Journal: Nature Cardiovascular Research

Article Title: Pharmacological blocking of neutrophil extracellular traps attenuates immunothrombosis and neuroinflammation in cerebral cavernous malformation

doi: 10.1038/s44161-024-00577-y

Figure Lengend Snippet: ( a ) Representative images of HBMVECs (shScramble (shScr), top and shCCM3 , bottom) stimulated with 500 ng/mL NET-enriched supernatant or control (Ctrl) media for 24 h showing changes in cell morphology (n = 3). ( b–g ) Representative western blot images of HBMVECs (shscramble or shCCM3) stimulated with 500 ng/mL NET-enriched supernatant or control media for 24 h (n = 3 biological replicates) ( B - C ) and quantified ( D - G ; in E-G normalization was done to α-tubulin or GAPDH). ( h ) Representative images of HBMVECs (shScramble, top and shCCM3 , bottom) stimulated with 500 ng/mL NET-enriched supernatant or control (Ctrl) media for 48 h showing showing changes in cell morphology (n = 6 biological replicates). ( i–n ) Representative western blot images of HBMVECs (shscramble or shCCM3) stimulated with 500 ng/mL NET-enriched supernatant or control media for 48 h (n = 6) ( J ) and quantified ( J - N ). ( o ) Representative images of HBMVECs (shScramble, left and shCCM3 , right) stimulated with NET-enriched supernatant (bottom) or control (Ctrl; top) media for 48 h stained for VE-cadherin (red; junction) and DAPI (blue; nuclei). White arrow head highlights areas with no VE-cadherin staining. The stains were repeated independently three times with similar results. ( p ) Representative images of HBMVECs (shScramble, top and shCCM3 , bottom) stimulated with 500 ng/mL NET-enriched supernatant or control (Ctrl) media for 72 h. The stains were repeated independently two times with similar results. In the graphs, the bar indicates the mean of each group, and the error bars represent the standard deviation. Statistical significance was determined using one-way ANOVA followed by a post-hoc Holm-sidak’s test.

Article Snippet: Endothelial-specific Ccm3 -deficient C57BL/6J mice ( Cdh5 (PAC)-Cre-ER T2 / Ccm3 flox/flox ) were generated by crossing Cdh5 (PAC)-Cre-ER T2 mice with Ccm3 fl/fl mice (Taconic Artemis).

Techniques: Control, Western Blot, Staining, Standard Deviation

Journal: Nature Communications

Article Title: Endothelial Piezo1 channel mediates mechano-feedback control of brain blood flow

doi: 10.1038/s41467-024-52969-0

Figure Lengend Snippet: a Crossbreeding scheme used to generate inducible, EC-specific Piezo1-GOF mice. Tamoxifen-treated Cre+ mice are used as GOF, and littermate Cre- mice are the control. b Representative traces of Piezo1 currents recorded from freshly isolated ECs from the somatosensory cortices of control and GOF mice. The cell-attached configuration was used, where EC patches were held at -50 mV in the absence or presence of Yoda1 (5 µM) in the pipette solution. C denotes closed channel. c Averaged data of the open probability (NP O ) of Piezo1 in the absence (n = 11 ECs/6 control; 10 ECs/4 GOF mice) or presence of Yoda1 (n = 10 ECs/6 control; 7 ECs/4 GOF mice). d Schematic of the LSCI setup for imaging blood flow through a thinned skull. ( e ) The Allen mouse brain atlas highlighting the somatosensory barrel cortex. f, g Differential maps of blood flow responses in a control ( f ) and a GOF ( g ) mouse. Whisker stimulation using air puffs (5 Hz, 30 s) evoked hyperemia in the contralateral somatosensory cortex (arrows). h Representative traces and summary data of the hyperemic response to 30 s-whisker stimulations in control ( n = 8) and GOF ( n = 9) mice. Right: area under the curve analyses ( n = 19 whisker stimulations/6 control; 20 stimulations/8 GOF mice). i Representative responses and scatter plots of FH evoked by 5 s air puffs (n = 7 control, n = 9 GOF mice). Area under the curve analysis was calculated from 41 and 43 stimulations from 7 control and 9 GOF mice, respectively. j Representative images demonstrating the staining of ECs (caveolin-1), basement membranes (collagen IV), and nuclei (DAPI) in brain slices of the cortex of GOF and control mice. Scale bar: 100 µm. Vascular densities and string vessel densities in Piezo1 cx/cx ; Cdh5 -Cre+ ( n = 12) and control ( n = 10) mice. k Representative images and averaged data as in j from brain slices of the hippocampus of Piezo1 cx/cx ; Cdh5 -Cre+ ( n = 12) and control ( n =10) mice. All statistical tests were unpaired Student’s t test (two-sided, * P < 0.05, ** P < 0.01, *** P < 0.001). All error bars are SEM. Scatter plot data in c, h, I, j, k are presented as mean ± SEM. Area under the curve data in h and i are presented as histograms. Source data are provided as a Source Data file.

