Journal: Cell stem cell

Article Title: Human pallial MGE-type GABAergic interneuron cell therapy for chronic focal epilepsy

doi: 10.1016/j.stem.2023.08.013

Figure Lengend Snippet: Samples that were sequenced included day 0 hESCs (n=1), day 14 MGE progenitors (n=1), and week 6 EOP cells (n=3 paired unsorted/sorted lots). (A,B) UMAP (Uniform Manifold Approximation and Projection) visualization of cell clusters in all the samples combined (A), and in each of the separate samples listed (B). (C) Quantification of sample composition by cluster. (D) Feature plots of gene expression. All cells are displayed in light gray, cells with detectable expression are displayed in purple, with darker shade corresponding to higher expression level. (E) Dot plot for key genes that define different cell categories, including general markers of neurons, GABAergic and GE neurons, MGE, MGE pINs, MGE subpallial neurons, POA, CGE, LGE, neuronal progenitors (including MGE progenitor marker NKX2-1), cell cycle, pluripotent, as well as genes associated with glial cells, glutamatergic neurons (Glu), dopaminergic neurons (DA), serotonergic neurons (5HT) and cholinergic neurons (Ach). (F,G) Comparison of in vitro-derived Day 14 progenitors and EOP INs with human developing GE (GW9-18), using the Shi et al.dataset78 as a reference. (F) Prediction scores between 0 and 1 are projected onto day 14 and EOP UMAP. (G) Heatmap showing percentage of cells in each in vitro cluster that are assigned to different GE categories based on prediction scores.

Article Snippet: Single cell RNA sequencing To objectively assess cell composition, scRNA-seq analysis was performed using the 10X Genomics platform on the hESCs prior to the start of differentiation, hESC-derived MGE-type VZ-like progenitors on day 14 of differentiation, and nine independent unsorted cell lots at the end of the differentiation process.

Techniques: Gene Expression, Expressing, Marker, Comparison, In Vitro, Derivative Assay

Journal: The EMBO Journal

Article Title: Single‐cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin‐driven circadian network

doi: 10.15252/embj.2021108614

Figure Lengend Snippet: A Schematic work flow for 10× Genomics scRNASeq of pooled SCN slices after 3 days in culture. For each sequencing run, ca. 17 organotypic SCN slices were pooled into a single sample and their cells dissociated using papain to obtain single‐cell suspension of ˜8,000 cells/µl. Dispersed single cells, along with barcoded 10× Genomics Chromium Single Cell 3′ v2 technology gel beads, were partitioned into water‐in‐oil droplets. Within these droplets, reverse transcription and amplification steps generated cDNA libraries for the barcoded single cells. B t‐SNE plot from 13,324 sequenced SCN cells collected at CT7.5, across three independent sequencing runs. Each sequenced cell marked with a unique barcode is represented as a single dot. This dimensionality reduction method aims to maintain both local and global structure of the data by clustering data points of highest similarity nearest to each other. Cell clusters recognised by the graph‐based clustering algorithm are further colour‐coded to highlight the cell types: putative SCN neurons, extra‐SCN hypothalamic neurons, astrocytes, oligodendrocytes, radial glia, microglia, ependymocytes and endothelial cells. C Heatmap of the top 5 up‐regulated genes that most distinguish the transcriptional clusters identified in B. The degree of up‐regulation is measured as the log 2‐fold change ratio of gene expression calculated for cells of each cluster and normalised to a size factor accounting for the total transcript count of each sequenced cell across each cluster. D t‐SNE plot from 16,996 SCN sequenced cells collected at CT15.5, across two independent sequencing runs. As for B, the cell types, putative SCN neurons, extra‐SCN hypothalamic neurons, astrocytes, oligodendrocytes, radial glia, microglia, ependymocytes and endothelial cells, were identified. E As C, but for cell clusters identified at CT15.5. Source data are available online for this figure.

Article Snippet: Codes used to analyse scRNASeq data are publicly available from 10× Genomics at “ https://support.10xgenomics.com/single‐cell‐gene‐expression/software/overview/welcome .” Viral reagents generated in this study have been deposited with Addgene, AAV1.pProk2.Cre.T2A.mCherry Plasmid #169013 and AAV1.pProkR2.Cre.T2A.Venus Plasmid #169014.

