Journal: Nature Communications

Article Title: A living biobank of ovarian cancer ex vivo models reveals profound mitotic heterogeneity

doi: 10.1038/s41467-020-14551-2

Figure Lengend Snippet: a Phase contrast images showing distinct morphologies of stromal and tumour cells. Scale bar, 200 µm. b Time-lapse imaging measuring confluency showing suppression of proliferation by 1 µM cisplatin and 100 nM paclitaxel. c Immunofluorescence images showing expression of PAX8 and Ki67. Scale bar, 50 µm. d Flow cytometry profiles quantitating the tumour markers EpCAM and CA125, and the stromal markers CD44 and CD105. Numbers represent percentage of cells in the quadrant. e Immunofluorescence images of Nutlin-3-treated cells (OCM.79) showing stabilisation of p53 in stromal cells but not tumour, and DNA sequence showing TP53 mutation in tumour cells (OCM.38a). Scale bar, 20 µm. Data in panels a and c are derived from analysis of OCM.79, while data in panels b and d are derived from analysis of OCMs 38a, and 66-5 respectively. Panels a , c and e are representative images from single experiments. Source data for panels b , c and d are provided as a Source Data file, including the gating/sorting strategy for panel d . See also Supplementary Figs. and .

Article Snippet: For immunofluorescence, cells were plated on collagen- or gelatin-coated 13 mm coverslips and incubated for 48 h. Cells were washed and fixed in 1% formaldehyde, quenched in glycine, then incubated for 30 min at room temperature using the following primary antibodies: rabbit anti-Mucin-16 (Merck Millipore cat#ABC240, 1:50); mouse anti-EpCAM (VU1D9) (Cell Signaling cat#2929, 1:800); rat anti-CD44 (Calbiochem cat#217594,1:200); rabbit anti-Vimentin (EPR3776) (Abcam cat#ab92547, 1:1,000); mouse anti-pan cytokeratin (C-11) (Abcam cat#ab7753, 1:500); mouse anti-Pax8 (Abcam cat#ab53490, 1:100); rabbit anti-Ki67 (Abcam cat#ab15580,1:2,000); and mouse anti-p53 (DO-1) (Santa Cruz Biotechnology cat#sc-126,1:1,000).

Techniques: Imaging, Immunofluorescence, Expressing, Flow Cytometry, Sequencing, Mutagenesis, Derivative Assay

Journal: Oncotarget

Article Title: PAX2 maintains the differentiation of mouse oviductal epithelium and inhibits the transition to a stem cell-like state

doi: 10.18632/oncotarget.20173

Figure Lengend Snippet: (A) OVE4, OVE16 and OVE22 cells express oviductal cell markers ( Cdh1 , Pax2 , Pax8 , Ovgp1 and FoxJ1 ). (B) TGFβ treatment (10ng/ml) for 7 days induced fibroblast-like phenotypes in OVE clonal cell lines and (C) inhibited cell proliferation in all OVE lines, as assessed after 7 days. Data presented in histograms are mean ± SEM. Scale bars indicate 100μm. All experiments were performed at least three times. In A, different letters denote values that are significantly different (p<0.05). In C, * indicates p<0.05; ** indicates p<0.01.

Article Snippet: After blocking in 5% goat serum, cells were probed with each antibody as follows: anti-mouse PAX2 (1:500, Santa Cruz), anti-mouse PAX8 (1:200, Santa Cruz), anti-rabbit E-cadherin (1:200, Abcam) and anti-rat CD44 (1:200, Abcam).

Techniques:

Journal: Oncotarget

Article Title: PAX2 maintains the differentiation of mouse oviductal epithelium and inhibits the transition to a stem cell-like state

doi: 10.18632/oncotarget.20173

Figure Lengend Snippet: (A) Formation of luminal structure by OVE cells cultured in matrigel starts with small spheres by day 4 and ends with spheroids with a lumen by day 14. (B) Immunofluorescence by Z-stack imaging starting from the top of spheroids formed by OVE4 and MCF10a cells in matrigel after 14 days of culture shows the lumen in the middle of each spheroid. (C) OVE4-derived luminal structures express E-cadherin and the oviductal markers, PAX2 and PAX8, as detected by immunofluorescence. (D) Knockdown of PAX2 (right panels) disrupts the formation of luminal structures by OVE4 cells (left panels), which is quantified in (E) . Data are presented as mean ± SEM (N=3). Scale bars in (A-C) indicate 100μm. * indicates p<0.05.