Article Snippet: Tamoxifen-inducible, Piezo1 gain-of-function (Piezo1 cx/cx ; Cdh5 -Cre+) mice were engineered by crossing Cdh5 -CreER T2 and knock-in Piezo1 cx/cx mice (Taconic Biosciences, Model No. 21617) in which floxed exons 45-51 are followed by a mutant Piezo1 copy (R2482H mouse mutation- equivalent to R2456H in humans) , .

Techniques: Control, Isolation, Transferring, Imaging, Whisker Assay, Staining

Journal: Nature Communications

Article Title: Endothelial Piezo1 channel mediates mechano-feedback control of brain blood flow

doi: 10.1038/s41467-024-52969-0

Figure Lengend Snippet: a Left : Example FH response to whisker stimulation showing the increase in CBF shortly after the stimulus starts (upstroke) and the return to baseline at the end of the stimulation (downstroke). Right : A schematic showing that neuronal activity increases blood flow (FH), and the resulting change in forces activates cationic influx by opening Piezo1 channels, thus acting as a mechano-feedback system to attenuate FH. b Experimental setup illustrating impalement of a freshly isolated brain EC using a sharp microelectrode. ECs were obtained from a Pdgfrb -Cre-TdTomato mouse, where pericytes are TdTomato+. Scale bar = 10 µm. c Traces and summary data of V m measurements showing the effect of Yoda1 (5 µM) on endothelial V m (-Yoda1 [n = 16 ECs/5 mice]; +Yoda1 [n = 23 ECs/4 mice]). d, e Representative traces and scatter plots of functional hyperemia responses to whisker stimulations in Piezo1 cx/cx ; Cdh5 -Cre+ (GOF) and Piezo1 flox/flox ; Cdh5 -Cre+ (knockout) mice, and their respective controls ( d : n = 6 GOF, 6 controls; e : n = 7 knockout, 5 controls). f Representative traces and plateau-followed-by-exponential fittings ( dotted ) to obtain time constant for downstroke (Tau), in a control mouse and a GOF mouse. Right : Scatter plot of downstroke Tau values in control ( n = 5) and GOF ( n = 6) mice. g Similar to f in knockout and control mice (n = 5 each). h Representative traces and plateau-followed-by-exponential fittings ( dotted ) to obtain upstroke Tau, in a control mouse and a GOF mouse. Right : Scatter plot of upstroke Tau values in control and GOF ( n = 6 mice each). i Representative traces and averaged data of upstroke Tau in control and knockout mice (n = 5 mice each). FH responses were measured through a cranial window using laser Doppler flowmetry. Statistical tests were unpaired Student’s t test (two-sided, c ), and Mann-Whitney test (one-sided in d – e , two-sided in f – i ; * P < 0.05, ** P < 0.01, *** P < 0.001). Error bars in c , d and e are SEM. Data in c , d , e are presented as mean values ± SEM, and data in f , g , h , i are presented as violin scatter plots. Source data are provided as a Source Data file.

Article Snippet: Tamoxifen-inducible, Piezo1 gain-of-function (Piezo1 cx/cx ; Cdh5 -Cre+) mice were engineered by crossing Cdh5 -CreER T2 and knock-in Piezo1 cx/cx mice (Taconic Biosciences, Model No. 21617) in which floxed exons 45-51 are followed by a mutant Piezo1 copy (R2482H mouse mutation- equivalent to R2456H in humans) , .

Techniques: Whisker Assay, Activity Assay, Isolation, Functional Assay, Knock-Out, Control, MANN-WHITNEY

Journal: Nature Communications

Article Title: Endothelial Piezo1 channel mediates mechano-feedback control of brain blood flow

doi: 10.1038/s41467-024-52969-0

Figure Lengend Snippet: a Novel object recognition test in control and Piezo1 cx/cx ; Cdh5 -Cre+ mice. Heat maps demonstrate the cumulative time spent with familiar (F) and novel (N) objects. b Scatter plot shows the discrimination index (DI%-calculated based on duration) for the two groups (n = 7 control, 9 Piezo1 cx/cx ; Cdh5 -Cre+ mice). DI of 50% ( dotted ) indicates equal time spent with novel and familiar objects. c Velocities of control ( n = 7) and Piezo1 cx/cx ; Cdh5 -Cre+ mice (n = 9) during the NOR test. d, e Similar to a and b , test results from Piezo1 cx/cx ; Slco1c1 -Cre+ and their controls ( n = 10 each). f Motor activity of Piezo1 cx/cx ; Slco1c1 -Cre+ mice and their controls (n = 10 each) during the NOR test. g Percent of alternation over all 6 trials during the spontaneous alternation T-maze was calculated as [(number of correct alternations/6)*100)] in Piezo1 cx/cx ; Slco1c1 -Cre+ and their controls ( n = 11 each). The dotted line represents the 50% chance level. h Choice latency in Piezo1 cx/cx ; Slco1c1 -Cre+ and control mice over trials 1 to 6. Asterisks highlight significantly higher choice latency in Piezo1 cx/cx ; Slco1c1 -Cre+ mice at T4, T5 and T6. Data from individual mice are shown, and bold lines and transparent shades are means and SEM, respectively (n = 11 mice each). Statistical tests were unpaired Student’s t test (two-sided, b, c, e, f, g ) and two-way ANOVA test in h (* P < 0.05, ** P < 0.01, *** P < 0.001). All error bars are SEM. Data in b, c, e-g are presented as mean ± SEM. Source data are provided as a Source Data file.

