Journal: Nature Neuroscience

Article Title: Placental endocrine function shapes cerebellar development and social behavior

doi: 10.1038/s41593-021-00896-4

Figure Lengend Snippet: a–h , Cerebellar sagittal 40-μm-thick sections through the vermis in males. a-b , FluoroMyelin (FM) green staining. c-d , NeuN-immunofluorescence (marker for neuronal nuclei). e-f , NeuroD1-immunofluorescence (marker for granule cells). g-h , Calbindin-immunofluorescence (Purkinje cell marker; in magenta) and DAPI (in blue). i , Cerebellar layer volumes were unchanged in plKO when compared with C mice. Data is presented as means ± SEM from 6 sections/animal. Two-way ANOVA with Sidak’s multiple comparisons test. n = 5 C and 7plKO (WM: p = 0.48; GCL: p = 0.98; ML: p = 0.22). j , Purkinje cell linear density in the lobule VI-VII of C and plKO males at P30. Data is presented as means ± SEM from 6 sections/animal. Two-tailed unpaired Student’s t test with Welch’s correction. n = 6 C and 7plKO (p = 0.556). Calb, calbindin; GCL, granule cell layer; ML, molecular layer; WM, white matter. Scale bar, 200 μm.

Article Snippet: Immunohistochemistry on brain or placenta sections was performed using the following antibodies: rabbit anti-MBP (Abcam, no. ab40390, 1:500), mouse anti-APC clone CC1 (EMD Millipore, no. MABC20, 1:500), rabbit anti-Olig2 (Abcam, no. ab9610, 1:500), mouse anti-neuronal nuclei (NeuN) (Millipore, no. MAB377, 1:500), goat anti-NeuroD1 (RD Systems, no. AF2746, 1:500), rabbit anti-calbindin (Swant, no. CB38, 1:1,000), rat anti-mouse PDGFRα (CD140a; BD Biosciences, 17-1401-81, 1:500) and chicken anti-GFP (Abcam, no. ab13970, 1:500).

Techniques: Staining, Immunofluorescence, Marker, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: Dynamics of the H3K27ac mark following NeuroD1 expression. (A) Schematic of analysing H3K27ac dynamics upon NeuroD1 expression. (B) Global dynamics of H3K27ac enrichment before and after NeuroD1 induction (12 h and 48 h). R1 and R2 are replicates. (C) Boxplots show average H3K27ac enrichment in each cluster (C1 to C7). Middle bars show median values, boxes show first to third interquartile ranges, whiskers show the minimum and maximum of data range and beyond that are outliers. (D) NeuroD1-bound and transcriptionally induced sites. (E) Motif enrichment profiles at transcriptionally active and inactive sites.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: Identification of potential NeuroD1 co-factors. (A) Schematic of ISMARA analysis for co-enriched TF selection. (B) Overlap of upregulated motifs predicted from ISMARA with bHLH transcription factors. (C) Activity profiles for selected motifs from the overlap of ISMARA motifs and bHLH factors. R1 and R2 are the replicates for each time point (0 h, 12 h and 48 h). Data are mean±s.d. (D) Expression of selected factors in different cortical layers. (E) In situ hybridisation images (Allen Brain Atlas) showing the expression of NeuroD1 and Tcf12 in developing cortex at E11.5, E13.5 and E15.5. (F) Bar chart showing the expression level of NeuroD1 in VZ, SVZ and CP regions. (G) Bar chart showing the expression level of Tcf12 in VZ, SVZ and CP regions. (H) Bar chart showing the expression level of Tcf12 in proliferating progenitors (PP), differentiating progenitors (DPs) and neurons (NNs). Data are mean±s.d. Statistical significance was calculated using a paired two-tailed Student's t -test. * P <0.05, ** P <0.01, *** P <0.001.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Selection, Activity Assay, Expressing, In Situ, Hybridization, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: NeuroD1 and Tcf12 are co-expressed in distinct subpopulations during cortical development. (A) Expression of NeuroD1 and Tcf12 in different brain cell types in E14.5 cortex shows the highest NeuroD1 expression in the migrating neuronal population and the Tcf12 expression levels. (B) Pseudo-timecourse trajectory of SVZ migrating cells. Numbers show the clusters arranged in developmental pseudotime. (C) Expression of progenitor, neurogenic and neuronal markers in a SVZ migrating subpopulation arranged by pseudotime, where we see increasing NeuroD1 expression levels.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Expressing

