Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-b) Correlation between Log2FC of gene expression in 12-month-old APP (a) and Tau (b) mice and human AD DEGs in astrocytes obtained from Grubman et al. Red and blue dots depict up and downregulated genes respectively in APP (a) or Tau mice (b). Grey dots are genes exclusively significant in human astrocytes. Complete gene list can be found in Supplementary Table 12. c) Heatmap showing Log2FC in synapse-related genes in astrocytes from postmortem human AD patients and in 12-month-old APP and Tau mice. Human data obtained from – . * padj <0.05. d) Immunostaining of postmortem human frontal cortex showing GPC5 protein expression in astrocytes marked with GFAP in layer 1. Scale bar = 20 µm. e-f) Representative image showing Gpc5 mRNA in situ hybridization in APP 12-month-old hippocampus (left, scale bar = 100 µm). Right: Zoom in panel showing Gpc5 mRNA levels (white), astrocytes (marked with s100b, magenta) and amyloid plaques (stained with 6e10, green). Scale bar = 20 µm (e). f) Quantification of Gpc5 mRNA signal (% area) in astrocytes associated with amyloid plaques (plaques) or non-associated with amyloid plaques (no plaques). Each datapoint is a mouse (open circles=female, close=male). N = 7 (4F, 3M). Paired t-test was performed for statistical analysis.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Expressing, Immunostaining, In Situ Hybridization, Staining

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-b) Diagram depicting a) AAV-PHP.eB expressing HA-Gpc5 or smFP as control under the astrocyte-specific minimal GFAP promoter; b) APP 2-month-old mice were retro-orbitally injected with AAV-HA-Gpc5 or AAV-smFP-HA as control, and at 4 months of age mice were collected for electrophysiology recordings. Same image is shown in . c-d) Representative image of 7-month-old hippocampi from mice injected with either HA-GPC5 or smFP-HA and stained with HA and s100b antibodies. Same images are shown in . Scale bar=20 µm. d) Quantification showing the percentage of s100b-positive astrocytes overexpressing the HA-Gpc5 or smFP-HA construct in the hippocampus. N=3 mice per group, statistical test: T test. e-h) Whole cell patch clamp recordings of spontaneous excitatory postsynaptic currents (sEPSC) in hippocampal CA1 pyramidal neurons. e) Diagram showing the hippocampal neurons recorded in CA1. f) Representative traces from different groups showing an increased frequency of sEPSC in the APP smFP group that is prevented APP GPC5 overexpressing group. g-h) Average frequency (g) and amplitude (h) of sEPSC events during 5-minute recordings. Each data point represents an independent neuron. n= WT smFP: 19 neurons, 9 mice, WT GPC5: 18 neurons, 10 mice, APP smFP: 15 neurons, 10 mice; APP GPC5: 16 neurons, 8 mice. Male (close circles) and female (open circles) mice were included for the analysis. Statistics: 2-way-ANOVA Tukey’s correction for multiple comparison run on neurons. * p <0.05, ** p <0.01, *** p <0.001. Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Expressing, Control, Injection, Staining, Construct, Patch Clamp, Comparison