Article Snippet: Tamoxifen-inducible, Piezo1 gain-of-function (Piezo1 cx/cx ; Cdh5 -Cre+) mice were engineered by crossing Cdh5 -CreER T2 and knock-in Piezo1 cx/cx mice (Taconic Biosciences, Model No. 21617) in which floxed exons 45-51 are followed by a mutant Piezo1 copy (R2482H mouse mutation- equivalent to R2456H in humans) , .

Techniques: Control, Activity Assay

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: PrE and nEnd differentiation competence of SGGC reporter cells and derivation from E3.5 ICMs, related to Figure 2 (A) Bright-field and immunofluorescence imaging of SGGC reporter cells in 2iLIF and day 7 of PrE differentiation. (B) Flow cytometry contour plots of (A). Bottom left quadrant: gating based on a negative control. (C) Flow cytometry contour plots of SGGC nEnd showing gating strategy for isolating GATA6-mCherry + and SOX2-GFP + populations for re-plating. (D) Flow cytometry contour plots of E14JU nEnd stained for PDGFRA-APC and PECAM-FITC followed by FACS for PDGFRA-APC + cells and analyzed by flow cytometry after 96 h. Bottom left quadrant: gating based on a negative control. (E) Quantification of 3D nEnd growth in size 24–144 h following FACS and seeding in AggreWells ( n = 200 spheroids per time point). (F) Quantification of total expression by flow cytometry of SGGC 3D nEnd 0–120 h following FACS for GATA6-mCherry + /SOX2-GFP − cells. (G) Bright-field images of nEnd derivation from E3.5 blastocysts. (H) Flow cytometry contour plots of embryo-derived nEnd stained for PDGFRA-APC and PECAM-FITC. Bottom left quadrant: gating based on a negative control. (I) Immunostaining of 3D nEnd from embryo-derived nEnd for indicated markers. Errors bars represent ± SD. Scale bars: 50 μm in (A) and (I); 100 μm in (G).

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Immunofluorescence, Imaging, Flow Cytometry, Negative Control, Staining, Expressing, Derivative Assay, Immunostaining

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: GATA6-expressing nEnd spontaneously undergoes de-differentiation to SOX2-expressing Epi-like cells (A) Bright-field image of nEnd with aggregate emerging from monolayer (white arrowhead). (B) Immunostaining of SGGC nEnd for OCT4 and DAPI with orthogonal projection. (C) Flow cytometry contour plots of SGGC nEnd 12–120 h following FACS for GATA6-mCherry + /SOX2-GFP − cells ( n = 4 biological replicates). Bottom left quadrant: gating based on a negative control. (D) Quantification of total expression by flow cytometry of SGGC nEnd 12–144 h following FACS for GATA6-mCherry + /SOX2-GFP − cells ( n = 4 biological replicates). (E) Immunofluorescence imaging of SGGC nEnd 7 days following isolation by FACS for GATA6-mCherry + or SOX2-GFP + cells. (F) Quantification of total expression of SGGC nEnd subpopulations across 4 consecutive rounds of FACS for GATA6-mCherry + nEnd. Time points collected 5 days after seeding. (G) Illustration of 3D nEnd cultured in AggreWells with endodermal outside cells (magenta) and Epi-like inside cells (yellow). (H) Bright-field image of 3D nEnd cultured in AggreWells. (I) Immunofluorescence imaging of SGGC 3D nEnd cultured in AggreWells. (J) Schematic of nEnd culture system. ESCs are differentiated toward PrE and following FACS for PDGFRA-APC expression, these are expanded as nEnd in adherent culture or 3D nEnd in suspension culture. Scale bars: 100 μm in (A) and (H); 50 μm in (B), (E), and (I). See also Figure S2 .