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: Tcf12-NeuroD1 complex induces active chromatin and expression of neuronal migration genes. (A) Co-IP experiment showing GFP-tagged NeuroD1 interacting with HA-tagged Tcf12 in vitro . This specific interaction was absent in control immunoprecipitates where we co-expressed HA-tagged Tcf12 with only GFP protein. (B) IP experiment showing the specific enrichment of endogenous Tcf12 by NeuroD1 only in the negative control siRNA (left panel). It was absent in Tcf12 knockdown conditions (right panel) during in vitro neurogenesis. (C) IP experiment from the E14.5 mouse cortex showing specific interaction of Tcf12 with NeuroD1 during cortical development in vivo . (D) ChIP qPCR results showing the enrichment of Tcf12 at NeuroD1 target sites after 12 h and 48 h of NeuroD1 induction. (E) ChIP Re-ChIP qPCR experiment showing the NeuroD1 and Tcf12 co-binding at the same target genes. (F) qPCR results showing a change in H3K27ac levels at NeuroD1 target sites upon Tcf12 knockdown. (G) qPCR results showing the expression of NeuroD1 target genes upon Tcf12 knockdown. All the ChIP experiments were performed as three independent biological replicates. The ChIP-Re-ChIP experiment was repeated twice. Data are mean±s.d. Statistical significance was calculated using a paired two-tailed Student's t -test. * P <0.05, ** P <0.01.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Expressing, Migration, Co-Immunoprecipitation Assay, In Vitro, Control, Negative Control, Knockdown, In Vivo, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: Development (Cambridge, England)

Article Title: Tcf12 and NeuroD1 cooperatively drive neuronal migration during cortical development

doi: 10.1242/dev.200250

Figure Lengend Snippet: A schematic showing how the functional cooperativity of Tcf12 and NeuroD1 in specific subpopulations of the developing cortex creates the gene regulatory program essential for neuronal migration. Both Tcf12 and NeuroD1 are highly co-expressed during the transition of cortical progenitors from proliferative to neurogenic divisions. During this phase, Tcf12 forms a complex with NeuroD1 and co-occupies a subset of NeuroD1 target loci. This Tcf12-NeuroD1 cooperativity is essential for a gain in active chromatin and expression of neuronal migration genes, and therefore for the correct migration of newborn neurons.

Article Snippet: The eluted fractions were immunoblotted with Tcf12 antibody (SCBT sc-28364 X) and NeuroD1 antibody (CST 4373S) (1:1000).

Techniques: Functional Assay, Migration, Expressing

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Representative images from IHC staining on human SCLC biopsies for given markers. One row = one tumour. Scale bar=25 μm. (b) Venn diagram depicting number of human SCLC biopsies (n=119 total) staining positive for ASCL1, NEUROD1, POU2F3, or lacking all markers (“Subtype-Neg”). (c) Representative images from co-IF staining on human SCLC biopsies for DAPI (nuclei, blue), ASCL1 (yellow), NEUROD1 (purple) and POU2F3 (green). Individual channels (top) and an overlay without DAPI (bottom) are shown. Scale bars=50 μm (n=28 biopsies stained). (d) Representative images from co-IF staining on patient-derived-xenografts (PDX, n=2 distinct models) for DAPI (nuclei, blue), ASCL1 (green), NEUROD1 (purple) and POU2F3 (red). Individual channels (left) and an overlay without DAPI (right) are shown. Yellow arrows and insets (a-b) emphasize co-expressing cells (bottom). Scale bars=75 μm.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Immunohistochemistry, Staining, Derivative Assay, Expressing