Article Snippet: After blocking in 5% goat serum, cells were probed with each antibody as follows: anti-mouse PAX2 (1:500, Santa Cruz), anti-mouse PAX8 (1:200, Santa Cruz), anti-rabbit E-cadherin (1:200, Abcam) and anti-rat CD44 (1:200, Abcam).

Techniques: Cell Culture, Immunofluorescence, Imaging, Derivative Assay

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) Time course RT-qPCR data for OSR1 and SALL1 over the period of the kidney organoid protocol. Dots and bars represent mean ± SEM, N = 2-4 independent organoid batches at each time point. (B) Representative immunofluorescence image of a renal organoid at day 24 stained for WT1 (green), LTL (white), CDH1 (red) and DAPI (blue). Scale bar is 30µm. (C) UMAP plot of 23,856 sc-RNA-seq and sn-RNA-seq cells harvested across 3 organoid batches, harvested at days 10, 14 and 24, coloured by batch. (D) Violin plots showing number of genes per batch. (E) Violin plots showing percentage mitochondrial reads per batch. Box center line, median; limits, upper and lower quartiles; whiskers, 1.5x interquartile range (F) UMAP plot of 23,856 sc-RNA-seq and sn-RNA-seq cells as in (C), coloured by epithelial or stromal lineage. (G) Heatmap showing top 10 genes that define the epithelial and stromal lineages. (H) UMAP plots of 23,856 sc-RNA-seq and sn-RNA-seq cells as in (C), coloured by PAX8 expression (top) and TWIST1 expression (bottom). (I) UMAP plot of 15,917 sc-RNA-seq and sn-RNA-seq cells corresponding to the epithelial lineage as in (F), re-projected and coloured according to time point. (J) UMAP plots of 15,917 6 sc-RNA-seq and sn-RNA-seq cells as in (I), coloured by MAFB expression (top left), HNF4A expression (top right), TFAP2B expression (bottom left) and GATA3 expression (bottom right). (K) UMAP plots of 15,917 6 sc-RNA-seq and sn-RNA-seq cells as in (I), coloured by cell cycle phase. (L) UMAP plots of 15,917 6 sc-RNA-seq and sn-RNA-seq cells as in (I), coloured by unsupervised clustering, annotated according to querying marker genes for each cluster with the Human Nephrogenesis Atlas ( https://sckidney.flatironinstitute.org ) and assessment of cell cycle phase. (M) Dot plot of gene expression for epithelial genes in 15,917 6 sc-RNA-seq and sn-RNA-seq cells as in (D) separated by time point. (N) Dot plot of gene expression for nephron marker genes in 15,917 6 sc-RNA-seq and sn-RNA-seq cells as in (D) separated by time point.

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: Quantitative RT-PCR, Immunofluorescence, Staining, RNA Sequencing Assay, Expressing, Marker

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) UMAP plot of 17,994 sc-RNA-seq cells from 6 human fetal kidney samples collected between post conception week (pcw) 7 and 16, split by epithelial and stromal lineages, coloured by cell type annotations . (B) UMAP plot of 17,994 sc-RNA-seq cells as in (A), coloured by gene expression of the stromal marker TWIST1 (left) or the nephron marker PAX8 (right). (C) Asymmetric integration of kidney organoid sc-RNA-seq data split by time point with human fetal kidney sc-RNA-seq data, coloured by point density of organoid data. (D) UMAP plots of 8,862 nephron epithelial cells from (A), isolated and re-projected, and coloured by gene expression of nephron marker genes (related to ).