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) APP and WT 2-month-old mice were retro-orbitally injected with AAV-HA-GPC5 or AAV-smFP as control. At 6 months of age mice were behaviorally tested in the open field and Barnes maze test, at 7 months brains were collected for immunohistochemistry analysis. b) Barnes maze memory test: mice were trained for 5 consecutive days (2 trials a day) to find an escape box. The next 3 days the escape box location is changed to the opposite hole to test cognitive flexibility. On days 6 and 10 a probe trial is performed when the box is removed. c) Representative trajectories used by mice to find the escape box during standard learning (day 2). d) Mean latency to find the target hole is plotted across different training days (mean of the 2 trials per day). Each panel shows different experimental groups. Statistics are calculated using 2-way ANOVA with Dunnett’s multiple comparisons test relative to day 1. WT smFP: N=27(13F, 14M); APP smFP: N=21 (10F, 11M); WT GPC5: N=24 (13F, 11M), APP GPC5: N=18 (7F, 11M). e) Learning slope between day 1 and day 2 (mean latency day 1-mean latency day 2). Statistical analysis is calculated using one-sample T test against hypothetical value of 0 and corrected for multiple comparisons. Each data point represents an independent mouse. (N same as in panel d). f-g) Representative tile scans stitched images of GluA2 immunostaining in the hippocampal CA3 region in 7-month-old APP and WT littermates overexpressing AAV-Gpc5 or smFP. Scale bar = 20 µm (f). Quantification of GluA2 coverage showed a significant increase in GPC5-overexpressing mice (2-way ANOVA treatment effect: * p = 0.02). WT smFP: N=13 (6F, 7M); WT GPC5: N=12 (6F, 6M); APP smFP: N=12 (5F, 7M); APP GPC5: N=11(5F, 6M). Scale bar= 100 µm. h-i) Pearson correlation between GluA2 area and number of smFP (h) or GPC5 (i)-overexpressing astrocytes showed a significant correlation ( p =0.04) only in the GPC5 overexpressing group (i). Statistics: * p <0.05, ** p <0.01, *** p <0.001, **** p <0.0001

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Injection, Control, Immunohistochemistry, Immunostaining

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) Venn diagram showing overlapping upregulated (left panel) and downregulated (right panel) DEGs in 12 month-old APP and Tau mice and human AD astrocytes obtained from Grubman et al. b) Single nuclear RNA sequencing studies from human postmortem patients of different neurodegenerative disorders showing GPC5 downregulation in the astrocyte cluster. Data obtained from – , , . PFC=prefrontal cortex, EC=entorhinal cortex, CTX=cortex, TH=Thalamus, FC=Frontal cortex. c) Synapse-regulating genes are downregulated in GFAP-high astrocyte clusters relative to other clusters in human AD postmortem brains. Data obtained from , , . d) Example image of immunostaining of postmortem human frontal cortex showing GPC5 protein expression in astrocytes marked with GFAP. Scale bar=100 µm. e) Barplot showing TPMs for Gpc5 expression at 4, 6 and 12 months in APP mice. Statistics show the padj value calculated using Deseq2 package in R.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: RNA Sequencing Assay, Immunostaining, Expressing

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: Immunohistochemistry analysis of WT and APP 7-month-old mice overexpressing HA-GPC5 or smFP-HA from 2 to 7 months after testing for memory performance in Barnes maze test. a) Left: Sagittal section of a 7-month-old mouse overexpressing HA-GPC5 for 5 months and immunostained with anti-GPC5 antibody showing GPC5 protein being overexpressed throughout the brain. Scale bar=1mm. Right: Magnification showing hippocampal astrocytes overexpressing GPC5 protein, scale bar=100 µm. Both images were stitched image from a tile scan. b-c) Representative hippocampal image (a) and quantification (b) of 7-month-old mouse overexpressing HA-GPC5 or smFP-HA showing colocalization between HA and the astrocyte marker s100b. Scale bar=20 µm. Same image and quantification were shown in main . N= 9-13 mice per group. Male (closed circles) and female mice (open circles) included in the analysis. Statistics: 2-way ANOVA. d-e) Representative hippocampal image showing lack of colocalization between HA and the neuronal marker NeuN in 7-month-old smFP and GPC5-overexpressing mice. e) Quantification showing less than 2% of neurons overexpressing smFP-HA or HA-GPC5. N= 3 mice per group. Statistical analysis: T test.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Immunohistochemistry, Marker