Techniques: Sequencing, Suspension, Reverse Transcription, Amplification, Generated, Gene Expression

Journal: The EMBO Journal

Article Title: Single‐cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin‐driven circadian network

doi: 10.15252/embj.2021108614

Figure Lengend Snippet: A Schematic view of populations of interest from Venn analysis of scRNAseq data from daytime SCN neurons. B–E Representative RNAScope in situ hybridisation images from SCN cryostat sections to reveal overlap/non‐overlap of neuropeptide/receptor‐expressing cells. (B) Cells expressing Prok2 (green) and/or Avp (red). (C) Cells expressing Vipr2 and/or ProkR2 . (D) Potential autocrine signalling in cells expressing Prok2 + and/or ProkR2 + . (E) Cells expressing Av p and/or Avpr1a . Left: 40×, right 63×, scale bar: 100 µm. For each gene set, panel A and B show magnified 63× images. White arrows highlight the overlap‐/non‐overlap of neuropeptide/ receptor‐expressing cells. F, G Inferred topology of neuropeptidergic signalling axes between identified neuronal sub‐populations in SCN from circadian day (F) or circadian night (G). Inter‐cluster signalling is unscaled as connections are weighted by highest expression count measured across the entire dataset. Clusters are numbered according to their size and are annotated in the overview t‐SNE for day (bottom left) and night (top right), respectively. Source data are available online for this figure.

Article Snippet: Codes used to analyse scRNASeq data are publicly available from 10× Genomics at “ https://support.10xgenomics.com/single‐cell‐gene‐expression/software/overview/welcome .” Viral reagents generated in this study have been deposited with Addgene, AAV1.pProk2.Cre.T2A.mCherry Plasmid #169013 and AAV1.pProkR2.Cre.T2A.Venus Plasmid #169014.

Techniques: RNAscope, In Situ, Hybridization, Expressing

Journal: The EMBO Journal

Article Title: Single‐cell transcriptomics of suprachiasmatic nuclei reveal a Prokineticin‐driven circadian network

doi: 10.15252/embj.2021108614

Figure Lengend Snippet:

Article Snippet: Codes used to analyse scRNASeq data are publicly available from 10× Genomics at “ https://support.10xgenomics.com/single‐cell‐gene‐expression/software/overview/welcome .” Viral reagents generated in this study have been deposited with Addgene, AAV1.pProk2.Cre.T2A.mCherry Plasmid #169013 and AAV1.pProkR2.Cre.T2A.Venus Plasmid #169014.

Techniques: Plasmid Preparation, Recombinant, Modification, RNAscope, Multiplex Assay, Software

Journal: bioRxiv

Article Title: The pericardium forms as a distinct structure during heart formation

doi: 10.1101/2024.09.18.613484

Figure Lengend Snippet: A-D . Anatomy of the heart and pericardium in zebrafish embryos and early larvae. A,B . Lateral confocal imaging of transgenic hand2:EGFP;myl7:DsRed zebrafish, anterior to the left. Embryo showing hand2:EGFP and myl7:DsRed (myocardium marker) co-expression in the heart tube and hand2:EGFP- expressing cell populations in the pericardium, posterior mesothelium, pectoral fin, and pharyngeal arches at 48 hpf ( A , 10x; B , 20x). C,D . Ventral confocal imaging ( C 72 hpf single Z-slice, D 96 hpf max projection, anterior to the top) of hand2:EGFP;myl7:DsRed embryo showing reporter co-expression in the atrium and ventricle myocardium and hand2:EGFP- expressing cells in the pharyngeal arches and pericardial sac surrounding the heart ( C ), with the pericardium acquiring a mesh-like squamous epithelial structure at 96 hpf ( D ). E . SPIM-based Mercator projection of double-transgenic hand2:EGFP;drl:mCherry embryo at 14 hpf, anterior to the top, showing the anterior-to-posterior extent of lateral-most hand2:EGFP -expressing LPM cells fated as mesothelial progenitors (mp), with the anterior-most extent of the bilateral stripes indicated by white arrowheads. Dotted box outlines region of interest for subsequent imaging panels. F-H . Dorsal confocal imaging of transgenic reporter combinations at the level of the heart field, anterior to the top, asterisks showing the midline where the heart field converges, arrowheads at lateral mesh-like cells. F . hand2:EGFP;drl:mCherry embryo at 16 hpf prior to medial heart field migration, showing hand2:EGFP -expressing cell populations comprising lateral-most LPM. G ) hand2:EGFP;nkx2 . 5:ZsYellow embryo with dual-marked emerging cardiac disk. H . hand2:EGFP;tbx1:mCerulean embryo with similar co-expression across bilateral mesh-like cells and the cardiac disk. I . SPIM-based live imaging stills from Movie 1 depicting a hand2:EGFP;tbx1:mCerulean embryo from 19-24 hpf, anterior to the top, showing hand2:EGFP- expressing lateral-most cells migrating away from the midline towards the front (migration front, yellow dashed line), while cardiac precursors form the heart tube towards the midline (white dashed line). White arrows for migration direction. pa, pharyngeal arches; pc, pericardium; ht, heart; v, ventricle; a, atrium; hg, hatching gland. Scale bars A-D 100 μm, E-H 200 μm, I 50 μm.

