Journal: bioRxiv

Article Title: A second wave of Notch signaling diversifies the intestinal secretory lineage

doi: 10.1101/2024.07.15.603542

Figure Lengend Snippet: A) Diffusion maps of rat intestinal epithelial cells, showing their differentiation as a measure of pseudotime. E1-E6 denote enterocytes of increasing maturation. B) Reclustered UMAP of cells arising from stem and secretory progenitor clusters. C) Diffusion maps of stem cells and secretory progenitors as a readout of pseudotime. Note the clear bifurcation between Goblet/Paneth and the EEC/Tuft/CHE lineages. D) Diffusion map color coded for Atoh1 expression. E) Atoh1 (magenta) and Goblet cell staining (Muc2; cyan). F) Atoh1 (magenta) and Best4 (cyan; CHE marker) costaining. G) Atoh1 (magenta) and Dclk1 (cyan; Tuft cell marker) costaining. H) Diffusion map of stem and secretory cells color coded for Prox1 expression. I) CFTR (cyan; CHE marker) and Prox1 (magenta) co-staining. J) ChgA (cyan; EEC marker) and Prox1 (magenta) costaining. Arrow marks Prox1+/ChgA+ cell, red arrowhead marks Prox1-/ChgA+ cell. K-K’) Dclk1 (cyan, Tuft cell marker) and Prox1 costaining (in magenta, K’). L) Proportion of Tuft cells and EECs in rat proximal jejunum that are Prox1+. n=2 animals. Error bars, SD. M) Diffusion map of stem and secretory cells color-coded for Meis1 expression. N) Meis1 (magenta) and CFTR (cyan, CHE marker) costaining. DAPI in white. O-Q) Meis1 (magenta) staining with O) Muc2 (cyan, Goblet cells), P) Dclk1 (cyan, Tuft cells) or Q) ChgA (cyan, EECs). All scale bars, 10 μm.

Article Snippet: The remaining primary antibodies are commercially available: Mouse anti-Meis1 (1:100 O/N, Invitrogen, MA5-27191), Mouse anti-Prox1 (1:100 O/N, Novus Biologicals, NBP1-30045), Goat anti-Notch2 (1:100 15min, R&D Systems, AF1190-SP), Rabbit anti-Muc2 (1:1000 O/N, Abcam ab272692), Rabbit anti-Dclk1 (1:1000 O/N, Abcam ab31704), Rabbit anti-ChgA (requires prefixation, 1:500 O/N, Abcam, ab254557), Sheep anti-Dll1 (1:100 O/N, R&D Systems, AF5026-SP), Rabbit anti-Ki67 (1:200,1hr, Abcam, ab15580), Rabbit anti-Phospho Histone3 (1:500, 1hr, Cell Signaling, 3377), Rabbit anti-Hes1* (1:2500 Cell Signaling, 11988S) * requires 7.5 min incubation with Tyramide Signal Amplification Kit (ThermoFisher, B40922) Secondary antibodies were used at 1:200 as follows, except for DAPI (5µg/mL, Invitrogen, D1306).

Techniques: Diffusion-based Assay, Expressing, Staining, Marker

Journal: bioRxiv

Article Title: A second wave of Notch signaling diversifies the intestinal secretory lineage

