EMBO/soda-nel huggingface dataset:
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease (2020)
Figure 4

Figure 4. Tissue-resident macrophages engraft into BALO alveolar-like regions, maintain their identity and drive epithelial cell differentiation

Gating strategy to define TR‑Mac from BAL of adult tdTomato+ mice.

Representative images of BALO after microinjection of tdTomato+ TR-Mac at day 14. TR-Mac are preferentially found in alveoli (right). Scale bar represent 100 μm (left) and 50 μm (right).

Fluorescence confocal images of CD206, Siglec-F, and isotype control staining 14 days after tdTomato+ TR‑Mac microinjection in a day 28 BALO. Scale bars represent 100 μm (left) and 25 μm (right).

Electron microscopy depicting filopodium of TR‑Mac (white dashed lines) with a characteristic actin filament bundle (yellow background) (left panel) in contact with AEC I within BALO alveolar-like structures. Uptake of surfactant by TR-mac is depicted by a (*). Scale bar indicates 1000nm (left, in insert: 500 nm) and 500 nm (right, in insert: 250 nm).

Representative confocal images of Cx43 staining in tdTomato+ TR-Mac monoculture in Matrigel and tdTomato+ TR‑Mac microinjected at day 14 and analyzed in a mature day 21 BALO. Scale bars represent 50 μm (overview) and 5 μm (close up).

Heat map (left) and tSNE plot (right) of the comparative analysis of digested day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac depicting six distinct clusters (C1, club/secretory cells, red; C2, basal cells, yellow; C3, rMC, green; C4, AEC II, blue; C5, AEC I, purple; and C6, ciliated cells, pink).

Expression data dot plots of genes found differentially regulated in cluster C1 between day 23 BALO with (C1+) and without (C1-) microinjected TR-Mac. The circle size illustrates the number of cells expressing a specific gene.

Percentage of terminally differentiated cells (AEC I and ciliated cells) in day 23 BALO cultures with (+) and without (-) microinjected TR‑Mac.

Data information: Bar and dot charts presented as the mean ± S.E.M. and probability determined using t-test (**P < 0.01).
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease (2020)
Figure 5

Figure 5. Manipulation of WNT signaling pathway during BALO morphogenesis by knock-down of miR142-3p gene expression recapitulates developmental defects observed in embryonic lung explants.

Representative images of E11.5 lung explants after treatment for 48 h with 100 μM Scra or mo142-3p reveals reduced size and branching morphogenesis after miR142-3p knockdown.

Organoid diameter (n=30-50 per group) in µm before addition of 4 μM Scra or mo142-3p at day 6 (control) and 5 days after treatment (at day 11 BALO culture) in n=3 biological replicates

C-D. Representative images of β-galactosidase staining in TOPGAL epithelium (C) and TOPGAL rMC (D) BALO cultures before (day 6, control) or 5 days after treatment with either 4 μM Scra or mo142-3p (day 11 of co-culture). BASC and rMC were isolated form the lung homogenate of TOPGAL mice. β-galactosidase+ rMC at day 11 of culture are indicated with arrows.

Representative transmission and confocal images after LysoTracker staining indicating branching and number of branching points in n=4 BALO 15 days after treatment with 4 μM Scra or mo142-3p (day 21 of co-culture) in n=3 biological replicates.

mRNA levels of epithelial and mesenchymal miR142-3p expression in Scra and mo142-3p-treated organoids 5 days after treatment (n=3-4 biological replicates with pooled cells from 4 cultures per replicate).

Data information: Scale bars=100 μm. Bar charts presented as the mean ± S.E.M. and probability determined using t-test ((*P < 0.05, **P < 0.01).
Multilineage stem cell-derived organoids recapitulate murine distal lung development and disease (2020)
Figure 6

Figure 6. BALO support influenza virus infection and allow modeling of lung infection and injury

Representative images of day 21 BALO after microinjection of SC35M-GFP IAV (green) into the central airway-like (*) after 0 and 12 h pi visualizes IVA spread to the alveolar-like regions. Dotted lines illustrate BALO-borders. Scale bars represent 100 μm.

Quantification of plaque forming units in supernatants from mock or SC35M IAV infected BALO 48 h after infection (n=5 biological replicates). N.D.: not-detectable.