Article Snippet: We conducted a 10xGenomics-based single-cell RNA-sequencing (scRNA-seq) analysis of zebrafish LPM cells at 10 hpf (tailbud stage, end of gastrulation) when the LPM has coalesced into its bilateral stripe architecture ( ).

Techniques: Imaging, Transgenic Assay, Marker, Expressing, Migration

Journal: bioRxiv

Article Title: The pericardium forms as a distinct structure during heart formation

doi: 10.1101/2024.09.18.613484

Figure Lengend Snippet: A . Representative confocal max projection of drl:mCherry ; hand2:EGFP double-transgenic embryo at 10 hpf as used for FACS-based isolation of post-gastrulation LPM for 10xGenomics-based single cell transcriptomics; anterior-posterior axis as indicated. B . UMAP plot of single cell transcriptomes of mCherry-sorted 10 hpf drl:mCherry ; hand2:EGFP zebrafish embryo cells showing 18 significant cell clusters, colored by identified subpopulation. C,D . Whole-mount mRNA in situ hybridization of representative pericardial/mesothelial genes sfrp5 ( C ) and jam2b ( D ), white dashed outlines to emphasize mesh-like spread of the expression domains lateral to the embryo axis; anterior to the top. E,F . UMAP plots of key myocardial ( E ) and pericardial genes ( F ) expressed across identified cluster identities. Cell representations are colored by scaled expression values using lower and upper 2%-quantiles as boundaries. G . Clustering analysis of bulk mRNA-sequenced left ventricle myocardium (Myo) and pericardium (Per) from neonatal rats for genes defining myocardium versus pericardium as identified from the zebrafish-based scRNA-seq analysis in B . Heatmap bins colored by row-scaled log2-normalized counts; columns (samples) split by tissue type; rows and columns ordered by hierarchical clustering (scaled expression values), sex of sample indicated on top. Scale bar A,B 200 μm.

Article Snippet: We conducted a 10xGenomics-based single-cell RNA-sequencing (scRNA-seq) analysis of zebrafish LPM cells at 10 hpf (tailbud stage, end of gastrulation) when the LPM has coalesced into its bilateral stripe architecture ( ).

Techniques: Transgenic Assay, Isolation, Single-cell Transcriptomics, In Situ Hybridization, Expressing

Journal: bioRxiv

Article Title: The pericardium forms as a distinct structure during heart formation

doi: 10.1101/2024.09.18.613484

Figure Lengend Snippet: A . Dotplot including key cell fate marker genes to annotate broad mesothelial, pericardial, and myocardial clusters, respectively. Dots colored by column-scaled mean expression (log-transformed library-size-normalized counts) and sized by expression frequency (fraction of cells with non-zero counts). B . Dorsal confocal imaging of representative dual-transgenic hand2:EGFP;nkx2 . 5:ZsYellow zebrafish embryo at 16 hpf showing marker co-expression and heterogeneity in prospective cardiac and pericardial progenitor cells around the heart field; anterior to the top. C-E Slingshot-based trajectory inference analysis of early LPM cells assigned using key marker genes to the public Zebrahub data set of single cell transcriptomes throughout zebrafish development. Inferred end points for myocardium and pericardium indicated as color-coded clusters ( C ). PCA plots of key myocardial ( D ) and pericardial genes ( E ) expressed across identities over time. Cells are colored by scaled expression values using lower and upper 2%-quantiles as boundaries. Scale bar B 200 μm.

Article Snippet: We conducted a 10xGenomics-based single-cell RNA-sequencing (scRNA-seq) analysis of zebrafish LPM cells at 10 hpf (tailbud stage, end of gastrulation) when the LPM has coalesced into its bilateral stripe architecture ( ).