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: RNA Sequencing Assay, Expressing, Marker, Isolation

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) UMAP of 9,147 cells from multi-ome batch 3 projected according to ATAC data, with clusters annotated according to epithelial or stromal lineage. (B) UMAP plots of multi-ome cells from batch 3, coloured by PAX8 gene expression (top) and TWIST1 gene expression (bottom). (C) Heat map of expression of marker genes for clusters in (A) determined by snRNA-seq with representative marker genes annotated (log2 FC > 1, FDR < 0.01, two-sided Wilcoxon rank-sum test). (D) Heat map of marker peaks of accessible chromatin for clusters in (A) determined by snATAC-seq (left, log2 FC > 1, FDR < 0.01, two-sided Wilcoxon rank-sum test). (E) Schematic of the principle by which correlation values, between transcription factor gene expression and corresponding motif accessibility, were used to classify transcription factors as either transcriptional repressors or activators. PCC, Pearson correlation coefficient. (F) Transcription factors plotted according to PCC of gene expression versus corresponding motif accessibility, and log2FC gene expression in the epithelial compared to the stromal lineage as determined by snRNA-seq. Thresholds used to colour points according to principle outlined in (D): PCC > 0.2, Log2FC epithelial lineage > 1.5 (red = activators), PCC < -0.2, Log2FC epithelial lineage > 1.5 (blue = repressors). See also Supplemental Figs S3 and S4.

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: Expressing, Marker

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) UMAP plots of 9,147 multi-ome cells from batch 3 showing gene expression (top) and motif accessibility (bottom) for PAX8 and PAX2, highlighting start of expression of PAX8 at day 10 and of PAX2 at day 12, but accessibility of the shared motif apparent at day 10. (B) Immunofluorescence analysis of representative organoids at days 8, 10, 12 and 14, stained for PAX8 (top) or PAX2 (bottom), scale bars 25µm. (C) Time course RT-qPCR data for PAX8 and PAX2 over the period of the kidney organoid protocol. Dots and bars represent mean ± SEM, N = 2-4 independent organoid batches at each time point. (D) RT-qPCR for PAX2 in organoids generated from PAX2-dCas9-KRAB iPSCs (top 20% sorted, see STAR METHODS) and harvested at day 14, either with no treatment (control, white bars) or following Dox treatment from day 4 (1μM Dox, green bars). N = 3 independent organoid batches. Dots represent data from each batch normalised to corresponding control, with bars representing mean ± SEM. Unpaired t-test, **** (p < 0.0001), compared to control. (E) Immunofluorescence of organoids generated from PAX2-dCas9-KRAB iPSCs and harvested at day 14, either with no treatment (control, top) or following Dox treatment from day 4 (1μM Dox, bottom), showing GFP (green), PAX2 (magenta), E-Cadherin/CDH1 (white) with DAPI as counterstain (blue, nuclei). Scale bars 25µm. (F) Schematic of strategy used to identify peak to gene links (related to ). (G) Violin plots of gene expression of CDH1 , CDH3, CDH4, CDH6 and CDH16 in 9,147 multi-ome cells from batch 3 split by time point (related to ). (H) Immunofluorescence analysis of representative organoids at day 10 showing co-localisation of PAX8 (white) and CDH6 (green). Scale bars 20µm (related to ).

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: Expressing, Immunofluorescence, Staining, Quantitative RT-PCR, Generated