Relative NP expression in PR8 IAV infected (HA+) or non-infected (HA-) epithelial cells isolated from BALO 48 h pi, FACS-sorted according to EpCAM and HA expression (n=3 biological replicates with pooled cells from 4 cultures per replicate).

Representative flow cytometric data showing the percentage of EpCAM+NP+ cells in mock- and SC35M IAV‑infected BALO at 48 h pi in n=3 biological replicates with pooled cells from 4 cultures per replicate.

Representative fluorescence images of a day 21 distal region in BALO generated from mTmG reporter mouse and infected by SC35M-Cre IAV after 0, 10, 14 and 25 h. Arrows indicate cell death. Scale bars represent 25 μm and 10 μm in the insert.

mRNA expression of Ifnb in mock and PR8 IAV-infected BALO at 48 h pi (n=3 biological replicates with pooled cells from 4 cultures per replicate).

Release of TNFα (6 h pi), IL-6 (48 h pi), IL-1β (48 h pi) detected by Bio-Plex® Multiplex Immunoassay in the supernatant of mock and IAV infected BALO cultures with and without TR-Mac (n=3 biological replicates).

Data information: Bar charts presented as the mean ± S.E.M. and probability determined using t-test (F) and one‐way ANOVA (G) (*P < 0.05, **P < 0.01).

Table 1. Adjusted p-value of genes in LIF and MYO clusters. Adjusted p-value of genes associated with MYO (C3) and LIF (C4) phenotypes including Pdgfrα, Tagln, Acta2, Eln, Axin2 and Apoe, Serpina3n, Gsn Gas6, and Fgf10, respectively.
Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer (2021)
Figure 2

Figure 2. CRC cell clusters are distinguished by signaling pathway activities.

A Transcriptional activity associated with key oncogenic traits and signals, by tumor-specific cell type and patient, as indicated. Red: High activity, blue: low activity. Significance was assessed by Kruskal Wallis Test (FDR corrected p<0.05), followed by a post-hoc analysis using a Wilcox-test of each group against all other groups, FDR corrected significance levels are shown (*=p<0.05; **=p<0.01; ***p<0.001).

B Visualization of signatures corresponding to oncogenic traits and signals in the tumor cell transcriptome UMAP.

C Cell cycle distribution of TC1-4 epithelial tumor cells, as inferred from single cell transcriptomes.

D Immunofluorescence of DNA-damage-associated nuclear protein PARP. Images show adjacent normal and tumor crypts of tissue P009T, marked by N and T, respectively, and tumor tissue of patients P008 and P009, as indicated. Scale bar 100µm. Significance was assessed by an unpaired t-test, after blinded analysis of 10 random images per tumor.
Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer (2021)
Figure 3

Figure 3: RNA metabolic labeling defines tumor cell trajectories in patient-derived organoids.

A Phenotypes of patient-derived organoid lines P009T and P013T.

B UMAPs of organoid single cell transcriptomes. Organoid lines and medium conditions as indicated. LGR5-ISC stem cell, enterocyte and Goblet cell signatures are visualized.

C Schematic representation of SLAM-Seq workflow to infer RNA dynamics ("RNA velocity"). In short, organoids were passaged and assigned to different medium conditions. After three days, nascent RNA was metabolically labelled for 2 h using 4sU. Organoids were harvested, dissociated, and fixed. RNA in single cells was alkylated, and cells were subjected to single cell sequencing. Reads were assigned to nascent or old RNA status, depending on diagnostic T-C mutational status.

D Developmental trajectories inferred from RNA metabolic labelling. Bold and thin arrows indicate high versus low directionality of RNA velocity. Colors below RNA velocity show latent time (yellow: early latent time; blue: late latent time).

E Activities of Wnt/β-catenin and MAPK target genes in organoid single cell transcriptomes, ordered along latent time.

F Activities of TFF3, FABP1, MKI67 and MMP7 in organoid single cell transcriptomes, ordered along latent time.

Data information: Color code for panels E and F: Red: high activity; blue: low activity.
Mitogen-activated protein kinase activity drives cell trajectories in colorectal cancer (2021)
Figure 4

Figure 4: MAPK activity is linked to CRC cell differentiation states.

A Gene expression of LGR5 and EPHB2, along activity gradients of LGR5-ISC or MAPK target gene signatures.