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a) Representative image (left) and quantification (right) showing colocalization of GFAP-positive astrocytes with HA tag in 4-month-old hippocampus overexpressing smFP-HA or HA-GPC5 for 2 months. Stainings were performed in hippocampal acute slices after electrophysiology recordings were completed. Scale bar=20 µm. Each data point represents an independent mouse. N=3 mice/group. b-c) Representative image (b) and quantification (c) of GFAP staining in the hippocampal CA1 region in WT and APP 4-month-old mice overexpressing HA-GPC5 or smFP control for 2 months. Scale bar=50 µm. Stainings were performed in hippocampal acute slices after electrophysiology recordings were completed. Each data point represents an independent mouse. N=4-6 mice/group. Male (close circles) and female (open circles) mice were included for the analysis. d-f) Electrophysiology recordings of CA1 hippocampal pyramidal neurons performed in 4-month-old APP and WT mice overexpressing HA-GPC5 or smFP showing: d) Resting membrane potential, e) Average decay time and f) average rise time (10-90%) of sEPSC events during 5 minutes recordings. Each data point represents an independent neuron. N= WT smFP: 19 neurons, 9 mice, WT GPC5: 18 neurons, 10 mice, APP smFP: 15 neurons, 10 mice; APP GPC5: 16 neurons, 8 mice. Male (close circles) and female (open circles) mice were included for the analysis. Statistics: 2-way-ANOVA Tukey’s correction for multiple comparison based on neurons. Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Staining, Control, Membrane, Comparison

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: a-c) Open field test consisting of 10 minutes of spontaneous exploration showed an increased total distance travelled in 6-month-old APP mice compared to WT independently of GPC5 overexpression (a) with no changes in mean speed (b) and time spent in the center of the arena (c). d-e) Barnes maze test: area under the curve for the standard learning curve (d) and time spent exploring the target hole during probe trial (e) revealed no differences between groups. Statistical analysis: 2-way-ANOVA. f-g) Area under the curve for the reversal learning curve in the Bares maze test revealed a significant GPC5 treatment effect (2-way-ANOVA, p =0.03) (f) and no differences in the time spent exploring the target hole during the reversal probe trial (g). h-i) Representative trajectories used by mice to find the escape box during reversal learning (day 2) (h). Mean latency to find the target hole across reversal learning days (mean of the 2 trials per day). Each panel shows different experimental group. Statistics are calculated using 2-way ANOVA with Dunnett’s multiple comparisons test relative to day 1 (i). Statistics: * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001. Each data point represents an independent mouse. Male (filled dots) and female (open dots) mice were included for the analysis. WT smFP N=27 (13F, 14M); APP smFP N=21 (10F, 11M); WT GPC5 N=24 (13F, 11M), APP GPC5 N=18 (7F, 11M). Graphs show the mean ± SEM.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Over Expression

Journal: bioRxiv

Article Title: Astrocyte transcriptomic analysis identifies glypican 5 downregulation as a contributor to synaptic dysfunction in Alzheimer’s disease models

doi: 10.1101/2024.10.30.621182

Figure Lengend Snippet: Immunohistochemistry analysis of WT and APP mice overexpressing HA-GPC5 or smFP-HA from 2 to 7 months after testing for memory performance in Barnes maze test. a-c) Representative tile scans stitched image (a) and quantification (b) of hippocampal amyloid plaques stained with the OC antibody in APP 7-month-old mice. Statistics: T-test. smFP N=11 (5F, 6M); GPC5 N=11 (4F, 7M). Scale bar=100 µm. c) Pearson correlation analysis between plaque load and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions N=10 smFP, 8 GPC5. d-f) Representative tile scans stitched image (d) and quantification (e) of hippocampal GFAP immunoreactivity in the entire hippocampus across different experimental groups. Pearson correlation analysis between GFAP area and number of HA-positive astrocytes in smFP overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (f). WT smFP N=13 (6F, 7M); WT GPC5 N=11 (6F, 5M) APP smFP N=11 (5F, 6M); APP GPC5 N=11 (4F, 7M). Scale bar=100 µm. g-i) Representative tile scans stitched images (g) and quantification (h) of hippocampal CA3 vGlut1 immunoreactivity across different experimental groups. Pearson correlation analysis between vGlut1 area and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (i). WT smFP N=13 (6F, 7M); WT GPC5 N=12 (6F, 6M); APP smFP N=12 (5F, 7M); APP GPC5 N=12 (5F, 7M). Scale bar=50 µm. j-l) Representative tile scans stitched image (j) and quantification (k) of hippocampal CA3 synaptoporin immunoreactivity across different experimental groups. Pearson correlation analysis between synaptoporin area and number of HA-positive astrocytes in smFP-overexpression (left panel) or HA-Gpc5 overexpression (right panel) conditions (l). WT smFP N=11 (5F, 6M); WT GPC5 N=12 (6F, 6M); APP smFP N=10 (4F, 6M); APP GPC5 N=11 (5F, 6M) Scale bar=50 µm. Graphs show the mean ± SEM. Each data point represents an independent mouse. Male (filled dots) and female (open dots) mice were included for the analysis. Statistics: 2-way ANOVA Tukey’s test for multiple comparisons, unless specified otherwise.