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Schematic depicting method to induce SCLC from basal cells in the RPM GEMM. (b) Survival of RPM mice infected with indicated cell type-specific Ad-Cre viruses. Number of mice indicated in the figure. Dashed lines indicate historical data . Mantel-Cox log-rank test comparing each cohort to K5-Cre (purple); **** p<0.0001; ns=not significant, p>0.05. (c) Hematoxylin and eosin (H&E) staining of RPM K5-Cre tumours. Representative whole lung lobes (left) and individual tumour morphology (right) depicted. Scale bars=1 mm (left) or 50 μm (right). (d) Representative IHC images from RPM tumours initiated by indicated Ad-Cre viruses for ASCL1, NEUROD1, and/or POU2F3. Scale bars=50 μm for main images, 10 μm for high magnification insets. (e) Quantification of number of POU2F3 + tumours (H-score >50) vs total tumour number per lung per mouse for indicated cell-type-specific or CMV-Cre adenoviruses. Each dot represents lungs of one mouse. Number of mice indicated in the figure. Error bars represent mean ± SEM. (f) H-score quantification of IHC on K5-Cre RPM tumours vs other cells of origin for indicated proteins. Each dot represents one tumour. For each marker, n=11-101 tumours quantified from n=4-19 mice per Ad-Cre group. Median (red bar) and upper and lower quartiles (dotted line) are indicated. (g) Representative co-immunofluorescent (co-IF) staining for DAPI (nuclei, blue), ASCL1 (green), NEUROD1 (purple), and POU2F3 (red) in RPM K5-Cre tumours. Tumour regions outlined with dashed line. High magnification insets of co-expressing cells (yellow arrows) are in the upper left corner of overlays. Scale bars=75 μm. (h) UMAP of scRNA-seq data from n=5 tumours initiated from NE cells (Cgrp-Cre, purple) from n=5 RPM mice, or n=4 tumours initiated from basal cells (K5-Cre, orange) from n=2 RPM mice. Cells coloured by sample in the same UMAP on right. (i) UMAP in (h) annotated by Leiden cluster (left) (Supplementary Table 1). Proportion of cells in Cgrp vs K5 tumours per Leiden cluster, represented as % of all cells per sample (right). (j) FeaturePlots depicting expression of indicated genes in UMAP, as in (h) (top). Split violin plot depicting mRNA expression of indicated genes by scRNA-seq for all cells in Cgrp (purple, left) and K5 (orange, right) tumours (bottom). Each dot is one cell. Student’s unpaired t-tests. **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05, ns=not significant, p>0.05. Unless otherwise noted, statistical tests are one-way ANOVA with Tukey’s correction (e,f). **** p<0.0001, ** p<0.006, * p<0.02, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Infection, Staining, Marker, Expressing

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Representative IHC images from RPM K5-Cre tumours for KRT5. A Sox2 LSL/LSL ;Nkx2-1 fl/fl ;Lkb1 fl/fl mouse lung squamous tumour is included as a positive control. (b) Representative IHC for YAP1 in RPM tumours initiated by indicated Ad-Cre viruses (left) and corresponding H-score quantification (right). Median (red bar) and upper and lower quartiles (dotted lines) indicated. One-way ANOVA with Tukey’s correction (e,f). *** p<0.0003, ** p<0.003. (c) Split violin plot depicting expression of SCLC archetype signatures per tumour cell by scRNA-seq in CGRP (purple, left) and K5 (orange, right) tumours in . (d) Split violin plot depicting transcriptional signatures of human ASCL1, NEUROD1, or POU2F3 + tumours derived from scRNA-seq data or (e) expression of ASCL1, NEUROD1, and POU2F3 target gene signatures derived from ChIP-seq data , , , (Supplementary Table 2) by scRNA-seq for all cells in CGRP (purple, left) and K5 (orange, right) tumours. Each dot is one cell. (f) Split violin plot depicting NE score per tumour cell by scRNA-seq in CGRP (purple, left) and K5 (orange, right) tumours. All scale bars=50 μm. Unless otherwise noted, statistical tests are Student’s unpaired t-tests. **** p<0.0001, ** p<0.01, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Positive Control, Expressing, Derivative Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Schematic depicting isolation, growth and transformation of basal cell-derived organoids from RPM mice followed by implantation into the flanks of scid/beige hosts. (b) Representative brightfield images of basal organoids pre- (wildtype) and post- (transformed) CMV-Cre. Scale bars=650 μm (left, low mag) or 275 μm (right, high mag). (c) Representative H&E staining of RPM basal-organoid-derived tumours isolated from scid/beige mouse flanks with more classic (left) or variant (right) histopathology. Scale bar=50 μm. (d) UMAP of scRNA-seq data from wildtype (orange) and transformed (purple) RPM basal organoids, plus basal-organoid-derived RPM allograft tumour cells (turquoise). Allograft sample includes n=5 distinct RPM basal allograft tumours. FeaturePlots depicting expression of gene signatures derived from normal basal versus NE cells (right) (Supplementary Table 2). (e) UMAP of scRNA-seq data from RPM allograft tumours only, annotated by Leiden cluster (left) (Supplementary Table 1), FeaturePlot expression of indicated genes (top, right), and corresponding violin plot expression of indicated genes per Leiden cluster (bottom, right). Red dashed circle outlines Cluster 9 enriched for Pou2f3 . (f) UMAP in (e) annotated by SCLC fate. Fates assigned based on enriched cell fate marker gene expression per Leiden cluster. (g) Violin plot of NE score per cell grouped by SCLC fate (left) from data in (f). UMAP of scRNA-seq data in (e) coloured by NE score (right). (h) Violin plot depicting ASCL1, NEUROD1, and POU2F3 ChIP target gene enrichment , , , (Supplementary Table 2) in tumour cells from (e), grouped by SCLC fate assignment. (i) Violin plot depicting gene set enrichment of normal NE, tuft, and basal cells (Supplementary Table 2) in tumour cells from (e), grouped by SCLC fate assignment. (j) Representative co-immunofluorescent (co-IF) staining for DAPI (nuclei, blue), ASCL1 (green), NEUROD1 (purple), and POU2F3 (red) in RPM basal allograft tumours. High magnification insets of co-expressing cells (yellow arrows) are in the upper right corner of overlays. Scale bars=75 μm. (k) Representative IHC of RPR2 basal-derived allograft tumours for H&E and indicated SCLC subtype markers (left) with corresponding H-score quantification for ASCL1 (A), NEUROD1 (N) or POU2F3 (P) compared to RPM basal allograft tumours (right). Scale bar=50 μm. Mann-Whitney two-tailed t-test. * p<0.02, *** p<0.0005. (l) UMAP of scRNA-seq data from basal-organoid-derived RPM (turquoise, n=5 tumours) and RPR2 (maroon, n=1) allograft tumour cells. (m) UMAP in (l) annotated by Leiden cluster (Supplementary Table 1) (left). Proportion of cells from RPM vs RPR2 allograft tumours in each Leiden cluster, represented as % of all cells per sample (right). (n) UMAP of scRNA-seq data in (l) coloured by NE score (left). Violin plot of NE score per cell in RPM vs RPR2 basal allograft tumour cells (right). Student’s unpaired t-test. ** p<0.01. Box-whisker overlays on all violin plots indicate median and upper and lower quartile. Unless otherwise indicated, statistical tests are one-way ANOVA with Tukey’s correction. **** p<0.0001, * p<0.03, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Isolation, Transformation Assay, Derivative Assay, Staining, Variant Assay, Histopathology, Expressing, Marker, MANN-WHITNEY, Two Tailed Test, Whisker Assay