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) Schematic of the dCas9-KRAB CRISPR interference gene perturbation system . (B, C) RT-qPCR for (B) PAX8 and (C) E-Cadherin/CDH1 in organoids generated from PAX8-dCas9-KRAB iPSCs and harvested at day 14, either with no treatment (control, white bars) or following Dox treatment from day 4 (1μM Dox, green bars). N = 3 independent organoid batches. Dots represent data from each batch normalised to corresponding control, with bars representing mean ± SEM. Unpaired t-test, * (p < 0.05), *** (p < 0.001), compared to corresponding control. (D) Immunofluorescence of organoids generated from PAX8-dCas9-KRAB iPSCs and harvested at day 14, either with no treatment (control, top) or following Dox treatment from day 4 (1μM Dox, bottom), showing GFP (green), PAX8 (magenta), E-Cadherin/CDH1 (yellow) with DAPI as counterstain (blue, nuclei). White brackets indicate PAX8 + CDH1 + cells confined to a GFP - region. Scale bars 25µm. (E) Immunofluorescence images showing projected z-stacks of whole organoids of PAX8-dCas9-KRAB organoids at day 14, either with no treatment (control, top) or following Dox treatment from day 4 (1 μM Dox, bottom), showing PAX8 (white), E-Cadherin (red), and DAPI (blue, nuclei) alongside volume render of corresponding E-Cadherin/CDH1 signal (middle), and GFP expression after Dox treatment (green, right bottom). Arrow points to GFP - region that contains PAX8 + CDH1 + cells. Scale bars 30µm. (F) Quantification of volume-rendered E-Cadherin/CDH1 signal in PAX8-dCas9-KRAB organoids at day 14 either with no treatment (Control, white bar) or following Dox treatment from day 4 (1μM Dox, green bar). n = 9 organoids across N = 2 independent batches. Dots represent data from individual organoids, with bars representing mean ± SEM. Unpaired t-test, **** (p < 0.0001). See also Supplemental Fig. S5.

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: CRISPR, Quantitative RT-PCR, Generated, Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) Immunofluorescence analysis of organoids showing PAX8 (white, top) with DAPI as counterstain (blue, nuclei), with corresponding expression of HNF1B and E-Cadherin/CDH1 (green and magenta, respectively, bottom) at day 10, 12 and 14. Scale bars 25µm. (B) Footprint analysis of PAX8 and PAX2 motifs using cluster annotations from . (C) GO terms overrepresented in the PAX8 regulon (full list in Supplemental Table 5). (D-F) Chromatin accessibility tracks for (D) CDH6 , (E) E-Cadherin/CDH1 and (F) HNF1B loci, split according to clusters annotated in , epithelium day 10, 12, 14 and stroma (all time points combined), with peak to gene links highlighted. Violin plots of gene expression determined by snRNA-seq (box center line, median; limits, upper and lower quartiles; whiskers, 1.5x interquartile range). Purple asterisks indicate overrepresentation of PAX8 motifs as determined by analysis with HOMER (see STAR METHODS). See also Supplemental Fig. S5.

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: Immunofluorescence, Expressing

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) UMAP plot of 3,040 cells from batch 2 coloured according to tube epithelial pseudotime trajectory, generated in ArchR . (B) Cells in batch 2 plotted according to tube epithelial pseudotime as in (A), and gene expression (left) or motif accessibility (right), of the transcriptional activators PAX8, PAX2 which show consistent motif accessibility along pseudotime, an example upregulated activator HNF1B, and example downregulated activators LEF1 and TCF7 . (C) Cells in batch 2 plotted according to tube epithelial pseudotime as in (A), and gene expression (left) or motif accessibility (right), of example downregulated transcriptional repressors SNAI2, and ZEB1, and example upregulated repressor FOXK1 .

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: Generated, Expressing

Journal: bioRxiv

Article Title: Identification of a core transcriptional program driving the human renal mesenchymal-to-epithelial transition