Article Snippet: For the overexpression of HA-tagged GPC5, cDNA for the coding sequence of mouse GPC5 (Origene # MR218095) was used.

Techniques: Immunohistochemistry, Staining, Over Expression

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 1 TRT enhanced by avian influenza A virus infection represses genes on the complementary DNA strand. A Gene profile of the averaged normalized expression levels of 5,052 genes in A549 cells at 24 h after H1N1/H5N1/H7N9 influenza virus infection or AF treatment. The gene bodies between the TSSs and TTSs were equally sized and scaled to 60 bins, and the gene flanking regions 4 kb upstream of the TSSs and 4 kb downstream of the TTSs were divided into 100-bp windows. B Numbers of TRT genes (FC in the expression level of the TRT region (H5N1/AF) > 5) at different times after H1N1/H5N1/H7N9 infection of A549 cells. C Spearman rank linear correlation coefficient between the upregulated expression levels of the TRT region and the downregulated expression levels of TRT-influenced genes following the trans-TRT and cis-TRT patterns, respectively, at different times after H5N1/H7N9 infection in A549 cells. D Numbers of trans-TRT-influenced genes (FC in the expression level of the trans-TRT gene (H5N1/AF) > 5 and FC in the expression level of the trans-TRT-influenced gene (H1N1/H5N1) > 1.5) at different times after H5N1/H7N9 infection in A549 cells. E Functional pathway enrichment analysis of trans-TRT-influenced genes in H5N1-infected A549 cells (two-tailed P < 0.05, Benjamini–Hochberg adjusted P < 0.05). Detection of F GLS-TRT or G IL23A-TRT in A549 cells by using fluorescence in situ hybridization (FISH). A549 cells were treated with AF/H1N1/H5N1 for 24 h [DAPI nuclear staining (blue) and FISH signals obtained using a Cy3-conjugated DNA probe (red)]. The fluorescence intensity was semiquantitatively assessed using the mean fluorescence intensity (MFI) of each cell. The data are shown as the means ± SEMs. *P < 0.05, **P < 0.01. RNA-seq coverage levels of H the GLS gene, trans-TRT region of GLS, and STAT1 gene, and I the IL23A gene, trans-TRT region of IL23A, and STAT2 gene 12 h after AF/H1N1/H5N1/H7N9 treatment of A549 cells. The gene bodies and intergenic regions, as well as the gene flanking regions 2 kb upstream of the TSSs, were divided into 50-bp windows. Only exon regions are shown in this graph. RNA-seq datasets were established in duplicate

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Virus, Infection, Expressing, Functional Assay, Two Tailed Test, In Situ Hybridization, Staining, RNA Sequencing