Techniques: Marker, Expressing, Transformation Assay, Imaging, Transgenic Assay

Journal: bioRxiv

Article Title: The pericardium forms as a distinct structure during heart formation

doi: 10.1101/2024.09.18.613484

Figure Lengend Snippet: A . UMAP projection of zebrafish cells in Zebrahub dataset, colored by anatomical ontology classes, highlighting the diversity of cell types within the embryo. B . UMAP projection of zebrafish embryonic cells with pericardium cells highlighted in red, showing their distribution across different clusters. C . UMAP projections highlighting specific cell types within the zebrafish embryo: pericardium cells, pectoral fin cells, floor plate cells, pharyngeal arch 1 cells, pharyngeal arch 2 cells, and myocardium cells, each marked in red to show their spatial organization within the clusters. D . UMAP projections of zebrafish embryonic cells, with cells colored by cluster (left) and by time point (right), demonstrating the developmental stages and the organization of clusters over time. E . UMAP projection of a subset of clusters identified in zebrafish embryonic cells, highlighting the specific lineage groups: undifferentiated lateral plate mesoderm (LPM) at different hours post-fertilization (hpf), pericardium, and myocardium cells. F . Principal component analysis (PCA) of zebrafish embryonic cells, showing the distribution of cells over time (time point). G . PCA projection of the subset clusters identified in F , demonstrating the developmental trajectory of these specific lineages over time. See also .

Article Snippet: We conducted a 10xGenomics-based single-cell RNA-sequencing (scRNA-seq) analysis of zebrafish LPM cells at 10 hpf (tailbud stage, end of gastrulation) when the LPM has coalesced into its bilateral stripe architecture ( ).

Techniques:

Journal: bioRxiv

Article Title: The pericardium forms as a distinct structure during heart formation

doi: 10.1101/2024.09.18.613484

Figure Lengend Snippet: A . UMAP projections of representative genes found among LPM, myocardial, and pericardial clusters from zebrafish embryonic cells across time as key cell fate markers used to annotate clusters. Cells are colored by scaled expression values using top and bottom 2%-quantiles as boundaries. B . PCA projections of LPM, myocardial, and pericardial clusters from zebrafish embryonic cells across time showing key cell fate markers used to annotate clusters. Cells are colored by scaled expression values using top and bottom 2%-quantiles as boundaries. See also .

Article Snippet: We conducted a 10xGenomics-based single-cell RNA-sequencing (scRNA-seq) analysis of zebrafish LPM cells at 10 hpf (tailbud stage, end of gastrulation) when the LPM has coalesced into its bilateral stripe architecture ( ).

Techniques: Expressing

Journal: bioRxiv

Article Title: The pericardium forms as a distinct structure during heart formation

doi: 10.1101/2024.09.18.613484

Figure Lengend Snippet: A,B . Metascape analysis of top-50 cluster-defining genes from early LPM scRNA-seq; gene ontology terms enriched in myocardial cluster ( A ), and pericardial cluster ( B ); note regulation of canonical Wnt signaling as significant in pericardial cells. C-E . Confocal max projections of representative transgene expression for hand2:EGFP (green) and 7xTCF:mCherry (magenta, broadly reading out canonical Wnt signaling activity) with colocalization (white overlay), embryonic axis as indicated. At 10 hpf, canonical Wnt signaling shows a graded activity from the posterior towards the anterior ( C , lateral view, asterisk). At the start of heart field convergence, various anterior LPM cells show heterogeneous canonical Wnt activity, with lateral-most putative pericardial progenitors strongly expressing the 7xTCF:mCherry reporter ( D , dorsal view, arrowheads). This pattern continues throughout to 24 hpf ( E , lateral view, white arrowheads). F,G . Expression of canonical Wnt signaling-associated genes across myocardial and pericardial/mesothelial cells at tail bud stage in zebrafish. Cropped UMAP plot to depict cell clusters of interest ( F ) and individual Wnt signaling-associated genes, with expressing cells colored by scaled expression values using lower and upper 2%-quantiles as boundaries ( G ). Scale bar C-E 200 μm.

Article Snippet: We conducted a 10xGenomics-based single-cell RNA-sequencing (scRNA-seq) analysis of zebrafish LPM cells at 10 hpf (tailbud stage, end of gastrulation) when the LPM has coalesced into its bilateral stripe architecture ( ).

Techniques: Expressing, Activity Assay