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Expressing, Immunostaining, Flow Cytometry, Negative Control, Immunofluorescence, Imaging, Isolation, Cell Culture, Suspension

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: OCT4 expression separates uncommitted from committed extra-embryonic endoderm cell types, related to Figure 3 (A) Immunostaining of E3.5–E4.5 embryos for indicated markers, where E3.5: n = 20 embryos; E4.0: n = 5 embryos; E4.25: n = 5 embryos; E4.5: n = 17 embryos. (B) Quantification of E3.5 and E4.5 embryos for OCT4 and GATA6 expression, where E3.5: n = 20 embryos and 107 cells; E4.5: n = 17 embryos and 179 cells. (C) t-distributed stochastic neighbor (tSNE) embedding of scRNA-seq of the mouse preimplantation embryo at E3.5–4.5, where color scale represents expression of Pou5f1 transcripts and shape indicates cell type. (D) Immunostaining of E14JU nEnd for indicated markers. (E) Flow cytometry contour plot of unstained OCT4-mCherry nEnd. Bottom left quadrant: gating based on a negative control. (F) Median fluorescence intensity (MFI) of OCT4 expression by immunostaining normalized to DAPI ( n = 1,495 cells [left] and 1,467 cells [right]). (G) Flow cytometry contour plot of OCT4-mCherry nEnd stained for PECAM-FITC and PDGFRA-APC, where gating of PECAM-FITC + cells (left) are visualized for OCT4-mCherry and PECAM-FITC co-expression (right). (H) RT-qPCR of Oct4 + isolated by FACS, 2iLIF, serum/LIF (SL), and EpiLC ESCs for indicated markers. (I) Flow cytometry contour plots for monitoring OCT4-mCherry, PECAM-FITC, and PDGFRA-APC expression from 24 to 96 h following FACS for PDGFRA-APC + cells. Bottom left quadrant: gating based on a negative control. (J) Quantification of total percentage of expression of OCT4-mCherry-positive (Oct4 + ), double negative (DN), PDGFRA-APC single-positive (Pdgfra + ), and double positive (DP) nEnd, based on flow cytometry following multiple consecutive rounds of FACS to isolate and expand the DP population. (K and L) Quantification of (K) cell size of Pdgfra + ( n = 333 cells) and DP ( n = 511 cells) nEnd based on E-cadherin immunostaining and (L) nuclear size of Pdgfra + ( n = 426 cells) and DP ( n = 400 cells) nEnd based on DAPI localization. Nuclear/cytoplasmic ratio: DP = 0.63; Pdgfra + = 0.67. (M) Immunostaining of OCT4-mCherry nEnd for indicated markers. (N) Immunostaining of 3D nEnd cultured in AggreWells for indicated markers. p values determined by unpaired t test, and error bars represent ± SD. Scale bars, 50 μm.

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Expressing, Immunostaining, Flow Cytometry, Negative Control, Fluorescence, Staining, Quantitative RT-PCR, Isolation, Cell Culture

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: OCT4/PDGFRA co-expressing nEnd represents uncommitted PrE capable of multi-lineage differentiation (A) Heatmap of scaled expression of indicated genes from scRNA-seq of the mouse preimplantation embryo across scVelo-defined latent time. Top bar: progression from E3.5 ICM (dark yellow) to E4.5 PrE (dark red). (B) Flow cytometry contour plot of OCT4-mCherry nEnd stained for PDGFRA-APC. Boxes highlight Pdgfra + , DP, and Oct4 + populations. Bottom left quadrant: gating based on a negative control. (C) Flow cytometry contour plots of Pdgfra + , DP, and Oct4 + nEnd 5 days following FACS for PDGFRA-APC + cells. Bottom left quadrant: gating based on a negative control in Figure S3 E. (D) Quantification of (C) for subpopulation composition in total population for Pdgfra + , DP, and Oct4 + nEnd (DN, double negative). (E) Schematic of dynamic equilibrium established in nEnd culture. (F) Quantification of total expression by flow cytometry of OCT4-Cherry nEnd 0–96 h following FACS for PDGFRA-APC + cells in Figure S3 I. (G) Immunostaining of OCT4-mCherry nEnd for indicated markers 5 days following FACS for PDGFRA-APC + cells. (H) RT-qPCR of Pdgfra + and DP nEnd isolated by FACS for indicated markers. (I) Immunostaining of E4.5 embryos injected with either OCT4-mCherry-H2B-Venus Pdgfra + (top), DP (middle), or Oct4 + (bottom) nEnd at the 8-cell stage for indicated markers. (J) Allocation of lineage contribution of OCT4-mCherry-H2B-Venus Pdgfra + ( n = 44 embryos), DP ( n = 36 embryos), and Oct4 + ( n = 25 embryos) nEnd in chimera embryos (NC, no contribution; TE pos., TE position). (K) Immunostaining of E6.5 embryos following clonal injection of OCT4-mCherry-H2B-Venus DP (top, n = 4 embryos) or Oct4 + (bottom, n = 3 embryos) cells at the 8-cell stage for indicated markers. (L) Schematic of blastoid generation from DP nEnd. (M) Immunostaining of a DP nEnd-derived blastoid for indicated markers. (N) Quantification of efficiency of blastoid formation defined as the presence of TE, PrE, and Epi-like cell types ( n = 304 individual blastoids/trophospheres). p values determined by unpaired t test, and error bars represent ± standard deviation (SD). Scale bars, 50 μm. See also Figures S3 – .