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 2 TRT inhibition by CRISPR interference enhances STAT1/STAT2 expression and cell viability. RT-qPCR analysis of the A GLS-TRT gRNA and B IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). RT-qPCR analysis of C STAT1 mRNA expression in the Ctrl gRNA and GLS-TRT gRNA groups and D STAT2 mRNA expression in the Ctrl gRNA and IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). Western blot analysis of E STAT1 protein expression in the Ctrl gRNA and GLS-TRT gRNA groups and F STAT2 protein expression in the Ctrl gRNA and IL23A-TRT gRNA groups at different times after infection with H5N1 (MOI = 4). β-Actin expression served as the reference control. MTS cell viability assay in the G GLS-TRT gRNA and H IL23A-TRT gRNA groups at 48 h after treatment with AF or infection with H5N1 (MOI = 4). RT-qPCR analysis of viral M2 expression levels in the I GLS-TRT gRNA and J IL23A-TRT gRNA groups at 24 h after infection with H5N1 (MOI = 4). The expression levels in I and J are normalized to the Ctrl gRNA group. Each experiment was repeated at least three times. The data are shown as the means ± SEMs. *P < 0.05, **P < 0.01

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Inhibition, CRISPR, Expressing, Quantitative RT-PCR, Infection, Western Blot, Control, Viability Assay

Journal: Cellular & molecular immunology

Article Title: Avian influenza viruses suppress innate immunity by inducing trans-transcriptional readthrough via SSU72.

doi: 10.1038/s41423-022-00843-8

Figure Lengend Snippet: Fig. 4 TRT is reduced and lung injury is ameliorated in SSU72 transgenic mice infected with the lethal H5N1 virus. A Western blot analysis of mouse SSU72 expression in mouse lung tissues at 3 days after treatment with AF/H1N1/H5N1. β-Actin expression served as an internal control. B Numbers of TRT genes (expression of the TRT region upregulated by more than 5 compared with the AF-treated condition) in lung tissues from control (n = 5) and SSU72 transgenic mice (n = 5) at 3 days after intratracheal infection with H5N1 (106 TCID50). The relative mRNA expression ratios of C mouse STAT1 and D STAT2 in lung tissues from control (n = 8) and SSU72 transgenic mice (n = 4) at 3 days after intratracheal infection with H5N1 virus (106 TCID50). Mouse β- actin expression served as the reference control. E Kaplan–Meier survival curves for control (n = 8) and SSU72 transgenic mice (n = 10) after intratracheal infection with H5N1 (106 TCID50). F–H Control and SSU72 transgenic mice were infected with AF or H5N1 (106 TCID50) via intratracheal instillation. F Viral titers in the lungs were assessed 4 days after infection with H5N1 in control (n = 7) and SSU72 transgenic mice (n = 3). G Wet-to-dry weight ratios of the lungs of control (n = 4) and SSU72 transgenic mice (n = 4) at 3 days after infection with H5N1. H Representative images of lung pathology in control and SSU72 transgenic mice at 3 days after H5N1 infection. The lung injury scores (means ± SEMs) and numbers of infiltrating cells per microscopic field (means ± SEMs) are shown in the bar graphs. N = 100 fields for control (n = 15) and SSU72 transgenic (n = 6) mice. Bar = 100 μm. *P < 0.05 and **P < 0.01. Each experiment except for RNA-seq analysis of lungs from mice with or without H5N1 infection was repeated at least three times

Article Snippet: Primary antibodies specific for the following proteins/peptides were used: SSU72 (1:1000; Cell Signaling Technology, cat. no. 12816) and STAT1 (1:500; Cell Signaling Technology, cat. no. 9172), STAT2 (1:500; Bethyl Laboratories, Inc., Montgomery, Texas, USA; cat. no. A303-512A-M), Flag tag (rabbit, 1:5000; MultiSciences; cat. no. LK-ab002-100), DDDDK tag (mouse, 1:5000; MBL Inc., Ottawa, ON, Canada; cat. no. M185-3 L), β-actin (1:10000; Sigma Aldrich, Saint Louis, MO, USA; cat. no. A5441), and CRISPR/Cas9 (polyclonal; Diagenode Inc., Denville, NJ, USA; cat. no. C15310258).

