Journal: Cell reports

Article Title: Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation

doi: 10.1016/j.celrep.2022.111768

Figure Lengend Snippet: (A) Deletion of the SBE1 and SBE5 enhancers leads to a loss of Shh expression and Shh signaling activity in the caudal diencephalon. Whole-mount RNA in situ hybridization for Shh (left) and the Shh-responsive gene Gli1 (right) in bisected heads from control (top) and ΔSBE1/5 embryos (bottom) at E12.5 shows reduced expression in the caudal diencephalon of mutant embryos (n = 3). Scale bar, 500 μm. A Shh bacterial artificial chromosome (BAC) transgene (Shh-GFP) expressing eGFP in place of Shh shows persistent reporter activity in the ZLI of ΔSBE1/5 and control embryos (center). (B) Schematic of the experimental study design. The caudal diencephalon of control and ΔSBE1/5 embryos was micro-dissected at E12.5, E14.5, E16.5, and E18.5 in three replicates per time point and genotype, fixed in methanol, and profiled with single-cell RNA-seq. (C) 3D UMAP representation of the consolidated single-cell gene expression space across all samples, colored by the genotype of the cells. Cells from control and ΔSBE1/5 embryos substantially overlap in the representation. (D) 3D UMAP representation of the single-cell transcriptomic atlas colored by the 23 cell populations identified in the clustering analyses. (E) Fraction of cells in each cell population for each of the four time points. Only cells from control mice were considered in this analysis. The observed expansion of glial cells starting between E14.5 and E16.5 is consistent with the transition between neurogenesis and gliogenesis at this stage of embryonic development. Cell populations are listed in the legend in the same order as they appear in the band plot. cTh.N, caudal thalamic neurons; rTh.N, rostral thalamic neurons; cTh.Pro, caudal thalamic progenitors; preTh.N, prethalamic neurons; MB, midbrain; ZI, zona incerta; preThE, prethalamic eminence; OPCs, oligodendrocyte precursor cells. See also and and .

Article Snippet: 3D UMAP visualization , This paper , Mendeley Data: https://doi.org/10.17632/kjpj9n66jg ( https://data.mendeley.com/datasets/kjpj9n66jg ).

Techniques: Expressing, Activity Assay, RNA In Situ Hybridization, Control, Mutagenesis, RNA Sequencing, Gene Expression

Journal: Cell reports

Article Title: Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation

doi: 10.1016/j.celrep.2022.111768

Figure Lengend Snippet: (A) UMAP representation of the mRNA expression data of E18.5 control cells from the post-mitotic cTh.N, rTh.N, ZI, habenula, and reticular complex cell populations. In total, this analysis identified 23 cell subpopulations with distinct transcriptomic profiles (bold labels). Using a spectral graph method, we split some of these populations into smaller transcriptional identities (italic labels). (B) Heatmap depicting the expression of the top differentially expressed genes in the cell populations from (A). (C) UMAP representation of the mRNA expression data of E12.5 control cells from the Olig3 + thalamic progenitor population. In total, three thalamic (rTh.Pro, cTh.Pro1, and cTh.Pro2), one epithalamic (epiTh.Pro), and one pretectal (preT.Pro) progenitor populations were identified with distinct transcriptomic profiles. (D) Heatmap depicting the expression of the top differentially expressed genes in the cell populations identified in (C). (E) Schematic showing the rostro-caudal organization of the identified progenitor cell populations in the developing thalamus as inferred from RNA in situ hybridization data. See ; and .

Article Snippet: 3D UMAP visualization , This paper , Mendeley Data: https://doi.org/10.17632/kjpj9n66jg ( https://data.mendeley.com/datasets/kjpj9n66jg ).