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Expressing, Flow Cytometry, Staining, Negative Control, Immunostaining, Quantitative RT-PCR, Isolation, Injection, Derivative Assay, Standard Deviation

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: nEnd contributes to TE in vivo and differentiates into a TE-like cell types in vitro , related to Figure 3 (A) Total number of OCT4-mCherry-H2B-Venus DP and Pdgfra + cells contributing to each embryo at E4.5 (DP: n = 36 embryos; Pdgfra + : n = 44 embryos). (B) Immunostaining of an OCT4-mCherry-H2B-Venus DP nEnd chimera embryo at E4.5 for indicated markers. (C) Bright-field images of nEnd and nEnd cultured in TSC medium. (D) Immunostaining of OCT4-mCherry nEnd and nEnd cultured in TSC medium for indicated markers. White arrowheads point to BRACHYURY/CDX2 co-expressing cells and white asterisk points to CDX2 single-positive cells. (E) Differentiation of nEnd in TSC medium followed by whole transcriptome analysis by scRNA-seq. (F) UMAP dimensional embedding of 6,094 nEnd cells in TSC medium. Top: coloring based on culture condition defined in (C); bottom: coloring based on Louvain clustering. (G) UMAP dimensional embedding showing single-cell expression of indicated markers. (H) Heatmap of candidate lineage markers expressed in log 2 normalized clustered data. Scaled by row. (I) Expression of indicated TE and mesoderm-specific markers in TE-like ( n = 357 cells) and Mes-like ( n = 221 cells) clusters. (J) Gene overlap analysis of TE-like and Mes-like clusters with scRNA-seq of the mouse preimplantation embryo. Gray points represent p > 0.05. (K) Immunostaining of DP nEnd-derived blastoids for indicated markers, imaged by widefield microscopy. (L) Bright-field and immunostaining of a DP nEnd-derived blastoid for indicated markers. (M) Immunostaining of DP nEnd-derived blastoids and trophospheres for indicated markers. Values for (H) and (I) in Table S2 . p values determined by unpaired t test, and error bars represent ± SD. Scale bars: 100 μm in (C) and (K); 50 μm in (B), (D), (L), and (M).

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: In Vivo, In Vitro, Immunostaining, Cell Culture, Expressing, Derivative Assay, Microscopy

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: XEN cells represent later-stage extra-embryonic endoderm lacking OCT4 expression that cannot be rescued upon culture in nEnd medium, related to Figure 3 (A) Schematic of E14JU ESCs in 2iLIF toward XEN cells, , followed by FACS for PDGFRA-APC + cells to purify endoderm population. (B) Flow cytometry contour plots of XEN cell derivation compared with 2iLIF ESCs and nEnd stained for PECAM-FITC and PDGFRA-APC. Bottom left quadrant: gating based on a no stain (NS) negative control. (C) Bright-field images of XEN derivation compared with 2iLIF ESCs and nEnd. (D) Immunostaining and bright-field images of nEnd and XEN cells for indicated markers. (E) Flow cytometry contour plots of XEN cells cultured in indicated conditions stained for PECAM-FITC and PDGFRA-APC expression. Bottom left quadrant: gating based on a negative control. (F) Immunostaining of cXEN cells for indicated markers. (G) RT-qPCR of XEN cells compared with nEnd isolated by FACS for PDGFRA-APC expression for indicated markers. p values determined by unpaired t test, and error bars represent ± SD. Scale bars: 100 μm in (C); 50 μm in (D) and (F).

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Expressing, Flow Cytometry, Staining, Negative Control, Immunostaining, Cell Culture, Quantitative RT-PCR, Isolation

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: JAK/STAT signaling is required to maintain uncommitted PrE in vitro and in vivo (A) Bright-field images of OCT4-mCherry nEnd in control and treated conditions. Arrowheads indicate aggregates containing putative reverted cells and white dashed line highlights epithelial nEnd with compact morphology. (B) Immunostaining of OCT4-mCherry nEnd in control and treated conditions after 2 passages for indicated markers. (C) Single-cell quantification for pSTAT3 and OCT4 staining in (B) normalized to DAPI. n values indicate total number of cells quantified. (D) Quantification of median fluorescence intensity (MFI) normalized to DAPI for (B), where +LIF: n = 1,629 cells; −LIF: n = 873 cells; JAKi: n = 726 cells. (E) Total MFI normalized to DAPI for (B). (F) Flow cytometry histogram of OCT4-mCherry expression in nEnd for control and treated conditions. Dashed line indicates gating based on negative control. (G and H) Total expression by flow cytometry of G PDGFRA-APC and H OCT4-mCherry for control and treated conditions. Gating based on quadrants in Figure S3 E. (I) Treatment regimen subjected to 8-cell embryos in (J)–(O). (J) Immunostaining of E4.5 embryos for indicated markers. Expression of GATA6 and NANOG was used for analysis performed in (K)–(N), and expression of CDX2 was used for analysis performed in (O). (K) ICM lineage allocation in treated embryos. (L) Ratio of Epi/PrE cells per embryo in treated embryos. (M) Total number of cells within the ICM of treated embryos. (N) Quantification of presence or absence of an ICM in treated embryos. (O) Total number of TE cells based on CDX2 expression in treated embryos (CV = coefficient of variance), where (A) n = 11 embryos; (B) n = 14 embryos. n values for treatment regimen outlined in (I) shown in (J)–(O) are (A) n = 23 and (B) n = 26 embryos. p values determined by unpaired t test, and error bars represent ± SD. Scale bars: 100 μM in (A); 50 μm in (B) and (J).