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Recombination PCR for RPM basal organoids for indicated alleles pre- and post-treatment with TAT-Cre recombinase or Ad-CMV-Cre at two concentrations (2.5e7 or 5e7 pfu). *Organoids subject to spinoculation with CMV-Cre virus. Red font indicates condition used for subsequent allografting. (b) Co-IF on basal organoids pre-treatment with Cre (No Cre) or following recombination (RPM, RPR2, RPMA) for DAPI (nuclei, blue), KI67 (proliferation, orange), and ASCL1 (NE cell, green). Positive controls for KI67 and ASCL1 (bottom panel) is an RPR2 SCLC lung tumour. Quantification via CellProfiler of KI67 positivity per organoid from indicated conditions (right). Number of organoids quantified per group is labeled. One-way ANOVA with Tukey’s correction. *** p<0.003, ** p<0.005, ns=not significant, p>0.05. Error bars represent mean ± SD. (c) Co-IF on basal organoids pre-treatment with Cre (No Cre) or following recombination (RPM, RPR2, RPMA): Left) DAPI (nuclei, blue), NEUROD1 (neuronal cell, green), DNP63 (basal cell, yellow) and KRT8 (luminal basal cell, red). Positive control (+) for NEUROD1 is an RPM olfactory neuroblastoma tumour and for basal markers is an SNL GEMM lung tumour . Right) DAPI (nuclei, blue), FOXJ1 (ciliated cell, purple), CCSP (SCGB1A1, club cell, green) and KRT8 (luminal basal cell, red). Positive control (+) control for FOXJ1 and CCSP is airway from a normal mouse lung, and for KRT8 is an SNL GEMM lung tumour. (d) Co-IF on basal organoids pre-treatment with Cre (No Cre) or following recombination (RPM, RPR2, RPMA) for DAPI (nuclei, blue) and POU2F3 (tuft cell, green). Positive control (+) is an RPMA olfactory neuroblastoma tumour . All co-IF scale bars=150 μm.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Virus, Labeling, Positive Control, Control