Techniques: Expressing, Control, RNA In Situ Hybridization

Journal: Cell reports

Article Title: Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation

doi: 10.1016/j.celrep.2022.111768

Figure Lengend Snippet: (A) UMAP representation and RNA velocity field of the single-cell RNA-seq data of the glutamatergic cell lineage in control embryos at each developmental stage. For reference, the same UMAP is also colored by the inferred cell differentiation pseudo-time and the gene expression levels of Sox2, Foxp2, and Cadm1 . The expression of Sox2 and Foxp2 genes separates differentiation trajectories into Sox2 + Foxp2 − (cTh.N1) and Sox2 − Foxp2 + (cTh.N2) populations. (B) Proportion of cells belonging to different glutamatergic thalamic nuclei at each developmental stage. Distinct thalamic subpopulations emerge at different time points. Late- and intermediate-emerging cell subpopulations are indicated by double asterisk (**) and single asterisk (*), respectively. (C) Whole-mount RNA in situ hybridization for Sox2, Foxp2, and Cadm1 in sagittal sections of the E13.5 diencephalon. The expression of these markers regionalizes the developing thalamus into distinct domains. Image credit: Allen Institute. (D) Glutamatergic thalamic IPCs are a heterogeneous population of cells with contributions from both cTh.Pro1 and cTh.Pro2 progenitors. The UMAP representation of the single-cell RNA-seq data corresponding to the glutamatergic thalamic IPC population from E14.5 control embryos is colored by the inferred cell differentiation pseudo-time and the gene expression levels of Olig2, Dbx1, Foxp2, and Sox2 . CPT, counts per thousand. See ; and .

Article Snippet: 3D UMAP visualization , This paper , Mendeley Data: https://doi.org/10.17632/kjpj9n66jg ( https://data.mendeley.com/datasets/kjpj9n66jg ).

Techniques: RNA Sequencing, Control, Cell Differentiation, Gene Expression, Expressing, RNA In Situ Hybridization

Journal: Cell reports

Article Title: Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation

doi: 10.1016/j.celrep.2022.111768

Figure Lengend Snippet: (A) UMAP representation of the mRNA expression data of E12.5 control and ΔSBE1/5 cells from the Olig3 + thalamic progenitor population (left). The bar plot shows the depletion or enrichment of cells between control and ΔSBE1/5 embryos in each progenitor cluster, indicating a strong depletion of rTh.Pro and cTh.Pro1 cells in ΔSBE1/5 embryos (****p < 0.0001, two-sided Fisher’s exact test, n = 1,789 cells). (B) Whole-mount RNA in situ hybridization for Nkx2–2, Olig2, Dbx1, and Rspo3 in E12.5 control and ΔSBE1/5 embryos, confirming the strong depletion of rTh.Pro and cTh.Pro1 cells in mutant embryos (n = 3). Scale bar, 500 μm. (C) UMAP representation of the glutamatergic thalamic cell lineage at E14.5 colored by the difference in the G2/M cell cycle gene expression score between control and ΔSBE1/5 cells. The analysis shows G2/M cell cycle genes are downregulated in thalamic progenitors and IPCs in ΔSBE1/5 embryos. (D and E) EdU incorporation and immunofluorescence staining for Olig2 on coronal sections of the thalamus in control and ΔSBE1/5 E13.5 embryos, showing a depletion of Olig2-expressing cells (cTh.Pro1 cells) and a reduction of cell cycle in IPCs (**p < 0.01, two-sided t test, n = 4 mice of each condition; error bars represent standard deviation). Scale bar, 100 μm. (F) The total number of Sox2 -expressing cells in the post-mitotic cTh.N1 and cTh.N2 transcriptomic cell lineages is largely depleted in ΔSBE1/5 compared with control embryos at E14.5. For reference, the location of the post-mitotic cTh.N1 and cTh.N2 clusters in the UMAP representation of the glutamatergic thalamic cell lineage is shown on the left (**p < 0.01, two-sided t test, n = 3 mice; error bars represent standard deviation). (G and H) Immunofluorescence staining for Sox2 and Foxp2 in anterior, medial, and posterior sections of E14.5 control and ΔSBE1/5 embryos. A depletion of Sox2 + cells and an expansion of Foxp2 + cells is observed in mutant embryos (*p < 0.05, **p < 0.01, ***p < 0.001, two-sided t test, n = 3 mice of each condition; error bars represent standard deviation). Scale bar, 100 μm. See .

Article Snippet: 3D UMAP visualization , This paper , Mendeley Data: https://doi.org/10.17632/kjpj9n66jg ( https://data.mendeley.com/datasets/kjpj9n66jg ).

