : UMAP plot of control and damaged neonatal AP combined. Right : UMAP plot of annotated cell clusters in the integrated neonatal AP samples (i.e., collective single-cell transcriptome datasets from control and damaged AP). ( F ) Bar plots depicting proportion of PIT1 + Ki67 + or GH + Ki67 + cells in PIT1 + or GH + cell population, respectively, 1 week after DT-induced damage (PD14) (mean ± SEM). Data points represent biological replicates ( n = 4 for Control, n = 3–8 for Damaged; unpaired t -test). ( G ) Left : Immunofluorescence staining of SOX2 (red) and GH (green) in control and damaged neonatal pituitary at indicated timepoints (PD7 and PD14) following DT-induced injury. Nuclei are stained with Hoechst33342 (blue). Boxed areas are magnified (scale bar, 100 µm). Right : Bar graph showing proportion of SOX2 + GH + cells in GH + cell population following DT-induced damage (PD7) (mean ± SEM). Data points represent biological replicates ( n = 4; paired t -test).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: In Vivo, Immunofluorescence, Staining, Control, Injection, Labeling

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: ( A ) Bar graphs showing absolute number of SOX2 + cells in anterior pituitary (AP) as indicated, or of SOX2 + Ki67 + cells in SOX2 + cell population following DT injection (PD7) (mean ± standard error of the mean [SEM]). Data points represent biological replicates ( n = 3). ( B ) Organoid development from control and damaged neonatal AP (P0). Left : Representative brightfield pictures of organoid cultures (scale bar, 500 μm). Right : Bar graph showing number of organoids formed per well (mean ± SEM). Data points represent biological replicates ( n = 4). ( C ) Dot plot displaying percentage of cells (dot size) expressing indicated marker genes with average expression levels (color intensity; see scales on top) in the collective neonatal AP samples (i.e., control and damaged neonatal AP). ( D ) Violin plots displaying mRNA expression level of Il6 in SC1, SC2, and MC of control and damaged neonatal AP. ( E ) Left : Heatmap displaying scaled expression of selected WNT-associated genes in control and damaged neonatal AP. Right : Bar plots depicting relative expression level of indicated genes in neonatal damaged AP, at indicated timepoints (PD14 and PD21) (relative to control [CTRL], set as 1 [dashed line]) (mean ± SEM) ( n = 3).

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Injection, Control, Expressing, Marker

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet: Overview of primers, used for RT-qPCR.

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques:

Journal: eLife

Article Title: Decoding the activated stem cell phenotype of the neonatally maturing pituitary

doi: 10.7554/eLife.75742

Figure Lengend Snippet:

Article Snippet: Differently labeled RNAscope probes (Advanced Cell Diagnostics) were used for mouse Sox2 (401041-C3), Rspo3 (483781-C2), Lgr4 (318321-C2), and Lgr6 (404961-C2).

Techniques: Isolation, Sequencing, RNAscope, Recombinant, Multiplex Assay, SYBR Green Assay, Software

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