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) UMAP of scRNA-seq data from wildtype (orange) and transformed (purple) RPM basal organoids (left) and annotated by Leiden cluster (right) (Supplementary Table 1). (b) Dot plot expression of genes marking major lung cell types, stem-like, proliferative, and tumour cells for wildtype versus transformed RPM organoids, grouped by Leiden cluster as assigned in (a). Colour indicates level of gene expression and dot size represents frequency of expression per cluster. (c) UMAP of RPM organoids pre- and post-CMV-Cre coloured by cell cycle phase. (d) UMAP as in of RPM organoids pre- and post-CMV-Cre and RPM basal allograft tumour coloured by cell cycle phase (left). Proportion of cells from WT and transformed (RPM) organoid samples and RPM allograft tumour in each phase, represented as % of all cells per sample (right). (e) Dot plot expression of genes marking indicated cell types, stem-like, proliferative, and tumour cells for RPM basal allograft tumour by Leiden cluster derived from scRNA-seq in . (f) Violin plot expression of SCLC archetype signatures in RPM basal allograft tumour cells by scRNA-seq as in , grouped by Leiden cluster. (g) Recombination PCR for RPR2 basal organoids for indicated alleles pre- and post-treatment with TAT-Cre recombinase or Ad-CMV-Cre at two concentrations (2.5e7 or 5e7 pfu). *Organoids subject to spinoculation with CMV-Cre virus. Red font indicates condition used for subsequent allografting. (h) UMAP and corresponding violin plots depicting expression of indicated SCLC subtype markers and Myc-family oncogenes in RPM versus RPR2 basal organoid allografts from . (i) Violin plot expression of SCLC subtype archetype signatures or (j) ChIP target genes signatures (Supplementary Table 2) per tumour cell in RPM vs RPR2 basal allograft tumour samples from scRNA-seq data in . A=ASCL1, N=NEUROD1, and P=POU2F3. Unless otherwise noted, statistical tests are Student’s unpaired t-test. **** p<0.0001.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Transformation Assay, Expressing, Derivative Assay, Virus

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Representative H&E staining of RPM (top) and RPMA (bottom) basal-organoid-derived tumours isolated from scid/beige mouse flanks. Scale bar=50 μm. (b) Representative IHC images from RPM and RPMA basal-organoid-derived tumours for indicated markers (left). H-score IHC quantification for indicated proteins (right). Each dot represents one tumour. For each marker, n=6-9 tumours quantified. Scale bars=50 μm. Student’s unpaired t-tests. **** p<0.0001, ** p<0.01, ns=not significant, p>0.05. Error bars represent mean ± SD. (c) Immunoblot depicting expression of indicated markers in RPM (n=2) vs RPMA (n=3) basal allograft tumours with HSP90 as a loading control. (d) Representative co-IF staining for DAPI (nuclei, blue), NEUROD1 (purple), and POU2F3 (green) in RPMA basal allograft tumours. Scale bars=75 μm. (e) UMAP of scRNA-seq data from basal-organoid-derived RPM (purple, n=5) and RPMA (orange, n=3) allograft tumours. (f) UMAP in (e) annotated by Leiden cluster (left). Proportion of cells from RPM vs RPMA allograft tumours in each Leiden cluster (Supplementary Table 3), represented as a % of all cells per sample (right). (g) Dot plot expression of genes marking indicated cell fates, stem-like, proliferative, and tumour cells for RPM and RPMA basal-derived allograft tumour cells, grouped by Leiden cluster as assigned in (f). Colour indicates level of gene expression and dot size represents frequency of expression per cluster. Genotypes indicate enrichment but not exclusive expression of each cluster. (h) UMAP of scRNA-seq data in (e) coloured by SCLC fate (left). Fates assigned based on enriched fate marker gene expression per Leiden cluster. Proportion of cells from RPM and RPMA allograft tumour samples in each fate, represented as % of all cells per sample (right). (i) UMAP of scRNA-seq data in (e) coloured by NE score according to legend (left). Violin plot of NE score per cell grouped by SCLC fate or genotype as indicated on the x-axis (right). (j) UMAP of scRNA-seq data in (e) coloured by ASCL1, NEUROD1, and POU2F3 ChIP target gene scores , , , (Supplementary Table 2) where red/dark purple is high and orange is low. Upper right insets are violin plots depicting expression of target gene scores, grouped by genotype. Student’s unpaired t-tests. **** p<0.0001. (k) UMAPs (top) and violin plots (bottom) depicting gene set enrichment of normal NE, tuft, and basal cells (Supplementary Table 2) in tumour cells from (h), grouped by fate assignment. (l) Violin plot expression of SCLC subtype archetype signatures (Supplementary Table 2) per tumour cell in RPM vs RPMA basal allograft tumour samples from data in (h), grouped by SCLC fate. A=ASCL1, N=NEUROD1, and P=POU2F3. Box-whisker overlays on all violin plots indicate median and upper and lower quartile. Unless otherwise indicated, statistical tests are one-way ANOVA with Tukey’s correction. **** p<0.0001, ** p<0.001, * p<0.01, ns=not significant, p>0.05.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Staining, Derivative Assay, Isolation, Marker, Western Blot, Expressing, Control, Whisker Assay