B Cell state distribution of SCN-aberrant CRC cells along gradients of LGR5-ISC or MAPK transcriptional signatures, as in A.

C Cell state distribution of SCN-aberrant CRC tumor cells along MAPK signature activity, as in B, per tumor. Correlation between cell state distributions and MAPK target gene was calculated using Pearson´s r. For correlations and significances, see Table EV6. Color code as in Fig. 4B.

D UMAP representations of single cell transcriptomes derived from P009T or P013T organoids, after MAPK blockade using MEK or combined MEK and EGFR inhibition. Color codes are treatment conditions or expression strength of signature, as indicated. Dashed line in P013T UMAP roughly separates control (DMSO) and MEK/EGFR inhibitor treated cells.
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium (2020)
Figure 1

Figure 1. Massive progenitor cell apoptosis without global loss of proliferation in rostral telencephalon of Rx-Dicer mutants.

A - Distribution of Casp3+ cells in rostral and ventral domains of an E11.5 Rx-Dicer mutant embryo. NCx, neocortex; OB, olfactory bulb; Spt, septum.

B,C - Marker analysis of Casp3+ cells in the rostral telencephalon of E12.5 Rx-Dicer mutant embryos. Most apoptotic cells are Pax6+ RGCs (solid arrowheads) and not Tbr1+ neurons (open arrowheads). N = 3 replicates per marker.

D,E - Distribution and abundance of apoptotic cells (Casp3+) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3 replicates per genotype and age.

F,G - Distribution and abundance of mitoses (PH3+) and neurons (Tuj1) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, dashed line apical surface. N = 3-4 replicates per genotype and age. No significant differences were found between control and mutant embryos in apical nor basal mitoses.

Data information: Histograms represent mean ± SEM; symbols in plots indicate values for individual embryos; X2-test (C), t-test (E,G); *p<0.05, ** p <0.01, *** p <0.001.

Scale bars, 200 µm (A), 40 µm (B), 25 µm (D,F).
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium (2020)
Figure 2

Figure 2. Formation of rosettes in the rostral telencephalon of Rx-Dicer mutants.

A - DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and Rx-Dicer mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line).

B - Expression pattern of Grm1 mRNA in OB (dotted line) of control and Rx-Dicer mutants.

C - Quantification of OB perimeter (mean ± SEM; symbols indicate values for individual embryos); t-test, ***P < 0.001. N = 14 replicates per genotype.

D - Immunostains of E17.5 control and Rx-Dicer mutant brains showing the distribution of progenitor cells (Ki67, red) and neurons (Tuj1, green). Arrowheads indicate rosettes. Sp, septum.

E - Detail of a rosette from an E17.5 Rx-Dicer mutant embryo displaying typical features: closed apical surface (dotted line) with PH3+ apical mitoses (white arrowhead) and basal mitoses, surrounded by Tuj1+ neurons (green). Dashed line indicates the basal border of the rosette.

F - Rostral half of an Rx-Dicer mutant E17.5 brain immunostained for Pax6, clarified and segmented to reveal rosettes (yellow). C, caudal; R, rostral.

G,H - BrdU incorporation and cell cycle re-entry analysis with the progenitor cell marker Ki67 at E17.5, in the rostral cortex of control embryos compared to rostral rosettes of Rx-Dicer mutants. Dotted lines indicate apical surface, dashed lines indicate basal border of VZ. Arrowheads indicate double-positive cells. Data in histograms are mean ± SEM, symbols indicate values for individual embryos; n ≥ 3 embryos; X2-test, *P < 0.05, ***P < 0.001.

I - Apical lumen of rosettes immunostained against apical complex proteins (Par3, β-Catenin), primary cilia (Arl13b), apical mitoses (PhVim) and neurons (Tuj1).

J - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1).

K-S - Analysis of the regional identity of rosettes in E14.5 Rx-Dicer mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of Ngn2, Tbr2 and Pax6 identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L-N,P); Gsx2, Dlx2 and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity (O,Q); Nkx2.1 identifies MGE rosettes as being normotopic (R); absence of Gsx2 in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L,M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium.

Data information: Scale bars, 500 µm (A,B,D,F), 25 µm (E,G,I,P-S), 200 µm (J-O).
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium (2020)
Figure 3

Figure 3. Disturbance of ventricular integrity precedes the formation of rosettes.

A - Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo displaying the three types of rosettes.

B - High magnification of rosettes in Rx-Dicer mutants at E12.5 (Type 1), E14.5 (Type 2) and E17.5 (Type 3), immunostained for PH3 and Tuj1. Dashed line indicates ventricular surface; arrowheads indicate basal mitoses; arrows indicate a stream of apical mitoses connecting the lumen of the rosette with the lumen of the telencephalic ventricle.

C,D - Abundance of rosette types at the indicated ages (C), and rostro-caudal distribution of total rosette abundance per age, independent of type (D). n ≥ 2 brains per age.

E - Schematic of the progression of rosette formation in the rostral telencephalon of Rx-Dicer mutant embryos.

F,G - Distribution of neurons (Tuj1+ cells) and apical adherens junction protein Par3 in the OB primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted lines indicate basal border, dashed lines indicate apical border. White arrowheads indicate ectopic neurons and/or absence of Par3. Arrow in (G) indicates accumulation of Par3 at the lumen of a nascent rosette.

Data information: Scale bars, 100 µm (A), 40 µm (B), 50 µm (F,G).
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium (2020)
Figure 4

Figure 4. Changes in expression of miRNAs and mRNAs upon early loss of Dicer.

A,B - Changes in expression levels of miRNAs between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Red dots indicate statistically differentially expressed miRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis) (B). Dashes lines indicate stringency limits in evaluation of results.

C - Gene ontology terms (top) and signalling pathways (bottom) associated with target genes of the miRNAs differentially-expressed between control and Rx-Dicer mutants.

D,E - Changes in mRNA expression levels between control and Rx-Dicer mutants at E11.5 in the rostral telencephalon. Statistically differentially expressed mRNAs according to FDR correction of P values based on the Wald statistic (DESeq2 analysis; Adj. P < 0.1 and FC > 1.25) are highlighted in red, and the three with the highest Adj. P value are identified by name. Blue dot indicates Irs2: FC=1.748, Adj. P = 5.95e-08.

F - Gene ontology terms (top) and signalling pathways (bottom) associated with mRNAs differentially-expressed between control and Rx-Dicer mutants.

G - Heatmaps of relative expression levels of DEGs related to apoptosis (top) and proliferation (bottom). Rows correspond to independent biological replicates. Indicated are some example genes.

H - Enrichment plots from GSEA for MSigDB Hallmark Neurogenesis (NES = 2.98; p = 0; Adj. P = 0.00045), Regulation of cell proliferation (NES = 2.40; p=0; Adj. P = 0.0033), Positive regulation of cell adhesion (NES = 2.06; p = 0.0058; Adj. P = 0.013), Regulation of response to stress (NES = 2.50; p = 0; Adj. P = 0.0023), Positive regulation of cell death (NES = 1.84; p = 0.0179; Adj. P = 0.0326), Apoptotic signaling pathway (NES = 1.59; p= 0.047; Adj. P = 0.084).

I,J - ISH stains of Irs2 mRNA, and qPCR for Irs2 mRNA and let-7-5p miRNA, in the rostral telencephalon of control and Rx-Dicer mutant embryos. Dashed line indicates basal border, dotted line indicates apical surface. Arrowheads indicate area with the greatest increase in Irs2 expression. BG, basal ganglia; H, hippocampus; NCx, neocortex; OB, olfactory bulb; Spt, septum. Histograms represent mean ± SEM (logarithmic scale); symbols in plots indicate values for individual embryos; t-test, *P <0.05, **P <0.01. N = 6-9 replicates per group. Scale bar, 100 µm.

K - Working model of two potential genetic mechanisms leading to rosette formation in Dicer mutants: red, direct effect of increased p53 pathway activity upon loss of let-7; green, Irs2 expression is massively increased by the combined effect of let-7 loss and increased p53-related cell stress signals, which enhances proliferation and decreases cell adhesion, leading to rosettes.
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium (2020)
Figure 5

Figure 5. Loss of p53 in Dicer mutants blocks the formation of rosettes.

A-C - Immunostains for activated p53 (red) in the rostral telencephalon of control and Rx-Dicer mutant embryos at E12.5, and quantification of positive cells (C). Dashed lines indicate apical side, dotted lines indicate basal side. N = 4 replicates per genotype. Scale bar, 50µm.