Techniques: Transgenic Assay, Infection, Virus, Western Blot, Expressing, Control, RNA Sequencing

Journal: Nature communications

Article Title: Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

doi: 10.1038/s41467-022-29311-7

Figure Lengend Snippet: Fig. 2 scRNA-sequencing of early LPM reveals a heterogeneous progenitor pool. A LPM-marking drl:mCherry-positive cells of zebrafish embryos at tailbud stage, FACS-isolated, and sequenced using CEL-Seq2. B SPIM projection of drl:mCherry embryo at tailbud, labeling the LPM. C UMAP plot displaying 15 LPM cell clusters, colored by subpopulation: cardiopharyngeal (1,2), heart field, anterior vasculature, hemangioblasts/kidney (1,2), head mesoderm, hatching gland, hand2-high (1–4), and endoderm (1–3) as distinct group. D Approximate anterior-to-posterior orientation based on cdx4 (RNA ISH at 5 ss). E Dotplot including key cell fate markers to annotate clusters. 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); rows and clusters ordered by hierarchical clustering of scaled expression values. F UMAP plots of several genes co-expressed with hand2 or among cluster-determining genes in four hand2-high clusters. Cells colored by scaled expression values using lower/upper 1%-quantile boundaries. G–K Whole-mount gene expression analysis of select transcripts enriched in hand2-high cells by fluorescent in situ hybridization (ISH) (G, H) and colorimetric mRNA ISH (I–K). Fluorescent ISH of meis3 (G), foxh1 (H) with hand2 at 4 ss, revealing overlap in posterior LPM (magnified regions from dashed boxes). meis3 ISH at 10 ss (I), gata5 at 12 ss (J), and sfrp5 at 18 ss (K) showing expression in lateral-most LPM sprawling outwards (arrowheads). Scale bar G, H 100 μm, I–K 250 μm.

Article Snippet: The sections were blocked by incubating with 2% BSA 1× PBS supplemented with 1% horse serum at room temperature and incubated with HAND2 antibody (Santa Cruz Biotechnology A-12, Sc-398167, at dilution 1:100) overnight at 4 °C.

Techniques: Sequencing, Isolation, Labeling, Expressing, Transformation Assay, Gene Expression, In Situ Hybridization

Journal: Nature communications

Article Title: Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

doi: 10.1038/s41467-022-29311-7

Figure Lengend Snippet: Fig. 4 Primordial germ cells migrate with, and home within, the hand2-positive LPM. A UMAP plots of cxcl12a, hand2, and pax2a. Cells are colored by scaled expression values using lower and upper 1%-quantiles as boundaries. B, C Snapshots of time-lapse movie (Supplementary Movie 1) showing drl:EGFP with primordial germ cell (PGC) marker mCherry-f′-nos3′UTR, with 3D renderings at 60% epiboly, 1 ss, and 10 ss (B) and maximum intensity Mercator projections for the same time points (C). The PGCs migrate from four different clusters during gastrulation into two bilateral clusters during somitogenesis (boxed regions). Note the sparse (lost) PGCs located within the endoderm (asterisks). Animal side is for 60% epiboly to the top, anterior for 1 ss and 10 ss (B) to the top and (C) to the left. D Imaging of hand2:EGFP with mCherry-f′-nos3′UTR showing a dorsal view at 24 hpf: the PGC clusters fall completely within the hand2:EGFP domain. The outlines of the yolk extension are highlighted. E Segmented 3D rendering of a hand2:EGFP; mCherry-f′-nos3′ UTR double-positive embryo stained with anti-Pax2a (yellow) and DAPI (blue) at 28 hpf. F A schematic transverse section of a 24 hpf embryo showing two bilateral PGC clusters (magenta) lateral of the developing gut, ventral-lateral of the Pax2a-positive developing pronephros (yellow) within the cxcl12a-high hand2-positive LPM/coelomic epithelium (green). Scale bar D, E 250 μm.

Article Snippet: The sections were blocked by incubating with 2% BSA 1× PBS supplemented with 1% horse serum at room temperature and incubated with HAND2 antibody (Santa Cruz Biotechnology A-12, Sc-398167, at dilution 1:100) overnight at 4 °C.

Techniques: Expressing, Marker, Imaging, Staining