differently labeled rnascope probes  (Advanced Cell Diagnostics Inc)

Journal: bioRxiv

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

doi: 10.1101/2022.01.18.476723

Figure Lengend Snippet: Neonatal pituitary stem cells show a pronounced WNT profile. (A) DEG-based GSEA plots of indicated WNT-related hallmarks in neonatal versus adult stem cell clusters (SC1, SC2, 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 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 ± SEM). Data points represent biological replicates. ** P ≤ 0.01. (D) Left: Projection on UMAP plot of selected WNT-associated genes’ expression in neonatal stem cell (SC1, SC2, 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). (E) Bar graph displaying relative gene expression level of indicated genes in neonatal AP-derived organoids (mean ± SEM). Data points represent biological replicates. (F) Organoid development from neonatal AP cells, cultured and exposed to WNT ligands as indicated (P0). Top: Representative brightfield pictures of organoid cultures. (Scale bar, 500 μm). Bottom: Bar graph showing number of organoids formed under conditions as indicated (mean ± SEM). Data points represent biological replicates. (G) Top: Immunofluorescence staining of Ki67 (red) in neonatal AP organoids formed under conditions as indicated (P0). Nuclei are stained with Hoechst33342 (blue). Arrowheads indicate Ki67 + cells. (Scale bar, 100 µm). Bottom: Bar graphs showing percentage of Ki67 + cells in organoids as indicated (relative to DMSO, set as 1 (dashed line)) (mean ± SEM). Data points represent individual organoids from 3 biological replicates. * P ≤ 0.05. (H) Top: Projection on UMAP plot of Rspo1 gene expression in neonatal stem cell (SC1, SC2, Prolif SC) and MC clusters, with indication of cell clusters. Bottom: Organoid development from neonatal AP cells formed in standard RSpECT medium (with RSPO1) or RSpECT medium in which RSPO1 was replaced with RSPO3 (P0). Representative brightfield images are shown. (Scale bar, 500 μm). (I) Organoid development from adult AP cells formed under conditions as indicated (P0). Top left: Representative brightfield pictures of organoid cultures. (Scale bar, 500 μm). Bottom left: Immunofluorescence staining of Ki67 (green) in adult AP organoids, formed under conditions as indicated. Nuclei are stained with Hoechst33342 (blue). (Scale bar, 100 µm). Middle: Bar graph showing percentage of Ki67 + cells in organoids as indicated (relative to PitOM, set as 1 (dashed line)) (mean ± SEM). Right: Violin plot showing diameter of organoids developed in conditions as indicated. Data points represent biological replicates. * P ≤ 0.05. (J) Left: UMAP plot of fetal human and neonatal mouse AP combined. Middle and right: UMAP plot of annotated cell clusters in human and mouse AP, respectively. Somato, somatotropes; Lacto, lactotropes; Cortico, corticotropes; Gonado, gonadotropes; Thyro, thyrotropes; Melano, melanotropes; SC1 and SC2, stem cell cluster 1 and 2; EC, endothelial cells; IC, immune cells; CT, connective tissue cells; PL, posterior lobe (pituicyte) cells; Gonado Prog, gonado progenitor cells; Gonado Prec, gonadotrope precursor cells; CC, cell cycle cells; RBC, red blood cells, Pro.PIT1, progenitor cells of PIT1 lineage; Pre.Gonado, precursor cells of gonadotropes. (K) Projection of Il6 gene expression on human fetal pituitary UMAP plot, with indication of cell clusters.

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, Quantitative RT-PCR, In Situ Hybridization, Staining, Derivative Assay, Cell Culture, Immunofluorescence