doi: 10.1101/2023.04.30.538857

Figure Lengend Snippet: (A) Immunofluorescence of organoids at day 12 from same batch as shown in (B) and in Main , showing expression of CDH1 (magenta), ZO1 (yellow) and HNF1B (green). Scale bars 20µm (top), 40µm (bottom). (B) Insets from of organoids treated with the indicated conditions between days 12 to 14, to show individual channels of CDH1 (magenta), ZO1 (yellow) and HNF1B (green). Scale bars 20µm. (C) Schematic of experiment to test reversibility of effect of sustained Wnt/β-Catenin signalling with CHIR washout between days 14 and 17. (D) Organoids harvested at day 17 following treatments indicated in (A), with light microscopy images (top, scale bar 400µm), and immunofluorescence images showing expression of HNF1B (green), E-Cadherin/CDH1 (magenta) and ZO1 (yellow). Dotted white boxes indicate positions of magnification panels below. Scale bars 50µm. (E) Schematic of renal organoid multi-ome profiling strategy for batch 3 – corresponding to schematic in Main but including including the two day 14 samples sequenced in parallel: one treated with DMSO from day 12 to 14 (analogous to normal protocol), and one treated with 3µM CHIR from day 12 to 14. (F) UMAP plot of 13,502 multi-ome cells from batch 3, of organoids harvested at day 10, day 12, and at day 14 treated either with DMSO or 3µM CHIR from day 12 to 14 (See ), coloured according to sample. (G) UMAP plots of 13,502 multi-ome cells from batch 3 as in (D), coloured according to gene expression of PAX8 (left) , EPCAM (centre) , or TWIST1 (right). (H) Gene ontology analysis of genes enriched in day 14 epithelium treated with DMSO from day 12-14 compared to 3µM CHIR, showing top 5 highest specific GO Molecular Function terms by log10 adjusted p value, full list in Supplemental Table 7.

Article Snippet: Primary antibodies used for protein detection, with their corresponding dilutions for immunofluorescence (IF) were as follows: rabbit anti-PKC σ (C-20) (Santa Cruz Biotech, sc-216, 1:200), mouse anti-Integrin beta 1 [12G10] (Abcam, ab30394, 1:200), goat anti-TCF-2/HNF-1 beta (R&D Systems, AF3330, 1:200), rabbit anti-ZO1 (Invitrogen, 40-2200, 1:200), mouse anti-E-Cadherin (BD Bioscience, 610181, 1:200), goat anti-E-Cadherin (R&D Systems, AF648, 1:200), rabbit anti-PAX8 [EPR13511] (Abcam, ab189249, 1:200), mouse anti-PAX8 (Proteintech, 60145-4-Ig, 1:200), mouse anti-PAX2 (Developmental Studies Hybridoma Bank (DSHB), PCRP-PAX2-1A7, 1:20), rabbit anti-GFP (Abcam, ab290, 1:200), rabbit anti-WT1 [CAN-R9(IHC)-56-2] (Abcam, ab89901, 1:200), sheep anti-Cadherin-6/KCAD (R&D Systems, AF2715, 1:200).

Techniques: Immunofluorescence, Expressing, Light Microscopy

Journal: Biology Open

Article Title: Lateral line placodes of aquatic vertebrates are evolutionarily conserved in mammals

doi: 10.1242/bio.031815

Figure Lengend Snippet: Lateral line placodes of Q-VD-OPh-treated mice reveal the specific molecular properties of posterior placodes. (A–C,G) Micrographs taken from serially sectioned mouse embryos, with their positions being shown in the preceding reconstructions that demonstrate ectoderm (light grey), otic pit (A,C) or otic vesicle (B) with detachment site (dark grey), epibranchial placodes (orange), lateral line placodes (blue), plane of sectioning (black line in reconstructions). (A) Pax8 immunopositivity is present in epibranchial placode 1, in the prospective anterodorsal lateral line placode as well as in remaining parts of the thickened PPA. (B) Close apposition of Pax8 + epibranchial placode 2 and Pax8 − posterior lateral line placode. (C) Anterodorsal lateral line and otic placodes spring from a common Sox10 + domain. (D) Neuromast primordium of an anterodorsal lateral line placode with Sox10 + mantle (m) and support cells (s). (E,F) During the peak period of PPA apoptosis, in utero -developed control embryos [embryonic day 9.5 (E9.5)] demonstrate disorganized Sox10 + (E) or predominantly Sox10 − ectodermal cells (F , but see arrow) as well as high numbers of apoptotic cells (arrowheads) in the positions of vestigial lateral line placodes, here shown for an anterodorsal placode. (G) Scattered Tbx3 + cells in a middle lateral line placode (boxed area enlarged in H). (I) Neuromast primordium of an anterodorsal lateral line placode with Tbx3 + mantle (m) and support cells (s). Images A–C are stitched from two micrographs using Corel Photo-Paint. All micrographs were adjusted for brightness (including slight gamma changes), colour balance, and sharpness. Scale bars: 20 µm in A–C,G, 5 µm in D–F,H and I. ad, m, p, anterodorsal, middle, and posterior lateral line placode, respectively; e1, e2, e3, epibranchial placodes 1, 2, 3, respectively; gg, geniculate ganglion; ot, otic anlage; ov, optic vesicle; pg, petrosal ganglion; PPA, posterior placodal area; Q-VD-OPh, pan-caspase inhibitor; wec, whole embryo culture.