doi: 10.1101/2024.07.15.603542

Figure Lengend Snippet: A) Dotplot from scRNA-seq data showing relative expression of factors associated with Notch signaling across intestinal epithelial cell types. B-C) Module scores for Notch-on (B) and Notch-off (C) gene signatures. D) Meis1 (magenta, CHEs) and Notch2 receptor (cyan) costaining. DAPI in white. E) Rat proximal jejunum stained for Best4 (cyan), Prox1 (yellow), and Notch2 (magenta). E’) triple positive CHEs in the differentiated villus region, E’’-E’’’) Notch2+/Prox1+ cells in crypts that do not express Best4. Color of insert box denotes approximate localization along the crypt-villus axis (left). DAPI in white. F) Rat proximal jejunum stained for Notch2 (magenta) and Meis1 (yellow) with CFTR (cyan). Color of insert box denotes approximate localization along the crypt-villus axis (left; F’-F”’). In F”’, magenta asterisk denotes a stromal non-epithelial expressing Meis1. G-G”) Notch2+ /Prox1+ cell expressing Ki67. Notch2 in yellow, Prox1 in magenta, Ki67 in cyan. H-H’) Notch2+/Prox1+ crypt cells in active mitosis. Note symmetric cell division for both Notch2 (yellow) and Prox1 (magenta). H’) Low level of phospho-histone 3 (in cyan) as cell is exiting mitosis. I-I’) Notch2+/Meis1 Low cells in active mitosis. Notch2 (magenta), Meis1 (yellow), DAPI in white. J-J’) Notch2+ cell lacking Meis1 in active mitosis. Notch2 (magenta), Meis1 (yellow), DAPI in white. All scale bars, 10 μm.

Article Snippet: The remaining primary antibodies are commercially available: Mouse anti-Meis1 (1:100 O/N, Invitrogen, MA5-27191), Mouse anti-Prox1 (1:100 O/N, Novus Biologicals, NBP1-30045), Goat anti-Notch2 (1:100 15min, R&D Systems, AF1190-SP), Rabbit anti-Muc2 (1:1000 O/N, Abcam ab272692), Rabbit anti-Dclk1 (1:1000 O/N, Abcam ab31704), Rabbit anti-ChgA (requires prefixation, 1:500 O/N, Abcam, ab254557), Sheep anti-Dll1 (1:100 O/N, R&D Systems, AF5026-SP), Rabbit anti-Ki67 (1:200,1hr, Abcam, ab15580), Rabbit anti-Phospho Histone3 (1:500, 1hr, Cell Signaling, 3377), Rabbit anti-Hes1* (1:2500 Cell Signaling, 11988S) * requires 7.5 min incubation with Tyramide Signal Amplification Kit (ThermoFisher, B40922) Secondary antibodies were used at 1:200 as follows, except for DAPI (5µg/mL, Invitrogen, D1306).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: A second wave of Notch signaling diversifies the intestinal secretory lineage

doi: 10.1101/2024.07.15.603542

Figure Lengend Snippet: Working model of CHE cell differentiation in which: 1) CHEs arise from the secretory lineage, which is initially Notch off, 2) Notch is reactivated along the secretory lineage as a potentially multipotent Notch2+/Prox1+ progenitor and 3) Meis1 is upregulated to promote the expression of differentiated CHE markers, including CFTR and Best4.

Article Snippet: The remaining primary antibodies are commercially available: Mouse anti-Meis1 (1:100 O/N, Invitrogen, MA5-27191), Mouse anti-Prox1 (1:100 O/N, Novus Biologicals, NBP1-30045), Goat anti-Notch2 (1:100 15min, R&D Systems, AF1190-SP), Rabbit anti-Muc2 (1:1000 O/N, Abcam ab272692), Rabbit anti-Dclk1 (1:1000 O/N, Abcam ab31704), Rabbit anti-ChgA (requires prefixation, 1:500 O/N, Abcam, ab254557), Sheep anti-Dll1 (1:100 O/N, R&D Systems, AF5026-SP), Rabbit anti-Ki67 (1:200,1hr, Abcam, ab15580), Rabbit anti-Phospho Histone3 (1:500, 1hr, Cell Signaling, 3377), Rabbit anti-Hes1* (1:2500 Cell Signaling, 11988S) * requires 7.5 min incubation with Tyramide Signal Amplification Kit (ThermoFisher, B40922) Secondary antibodies were used at 1:200 as follows, except for DAPI (5µg/mL, Invitrogen, D1306).

Techniques: Cell Differentiation, Expressing