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: In Vitro, In Vivo, Control, Immunostaining, Staining, Fluorescence, Flow Cytometry, Expressing, Negative Control

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: Clustering annotation strategy for 2iLIF, nEnd, and 3D nEnd scRNA-seq, related to Figure 5 (A) scRNA-seq strategy for nEnd and 3D nEnd. (B) Allocation of each culture condition per cluster in Figure 5 A. (C) Differential expression of candidate 2C markers across all clusters. (D) Heatmap showing expression of genes related to apoptosis in log 2 normalized clustered data. (E) GO analysis of selected biological processes of upregulated genes in the putative apoptotic cluster. (F) PCA of scRNA-seq dataset for 2iLIF, nEnd, and 3D nEnd. Left: coloring based on culture condition; right: coloring based on Louvain clustering. (G) Heatmap of candidate lineage markers in log 2 normalized clustered data. Scaled by row. (H) Heatmap of scaled expression of candidate early ( Pou5f1 , Tbx3 , Fgfr2 , Klf5 , Idh1 , and Gata6 ) and late ( Gata4 , Col4a1 , Hnf4a , Foxa2 , Vegfa , and Cited1 ) PrE genes from scRNA-seq of ICM to PrE in vivo across pseudotime. Scaled by row. (I) PAGA of integrated in vitro nEnd, 3D nEnd and nEnd to TSC (this study), and somatic cell reprogramming to iPSCs from a XEN-like intermediate datasets using SCVI. Coloring based on Louvain clustering (XEN-like 1–3, Pdgfra + 1–5, DP, Oct4 + , Mes-like, and TE-like) or reprogramming stage (XEN, SII D8, SII D12, SIII D3, SIII D6, SIII D8, SIII D10, SIII D15, and SIII D21). Thicker lines indicate highly connected regions and thinner lines indicate regions with lower confidence. Values in Table S3 .

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Expressing, In Vivo, In Vitro

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: nEnd contains subpopulations mirroring stages of mouse preimplantation development (A) UMAP dimensional embedding of 14,788 2iLIF, nEnd, and 3D nEnd cells by scRNA-seq. Top: coloring based on culture condition shown above, including representative immunostaining for SOX2 and GATA6; bottom: coloring based on Louvain clustering. (B) UMAP dimensional embedding showing single-cell expression of indicated markers. (C) Heatmap of candidate lineage markers in log 2 normalized clustered data for early and late PrE in vivo, defined in Figure S6 H. Scaled by row. (D) GO analysis of selected biological processes of up- and downregulated genes in the DP cluster. (E) Gene overlap analysis of Oct4 + , DP, and Pdgfra + 1–5 clusters with 700 downstream targets of Oct4. Color scale is based on odds ratio and grids labeled with p value indicating significance of the respective odds ratio. (F) Venn diagram showing overlap of Oct4 + , DP, and 2iLIF clusters based on differential upregulation of 1,361 pluripotency and Epi-specific genes. , , , (G and H) Heatmap of scaled expression of indicated genes from scRNA-seq of (G) DP and Oct4 + nEnd clusters in vitro and (H) ICM to PrE in vivo across latent time. Top bar shows progression from (G) DP (yellow) to Oct4 + (pink) and (H) E3.5 ICM (ochre) to E4.5 (burgundy). (I) UMAP dimensional embedding of integrated in vitro nEnd and 3D nEnd with in vivo data using scVI. Top: coloring based on culture condition (nEnd, 3D nEnd) or developmental stage (E3.5, E4.5). Bottom: coloring based on Louvain clustering. (J) PAGA of integrated scRNA-seq dataset shown in (I). Dark gray lines: highly connected regions; light gray lines: regions with lower confidence. Values in Table S3 . See also Figures S6 and .