Article Snippet: Primary antibodies included: mouse anti-Atonal homolog 1 (Atoh1; Atoh1 supernatant, Developmental Studies Hybridoma Bank, Iowa City, USA, lot 6/20/13, RRID: AB_10805299; 1:50, overnight, 4°C) ( ; ), mouse anti-β-Tubulin-III (Tubb3; clone SDL.3D10, T8660, Sigma-Aldrich, lot 073K4835, RRID: AB_477590; 1:8000, overnight, 4°C) , rabbit anti-cleaved caspase-3 (9661; Cell Signaling Technology, lot 37, RRID: AB_2341188; 1:8000, overnight, 4°C) ( , ), mouse anti-Islet 1 (Isl1; 39.4D5 ascites fluid, Developmental Studies Hybridoma Bank, lot 2/12/09, RRID: AB_2314683; 1:2000, overnight, 4°C) , goat anti-Neurogenin1 (Ngn1; sc-19231, Santa Cruz Biotechnology, lot C1215, RRID: AB_2298242; 1:100, 4 h, 37°C) , mouse anti-Neurogenin2 (Ngn2; clone 7G4, MAB3314, R&D Systems, Minneapolis, USA, lot WWI01, RRID: AB_2149520; 1:20,000, overnight, 4°C) , goat anti-Neurogenic differentiation 1 (NeuroD1; sc-1084, Santa Cruz Biotechnology, lot A0616, RRID: AB_630922; 1:800, 2 h, 37°C) , rabbit anti-Paired homeobox (Pax) 2 (71-6000; Thermo Fisher Scientific, lot 1117672A, RRID: AB_2533990; 1:4000, overnight, 4°C) , mouse anti-Pax8 (ACI 438; Biocare Medical, Pacheco, USA, lot 051712, RRID: AB_10922964; 1:100, overnight, 4°C) , rabbit anti-phospho-Histone H3 (pHH3; 9701, Cell Signaling Technology, lot 13, RRID: AB_331535; 1:1000, 2 h, 37°C) , rabbit anti-Sine oculis homeobox 1 (Six1; HPA001893, Atlas Antibodies, Stockholm, Sweden, lot A00939, RRID: AB_1079991; 1:800, overnight, 4°C) , mouse anti-Six1 (clone CLO185, AMAb90544, Atlas Antibodies, lot 02852, RRID: AB_2665581; 1:100, overnight, 4°C) that, compared to rabbit anti-Six1, was raised against the same immunogen and shows identical reactivities (present study), mouse anti-Sex determining region Y-box (Sox) 2 (clone 245610, MAB2018, R&D Systems, lots KGQ0311111, KGQ0315062, RRID: AB_358009; 1:200, overnight, 4°C) , goat anti-Sox10 (sc-17342, Santa Cruz Biotechnology, lot L2908, RRID: AB_2195374; 1:800, overnight, 4°C) , mouse anti-Sox10 (sc-365692, Santa Cruz Biotechnology, lot I0516, RRID: AB_10844002; 1:1600, overnight, 4°C) that, compared to goat anti-Sox10, produced identical staining patterns in mice (present study) and rats , and rabbit anti-T-box transcription factor (Tbx) 3 (42-4800, Thermo Fisher Scientific, lot SI256120, RRID: AB_2533526; 1:200, 2 h, 37°C) ( ).

Techniques: In Utero, Embryo Culture