Journal: bioRxiv

Article Title: Basal cell of origin resolves neuroendocrine–tuft lineage plasticity in cancer

doi: 10.1101/2024.11.13.623500

Figure Lengend Snippet: (a) Survival of RPP mice infected with K5-Cre or Cgrp-Cre compared to RPM mice infected with K5-Cre. Dashed line indicates historical data . Number of mice indicated in the figure. Mantel-Cox log-rank test; **** p<0.0001, ns=not significant, p >0.05. (b) H-score quantification of ASCL1 and NEUROD1 in RPP GEMM tumours initiated by indicated Ad-Cre viruses. Each dot is one tumour. N=35-40 tumours from n=5-8 mice per cohort. One-way ANOVA with Tukey’s correction. ns=not significant, p>0.05. (c) Immunoblot analysis of human SCLC cell line, H1048, for indicated markers after LentiCRISPRv2 infection with non-targeting ( sgNTC ) or sgPTEN sgRNAs. (d) Immunoblot analysis of H1048 for indicated markers in parental cells versus cells with ectopic myristoylated-AKT (myrAKT) with HSP90 as a loading control.

Article Snippet: Primary antibodies included: anti-mouse ASCL1 (BD Pharmingen cat#556604) 1:25; anti-rabbit ASCL1 (Abcam cat#211327) 1:100; anti-goat NEUROD1 (R&D Systems cat# AF2746) 1:50; anti-rabbit NEUROD1 (Abcam cat#109224) 1:200; POU2F3 (Sigma cat# HPA019652) 1:100; anti-rabbit CCSP/SCGB1A1 (Millipore Sigma cat#07-623) 1:75; anti-rat KRT8 (DSHB cat# TROMA-I) 1:100; anti-mouse FOXJ1 (eBioscience cat#14-9965-80) 1:100; anti-mouse KI67 (BD Pharmingen cat# BDB556003) 1:100; anti-goat DNP63 (R&D cat# AF1916) 1:40.

Techniques: Infection, Western Blot, Control

Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: RNA-seq identifies altered neurodevelopmental genes and functions. (a) IPA analysis of DEGs showing the top five functional categories having differential expression of genes related to development (based on −log(P) calculated by Fisher’s exact test. (b) Gene network showing interactions of DEGs, colored in yellow (upregulation) and blue (downregulation), involved in neuronal development and relevant to HD pathogenesis. Several key regulatory pathways have been added to show interactions and possible contribution to the findings, including REST and miR-124, which are predicted upstream regulators with z-scores of 4.038 and −3.638, respectively. HTT is highlighted in orange and connects several critical gene pathways. NEUROD1 manifests as a major node. See Supplementary Figure 2c for symbol legend.

Article Snippet: NEUROD1 knockdown The HD iPSC derived EZ-spheres were differentiated in 24-well plates coated with Matrigel (BD), transduced with pGFP-sh lentiviral particles carrying vectors encoding either scrambled control or NEUROD1 shRNA (KD) (Origene) for 24 h, and then transferred to neural induction medium (NIM) without additives or supplied with 20 μM Isx-9 for 48 h. Lentivirus was used at concentration 100 ng/ml.