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Immunostaining, Expressing, In Vivo, Labeling, In Vitro

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: Gene expression characterization and validation in OCT4-mCherry and Oct4 LOF nEnd scRNA-seq datasets, related to Figures 5 and (A) Volcano plot of DEGs between DP and Pdgfra + clusters representative of log 2 fold change > 0.25 and p < 0.05. (B) Flow cytometry contour plots gated for PDGFRA-APC expression of OCT4-mCherry nEnd cultured in 2DG or etomoxir for 5 days. (C) Quantification of PDGFRA-APC expression in nEnd treated with 2DG or etomoxir compared with control. (D) Volcano plot of DEGs between Oct4 + and DP clusters representative of log 2 fold change > 0.25 and p < 0.05. (E and F) (E) RT-qPCR and (F) western blot of Oct4 LOF ESCs cultured in 2iLIF for Oct4 /OCT4 ± 4OHT at indicated time intervals. rpS6 used as a loading control in (F). (G) Flow cytometry contour plot of Oct4 LOF PrE following 7 days of differentiation in RACL. (H) PCA of scRNA-seq dataset for Oct4 LOF nEnd ± 4OHT at 120 h, where coloring is based on Louvain clustering. (I) Allocation of each culture condition per cluster in (H). (J) Violin plots showing expression of indicated markers across Louvain clusters in scRNA-seq dataset in (H). (K) Violin plots showing expression of select genes related to FGF/ERK signaling from Figure 6 G across PrE-like and Epi-like clusters defined in (J). p values determined by unpaired t test, and error bars represent ± SD.

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Quantitative RT-PCR, Western Blot

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: OCT4 regulates nEnd plasticity and ESRRB is a gatekeeper of nEnd de-differentiation (A) Oct4 depletion in Oct4 LOF nEnd performed for (B)–(G). PDGFRA-APC + nEnd isolated by FACS and cultured +4OHT (−Oct4) or −4OHT (+Oct4) for 48 h, followed by an additional 72 h in normal RACL medium. (B) Immunostaining of Oct4 LOF nEnd ± 4OHT at 120 h for indicated markers. (C) Flow cytometry contour plots of Oct4 LOF nEnd −4OHT (left) and +4OHT (right) stained for PECAM-FITC and PDGFRA-APC. Quadrants: gating based on a negative control. (D) Quantification of proportions of PECAM-FITC + and PDGFRA-APC + cells based on total expression by flow cytometry in (C). (E) PCA of scRNA-seq dataset for Oct4 LOF nEnd ± 4OHT at 120 h. Coloring indicates treatment and black arrows indicate inferred trajectories of dataset variance. (F) UMAP dimensional embedding showing single-cell expression of indicated markers. (G) Heatmap of candidate lineage markers in log 2 normalized clustered data for FGF/ERK-related genes. Scaled by row. (H) Esrrb induction with EKOiE nEnd. PDGFRA-APC + EKOiE nEnd isolated by FACS and cultured in +Dox (+Esrrb) or −Dox (−Esrrb) for 7 days. (I) Quantification of total expression of PECAM-FITC in EKOiE nEnd −Dox. (J) Immunostaining of EKOiE nEnd ±Dox for indicated markers. (K and L) Quantification of (J) for OCT4 and ESRRB in cells grouped by GATA6 expression for (K) single cell and (L) total expression. (M) RT-qPCR of PDGFRA-APC + EKOiE cells ±Dox isolated by FACS for indicated markers. p values determined by unpaired t test, and error bars represent ± SD. Scale bars, 50 μm. See also Figures S7 .

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Isolation, Cell Culture, Immunostaining, Flow Cytometry, Staining, Negative Control, Expressing, Quantitative RT-PCR

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: Chromatin states in nEnd subpopulations demonstrate specific developmental characteristics, related to Figure 7 (A) Profiles of H3K4me1 and H3K27ac for Oct4 + , DP, and Pdgfra + nEnd upstream of Nanog compared with defined pluripotency (yellow) enhancers, highlighted in gray with −5 and −45 Nanog SE annotated in blue. Bigwigs generated from 3 biological replicates. (B) Profiles of H3K4me1 and H3K27ac for Oct4 + , DP, and Pdgfra + nEnd at Col4a1 and Col4a2 loci compared with defined pluripotency (yellow) and nEnd (magenta) enhancers. Bigwigs generated from 3 biological replicates. (C) Histone mark categories for promoter states. Active: H3K27ac and H3K4me1; poised: H3K27me3 only; repressed: H3K27me3 and H3K9me3. (D) Unbiased hierarchical clustering of bulk RNA-seq dataset for Oct4 + , DP, and Pdgfra + nEnd biological replicates. (E) PCA of bulk RNA-seq dataset for Oct4 + , DP, and Pdgfra + nEnd replicates. (F and G) Scatterplots showing correlation between DEGs (log 2 fold change > 1.5; adjusted p < 0.05) in bulk RNA-seq compared with scRNA-seq for (F) DP vs. Oct4 + nEnd and (G) DP vs. Pdgfra + nEnd. (H) Quantification of expression for mean normalized bulk RNA-seq counts in DP, Oct4 + , and Pdgfra + nEnd across promoter categories outlined in (C). (I) Gene overlap analysis of promoter peak categories defined in (C) annotated to the nearest TSS against DEGs from scRNA-seq data of the mouse preimplantation Epi and PrE in vivo . Gray points represent p > 0.05. (J) Position of active Oct4 + peaks annotated to the nearest TSS (±20 kb) relative to genes selected for Figure 7 F and other downstream targets of Oct4. (K) GO analysis of selected biological processes for primed DP peaks. (L) Euler diagrams showing overlap between annotated genes within ±20 kb of primed and bivalent DP peaks and DEGs in scRNA-seq for Oct4 + , Pdgfra + , and TE-like clusters to DP nEnd. (M) Position of bivalent DP peaks annotated to the nearest TSS (±20 kb) relative to selected genes related to TE fate. Values in Tables S4 and .

