Journal: Investigative Ophthalmology & Visual Science

Article Title: Ontogenesis of the Mouse Ocular Surface Lymphatic Vascular Network

doi: 10.1167/iovs.64.15.7

Figure Lengend Snippet: Lymphvasculogenesis constitute a cellular process for ocular lymphatic vessel development. ( A ) Visualization of the presence of a LYVE-1+ Prox-1+ cell cluster ( box ) at the P0 ocular surface after whole mount immunofluorescent staining of the eye anterior segment and flat-mounting. The yellow asterisks mark the location of two lymphatic sprouts. The green arrows point to some isolated LYVE-1–positive cells that correspond to macrophages. ( B , C ) Imaging of the boxed LYVE-1+ Prox-1+ lymphatic cell cluster at a higher magnification. ( D ) LSFM imaging of some LYVE-1-positive lymphatic vessel fragments ( yellow arrows ), which have formed at the corneal and corneolimbal surfaces of a P1 neonate. Co, cornea. ( E ) LYVE-1 immunofluorescence staining image obtained at P1, allowing the view of both lymphangiogenic sprouting (sprout extremities are pointed by white arrows ), and a lymphatic vessel fragment, which could have been formed by lymphvasculogenesis ( yellow arrow ).

Article Snippet: Goat anti-mouse LYVE-1 (AF2125), rat anti-mouse LYVE-1 (MAB2125), goat anti-mouse Prox-1 (AF2727), and goat anti-mouse CD80 (AF740) were obtained from Biotechne (Minneapolis, MN, USA).

Techniques: Staining, Isolation, Imaging, Immunofluorescence

Journal: Investigative Ophthalmology & Visual Science

Article Title: Ontogenesis of the Mouse Ocular Surface Lymphatic Vascular Network

doi: 10.1167/iovs.64.15.7

Figure Lengend Snippet: Evidence for the existence of mixed Prox-1– and CD206-positive cells in lymphatic neovessels. Whole mount double immunofluorescence staining with LYVE-1 and CD206 antibodies ( A–C ) or with Prox-1 and CD206 antibodies ( D–F ), of the developing ocular surface lymphatic vasculature at P1. The yellow asterisk marks a CD206-positive macrophage displaying a low LYVE-1 expression when compared to the LYVE-1 expression level of cells constituting the lymphatic neovessel. The white arrows point to the CD206 immunoreactivity in some LYVE-1– ( upper panels ) or Prox-1–positive cells ( lower panels ) of the lymphatic neovessel.

Article Snippet: Goat anti-mouse LYVE-1 (AF2125), rat anti-mouse LYVE-1 (MAB2125), goat anti-mouse Prox-1 (AF2727), and goat anti-mouse CD80 (AF740) were obtained from Biotechne (Minneapolis, MN, USA).

Techniques: Double Immunofluorescence Staining, Expressing

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

Journal: Hippocampus

Article Title: Neuronal activity-related transcription is blunted in immature compared to mature dentate granule cells

doi: 10.1002/hipo.23515

Figure Lengend Snippet: Reduced immediate early gene (IEG) protein expression in immature DGCs. (a) Experimental timeline. Mice received tamoxifen (TAM) gavage for 7 days to label DGCs and were exposed to a novel environment (NE) to stimulate activation 5 or 13 weeks (wk) later. Untreated littermates collected directly from the home cage (HC) were used as a control. (b) Sample of durable GFP labeling 13 weeks after TAM in the DG. Scale bar: 20 μm. Yellow arrows indicate a cell triple labeled for GFP, ARC, and FOS. Dashed yellow line indicates the boundaries of the granule cell layer (GCL). (c) NE exposure induces significantly more FOS and ARC expression than the home cage control condition (n’s: HC = 6, 5-wk NE = 7, 13-wk NE = 7). (d) Quantification of active 5-week and 13-week DGCs in coronal tissue sections shows greater IEG expression in more mature cells (n’s: 5-wk = 7, 13-wk = 7). (e) Quantification of active 5-week and 13-week DGCs via flow cytometry (FACS) also shows greater IEG expression in more mature cells (n’s: Arc 5-wk = 7, Arc 13-wk = 7, Fos 5-wk = 10, Fos 13-wk = 9). (f) Co-expression of DG marker proteins PROX1 and CTIP2 occurs in the majority of GFP+ nuclei assessed by FACS, but co-expression continues to increase between 5 and 13 weeks (n’s: 5-wk = 10, 13-wk = 9). (g) Sample FACS gating strategy for identifying active immature DGCs. DGCs were identified by co-labeling with marker proteins PROX1 and CTIP2. Cell age was identified by GFP. Cell activation was assessed with ARC or FOS. (h) Experimental timeline for seizure treatment. Mice received TAM gavage for 7 days to label DGCs and were given pentylenetetrazole (PTZ) 5 or 13 weeks later. PBS-injected littermates served as staining controls. Mice were perfused following a 90-min observation period after the injection. (i) Top: Sample of FOS labeling following either PBS (left) or PTZ (right). Scale bar: 50 μm. Bottom: Sample of cell classification in PTZ-treated mice. Turquoise arrows indicate a cell triple labeled for GFP, CTIP2, and FOS. Yellow arrows indicate a cell double labeled for GFP and FOS, negative for CTIP2. Dashed yellow line indicates GCL boundaries. Scale bar: 20 μm. (j) Quantification reveals that CTIP2−GFP+ immature cells activate at significantly lower rates than CTIP2+GFP+ immature cells. CTIP2+GFP+ immature cells still have reduced FOS relative to mature CTIP2+GFP− cells at 5 weeks after TAM even under seizure conditions (n = 4, within-subject comparison). Points and n’s in dot plots represent mice. Error bars are mean ± S.E.M. *p < .05, **p < .01, ***p < .001, ns = not significant.

Article Snippet: Mouse anti-PROX1 (EMD Millipore #MAB5654, RRID:AB_2170714).

Techniques: Expressing, Activation Assay, Labeling, Flow Cytometry, Marker, Injection, Staining, Comparison