Techniques: RNA Sequencing Assay, Functional Assay, Expressing

Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: Gene expression changes reflect altered striatal development. (a) Heat map of genes common to mouse microarray analysis and human RNA-seq data. Values are in s.d. normalized by gene separately by set (human and mouse) (yellow, +3; blue, −3). Hierarchical clustering analyses with average linkage for sample, and genes are displayed with dendrograms; clusters were obtained automatically with Genesis Software. Cluster A (violet) includes germinal zone samples and HD human samples, whereas cluster B (green) contains human non-disease samples and mantle zone mouse samples. (b) Gene Ontology (GO) biological process (BP) terms with enrichment of all 679 common genes depicted in the heat map. For GO enrichment, a hypergeometric test was used with P < 0.05 adjusted by Benjamini-Hochberg correction. (c) Summary of the most enriched ‘analyzed network’ result from Metacore using genes from cluster I, which contains NeuroD1. Metacore’s Direct Interactions network was obtained from the 679 common human and mouse genes. The network depicted is filtered to include only transcription factors, protein kinases, receptors with kinase action and ligands. See Supplementary Figure 2c for symbol legend.

Article Snippet: NEUROD1 knockdown The HD iPSC derived EZ-spheres were differentiated in 24-well plates coated with Matrigel (BD), transduced with pGFP-sh lentiviral particles carrying vectors encoding either scrambled control or NEUROD1 shRNA (KD) (Origene) for 24 h, and then transferred to neural induction medium (NIM) without additives or supplied with 20 μM Isx-9 for 48 h. Lentivirus was used at concentration 100 ng/ml.

Techniques: Expressing, Microarray, RNA Sequencing Assay, Software

Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: Isx-9 induces expression of NEUROD1. (a) Total HTT silencing increases NEUROD1 gene expression in HD iPSC-derived neural cultures. High CAG repeat (109Q) cultures were treated with HTT ASOs and show enhanced NEUROD1 transcript levels. For qPCR analysis, a statistical difference in gene expression was determined using an unpaired two-tailed t-test in GraphPad Prism. HTT: P = 0.0004, t = 8.322, d.f. = 5; NEUROD1: P = 0.0331, t = 5.362, d.f. = 3. (b) NEUROD1 overexpression enhances neuronal gene expression in high CAG repeat lines. HD iPSC-neural cultures were transduced with either human NEUROD1 overexpression lentivirus or pFUGW-eGFP vector control. NEUROD1 overexpression lentivirus (Applied Biological Materials) was used at 1 × 106 infectious units/ml for transduction. Subsequent qPCR demonstrated increased expression of NEUROD1, CALB1 and CAMK4. NEUROD1 (21Q: P = 0.0016, t = 11.03, d.f. = 3; 109Q: P = 0.0002, t = 21.46, d.f. = 3); CALB1 (21Q: P = 0.07; t = 2.456, d.f. = 4; 109Q: P = 0.0065, t = 5.212, d.f. = 4) POU4F2 (21Q: P = 0.775, t = 0.3122, d.f. = 3; 109Q: P = 0.269, t = 1.515, d.f. = 2); CAMK4 (21Q: P = 0.0001, t = 15.33, d.f. = 4; 109Q: P = 0.0004, t = 10.80, d.f. = 4). (c) Isx-9 enhances NEUROD1 expression. qPCR was performed on iPSC-neural cultures after treatment with 20 μM Isx-9. Increased NEUROD1 mRNA was detected in one non-disease (21Q) and both HD (60Q and 109Q) lines. Between vehicle and Isx-9 treatment: 21Q: P = 0.0001, t = 14.89, d.f. = 4; 109Q: P = 0.0006, t = 9.804, d.f. = 4. (d) Isx-9 western analysis. NEUROD1 protein levels increased after treatment with 20 μM Isx-9 in the absence of BDNF. Conversely, BDNF did not increase NEUROD1 protein expression in the absence of Isx-9. Densitometric quantification was normalized to actin. Significance between Isx-9 and untreated cells was determined by one-way ANOVA. 21Q plus Isx-9 (P = 0.000171, n = 4 independent differentiations, d.f. = 1, F = 68.07). 109Q plus Isx-9 (P = 0.0001, n = 4 independent differentiations, d.f. = 4, F = 217.4). Full blots in Supplementary Figure 8. Plots show mean ± s.d.

Article Snippet: NEUROD1 knockdown The HD iPSC derived EZ-spheres were differentiated in 24-well plates coated with Matrigel (BD), transduced with pGFP-sh lentiviral particles carrying vectors encoding either scrambled control or NEUROD1 shRNA (KD) (Origene) for 24 h, and then transferred to neural induction medium (NIM) without additives or supplied with 20 μM Isx-9 for 48 h. Lentivirus was used at concentration 100 ng/ml.