Techniques: Expressing, Control, RNA In Situ Hybridization, Mutagenesis, Gene Expression, Immunofluorescence, Staining, Standard Deviation

Journal: Cell reports

Article Title: Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation

doi: 10.1016/j.celrep.2022.111768

Figure Lengend Snippet: (A) UMAP representation of the single-cell RNA-seq expression data from control and ΔSBE1/5 embryos at E18.5 corresponding to post-mitotic cTh.N, rTh.N, ZI, habenula, and reticular complex cell populations (left). The UMAP is colored by the amount of depletion or enrichment in the number of cells of each progenitor subpopulation between control and ΔSBE1/5 embryos (right). (B) RNA in situ hybridization for Neurog2, Cbln1, and Rorα on coronal sections through the thalamus of control and ΔSBE1/5 mice at P0 showing reduced expression in AV/AM/AD, VA/VL/VM, DLG, and VPM/VPL thalamic nuclei, consistent with the results of the single-cell RNA-seq analysis (**p < 0.01, ***p < 0.001, ****p < 0.0001, two-sided t test; n = 3 mice of each condition for Cbln1 and Rorα ; n = 4 mice of each condition for Neurog2 ; error bars represent standard error of the mean). (C) Locomotor behavior is compromised in adult ΔSBE1/5 mice as determined by force plate actometer (top four graphs) and rotarod (bottom) assays (*p < 0.05, **p < 0.01, ****p < 0.0001, two-sided t test; n = 8–9 mice of each condition for force plate; n = 10 mice of each condition for rotarod; error bars represent standard error of the mean). See .

Article Snippet: 3D UMAP visualization , This paper , Mendeley Data: https://doi.org/10.17632/kjpj9n66jg ( https://data.mendeley.com/datasets/kjpj9n66jg ).

Techniques: RNA Sequencing, Expressing, Control, RNA In Situ Hybridization

Journal: Cell reports

Article Title: Developmental trajectories of thalamic progenitors revealed by single-cell transcriptome profiling and Shh perturbation

doi: 10.1016/j.celrep.2022.111768

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 3D UMAP visualization , This paper , Mendeley Data: https://doi.org/10.17632/kjpj9n66jg ( https://data.mendeley.com/datasets/kjpj9n66jg ).

Techniques: Imaging, Software, Microscopy

Journal: bioRxiv

Article Title: Simultaneous assessment of eight phosphorylated STAT residues in T-cells by flow cytometry

doi: 10.1101/2021.11.22.469156

Figure Lengend Snippet: Naïve CD4+ T-cells were isolated and polarized as indicated in the methods section. Polarizations were done in triplicates. Cells were assessed for the expression of pSTATs after 30 minutes, 24 hours and 96 hours of polarization. ( A) Each panel shows the expression of the pSTATs in the different polarization groups at the different time points. ( B) Each panel shows the polarization frequencies for each pSTAT at different time points. ( C) The eight pSTAT parameters were reduced to two dimensions by UMAP and assessed for clusters by Phenograph. The concatenated UMAP of the 96-hour timepoints is shown in the large panel, with the Phenograph clusters labeled by number. The 96-hour UMAPs of each polarization group are shown in the smaller panels. ( D) The expression profiles for each pSTAT in each of the Phenograph clusters (separate panels) is plotted. Each cell belonging to the corresponding Phenograph cluster is plotted with its value on the x-axis representing the MFI value for the corresponding y-axis parameter. The MFI values for the corresponding FMO for each pSTAT are shown as black dots. ( E) The frequencies of the polarization groups in each Phenograph cluster are plotted. Results shown are representative of two independent experiments.

Article Snippet: Phenograph , UMAP and ClusterExplorer plugins for FlowJo were used for clustering and cluster composition analysis.

Techniques: Isolation, Expressing, Labeling

Journal: bioRxiv

Article Title: Simultaneous assessment of eight phosphorylated STAT residues in T-cells by flow cytometry

doi: 10.1101/2021.11.22.469156

Figure Lengend Snippet: Naïve CD4+ T-cells were cultured in different polarizing conditions. ( A) Cultures were evaluated by intracellular flow cytometry at the indicated time points for frequencies of IFNγ (leftmost panel), IL-4 (middle panel) and FOXP3 (rightmost panel) expressing cells. The “None” group (i.e. cells with only CD3/CD28 activation) are shown in black, the Th1 polarization samples in orange, Th2 polarized samples in blue, Th17 polarized samples in red, and iTreg polarized samples in purple. Polarizations were performed in triplicate. ( B) All timepoints and polarizations were concatenated and reduced to two dimensions by UMAP. Phenograph clustering was overlayed as a third, color dimension. Subplots of each timepoint are also shown. Results shown are representative of two independent experiments.

Article Snippet: Phenograph , UMAP and ClusterExplorer plugins for FlowJo were used for clustering and cluster composition analysis.

Techniques: Cell Culture, Flow Cytometry, Expressing, Activation Assay