D-L - Rostral telencephalon of E12.5 embryos of the indicated genotypes immunostained for the detection of apoptotic cells (Casp3), neurons (Tuj1) and the apical complex protein Par3, and quantification of cells positive for Casp3 (H). N = 4-7 replicates per genotype. Scale bar, 50 µm.

M-O - Sagittal sections through the rostral telencephalon of E17.5 embryos of the indicated genotypes. Note the rosettes and general disorganization in Rx-Dicer single mutants, and complete rescue of this phenotype in Rx-Dicer-p53 double mutants. Images in panels M,N are re-used from Fig 2A. NCx, neocortex; OB, olfactory bulb. Scale bar, 1 mm.

Data information: Data in histograms are mean ± SEM, symbols indicate values for individual embryos; t-test; ns, not significant; *P < 0.05, ***P < 0.001, ****P < 0.0001.
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium (2020)
Figure 6

Figure 6. Rosettes form by upregulation of Irs2 expression.

A - Relative Irs2 mRNA levels in HEK cells upon transfection with Gfp- or Irs2-encoding plasmids. Mean ± SEM; t-test, ****P < 0.0001. N = 6 replicates per group.

B-D - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 or E17.5 mouse embryos, as indicated, electroporated at E12.5 with the indicated plasmid combinations. Dashed line indicates the apical surface. Insets show the same field of view, with location and extent of electroporations. Note the overlap of rosettes (arrows) with ectopic neurons at the apical surface (arrowheads) and increased basal mitoses, upon overexpression of Irs2 (C). Asterisk indicates absence of malformation in nearby cortex. At E17.5 (D) GFP+ rosettes remain in deep cortical layers (arrowheads).

E-H - Details of rosettes (large arrows in E; dashed lines in F-H) from Irs2+Gfp-overexpressing E14.5 embryos immunostained as indicated. Small arrows indicate apical mitoses, arrowheads indicate basal mitoses (F,G) and Par3+ apical surface at the centre of rosettes (H), dotted lines indicate apical surface, dashed lines indicate basal side of rosettes. Inset in (H) is at the same scale.

I-M - Distribution of neurons (Tuj1, red), GFP (green) and mitotic (PH3, white) or apoptotic (Casp3, white) cells in the rostral telencephalon of wild-type E13.5 embryos electroporated at E12.5 with the indicated plasmids, and quantification of Casp3+ cells (M). Dashed line indicates apical surface, dotted lines indicate borders of the cortical plate (CP). Nascent rosettes (arrows) are next to ectopic ventricular neurons (arrowheads in K') and basal mitoses, without significant apoptosis in VZ (L',M). Mean ± SEM; t-test; ns, not significant, *P < 0.05. N = 3-6 replicates per group.

N - Distribution of neurons (Tuj1, red) in the rostral telencephalon of wild-type E14.5 embryos electroporated at E12.5 with a combination of plasmids encoding Irs2, let-7a, let-7b and let-7c, and Gfp. Dashed line indicates apical surface. Formation of rosettes in Irs2-expressing embryos is rescued by overexpression of let-7. Inset shows the same field of view, with location and extent of electroporation.

Data information: Scale bars, 150 µm (B-D,N), 50 µm (E-H), 100 µm (I-L).
Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium (2020)
Figure 7

Figure 7. Rosettes form by loss of let-7 and upregulation of Irs2 expression.

A - Frequency of rosette formation in the experimental conditions indicated. n, number of embryos per condition.

B-G - Distribution of neurons (Tuj1, red), GFP (green) and mitoses (PH3, white) in the rostral telencephalon of wild-type E14.5 mouse embryos electroporated at E12.5 with the indicated plasmid combinations. Inset shows the same field of view, with location and extent of electroporations. In (C) and (E), arrows indicate rosettes and arrowheads indicate neuronal ventricular ectopias. (D) and (F) are high magnification details of individual rosettes (dashed lines), where arrowheads indicate apical and basal mitoses. Loss of endogenous let-7 drives the formation of rosettes and ventricular neuronal ectopias (C), which is rescued with the additional loss of Irs2 (G).

Data information: Scale bars, 150 µm (B,C,E,G), 50 µm (D,F).