Techniques: Expressing, Derivative Assay, Two Tailed Test, Over Expression, Transduction, Plasmid Preparation, Western Blot

Journal: Nature neuroscience

Article Title: Developmental alterations in Huntington’s disease neural cells and pharmacological rescue in cells and mice

doi: 10.1038/nn.4532

Figure Lengend Snippet: Isx-9 treatment improves CAG repeat-associated phenotypes. (a) Nuclear condensation assay. Differentiated neural cultures were treated with Isx-9 or BDNF. Cell death, by nuclear condensation, was reduced in the HD 109Q line treated with Isx-9. One-way ANOVA (+BDNF +ISX9 versus −BDNF −ISX9, *P = 9 × 10−6; −BDNF −ISX9 versus −BDNF +ISX9, *P = 0.001; n = 7, d.f. = 7, F = 7.011). (b) NEUROD1 knockdown. The HD iPSC-derived neural cells were transduced with lentiviral particles carrying vectors encoding either scrambled (Scrbl) or NEUROD1 shRNA (KD) (shRNA and pFUGW-eGFP lentiviruses at 100 ng/ml for transduction) for 24 h, and then transferred to neural induction medium without additives or supplied with 20 μM Isx-9 for 48 h. NT, non-transduced control. **P < 0.005 versus scrambled control shRNA. One-way ANOVA (P = 1 × 10−10, n = 9, d.f. = 15, F = 11.15). (c) Isx-9 increases survival of HD i-neurons (neural cells derived from iPSCs). Images were used to follow individual cells over time and cell death was recorded to assess the effect of Isx-9 on HD and control cells. Left: cumulative risk of death of the pooled 46Qn1 and 46Qn10 (46Qn1/n10) lines is greater than that of the 18Qn2/n6 controls (hazard ratio (HR) = 1.34, *P = 0.0281). Isx-9 decreased the cumulative risk of death of all 46Qn1/n10 (HR = 0.74, ***P = 7.9 × 10−7) and also of the controls (HR = 0.75, P = 0.08). Control 18Qn2/n6 + DMSO, n = 124 cells, control 18Qn2/n6 + Isx-9, n = 143 cells (4 experiments); 46Qn1/n10 + DMSO, n = 702 cells, 46Qn1/n10 + Isx-9, n = 783 cells (6 experiments). Right: cumulative risk of death of 46Qn1/n10 is significantly greater than that of the control neural cells 18Qn2/6 (HR = 1.3, **P = 0.00209). Isx-9 (20 μM) rescued the survival deficit of 53Qn3 (HR = 0.78, ***P = 9.2 × 10−7). Isx-9 did not significantly change cumulative risk of death for the control Q18n2/6 (HR = 0.97, P = 0.685). All P values are reported from the log rank test, but HR values are from the Cox proportional hazards model. Control Q18n2/6 + DMSO, n = 490 cells, Data shown are all experiments combined into one curve. Individual survival curves are shown in Supplementary Figure 10. (d) HD cells have longer neurite-like process length than controls, and process length is reverted by Isx-9. HD and control iPSCs were differentiated as above. HD cells have longer neurite-like process length than controls; length is restored by Isx-9. HD and control iPSCs were differentiated as above. The HD lines Q46n1, Q46n10, Q53n3, Q53n5, Q109n4 (****P < 0.0001 in all comparisons for these five lines) and Q109n5 (P = 0.0069, P = 0.0018, P < 0.0001) had significantly longer processes compared to control cell lines Q18n2, Q18n6 and Q28n6, respectively. Isx-9 rescued the abnormal increased process length in HD lines Q46n1 (****P < 0.0001), Q46n10 (**P = 0.0067), Q53n3 (****P < 0.0001), Q53n5 (***P = 0.0004), Q109n4 (***P = 0.0005) and Q109n5 (****P < 0.0001) but did not alter process length in control cell lines Q18n2 (P = 0.999), Q18n6 (P = 0.939) and Q28n6 (P > 0.999). Error bars represent the s.d. of the mean.

Article Snippet: NEUROD1 knockdown The HD iPSC derived EZ-spheres were differentiated in 24-well plates coated with Matrigel (BD), transduced with pGFP-sh lentiviral particles carrying vectors encoding either scrambled control or NEUROD1 shRNA (KD) (Origene) for 24 h, and then transferred to neural induction medium (NIM) without additives or supplied with 20 μM Isx-9 for 48 h. Lentivirus was used at concentration 100 ng/ml.

Techniques: Derivative Assay, Transduction, shRNA