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Generated, RNA Sequencing Assay, Expressing, In Vivo

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet: Status of lineage-specific regulatory elements supports DP nEnd plasticity and predicts future differentiation competence (A and B) H3K4me1 occupancy at defined (A) pluripotency and (B) nEnd enhancers in Oct4 + , DP, and Pdgfra + nEnd. (C) Histone mark categories for enhancer states. Active: H3K27ac and H3K4me1; primed: H3K4me1; bivalent: H3K4me1 and H3K27me3; repressed: H3K9me3. (D and E) Heatmap of log 2 normalized peaks overlapping with annotated (D) pluripotency and (E) nEnd enhancers. Scaled by row. (F) Position of DP-primed peaks annotated to the nearest TSS (±20 kb) of select downstream target genes of Oct4. (G) Profiles of H3K4me1, H3K27ac, and H3K4me3 for Oct4 + , DP, and Pdgfra + nEnd upstream Oct4 compared with defined 2iLIF (yellow) and EpiLC (magenta) enhancers. The proximal enhancer (PEnh) and distal enhancer (DEnh) of Oct4 are highlighted in blue. Bigwigs generated from 3 biological replicates. (H) PCA of global naive (2iLIF) to primed (EpiLC) enhancer patterns across indicated conditions. (I) Heatmap of scRNA-seq data from log 2 normalized DEGs in Oct4 + , Pdgfra + , and TE-like clusters compared with genes with a TSS ±20 kb of primed and bivalent DP peaks. Annotations include lineage-specific genes for Epi, endoderm, and TE. (J) Gene overlap analysis of bivalent enhancer peaks annotated to the nearest TSS against DEGs from scRNA-seq data of the mouse preimplantation embryo in vivo . Gray points: p > 0.05. (K) Ternary plots of motif enrichment across Oct4 + , DP, and Pdgfra + nEnd in the active, primed, and bivalent categories. p < 0.05 for at least one population for all motifs, axis represents −log 10 ( p value). Gray dots indicate all motifs and colored dots highlight motifs of interest. Values in Table S4 . See also Figure S8 .

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Generated, In Vivo

Journal: Cell

Article Title: The primitive endoderm supports lineage plasticity to enable regulative development

doi: 10.1016/j.cell.2024.05.051

Figure Lengend Snippet:

Article Snippet: C57BL/6N (Taconic), Rosa mT/mG and Pdgfra-CreER T2 mice were housed under 12h light/dark cycles at room temperature (RT) of 22°C (± 2°C) and a humidity of 55% (±10%), with the air in the room changed 8-10 times per hour.

Techniques: Recombinant, Reverse Transcription, Multiplex Assay, Software, Microscopy

Journal: Current opinion in hematology

Article Title: Mouse models of vascular development and disease

doi: 10.1097/MOH.0000000000000649

Figure Lengend Snippet: Genetic mouse models useful to study vascular malformations. Non-exhaustive list of mouse models of Arteriovenous Malformations (AVMs), Brain AVMs (BAVMs), Lymphatic malformations (LMs), Hereditary Hemorrhagic Telangiectasias (HHTs) and Cerebral Cavernous Malformations (CCMs). Table adapted from Nielsen et al., 2016 [ 49 ], Tual-Chalot et al, 2015 [ 34 ], Zeng et al., 2019[ 50 ] and McDonald et al., 2011[ 39 ]. (Endothelial cell, EC; lymphatic endothelial cell, LEC; overexpression, OE; vascular endothelial growth factor, VEGF; vascular smooth muscle cell, vSMC).

Article Snippet: Cdh5-CreER T2 (VE-cad) , Tg(Cdh5-cre/ERT2) 1Rha Taconic # 13073-M , Pan EC , E9.5 , yes , [ 8 ].

Techniques: Over Expression, Mutagenesis, Knock-Out, Expressing, Knock-In

Journal: iScience

Article Title: A murine model of cerebral cavernous malformations with acute hemorrhage

doi: 10.1016/j.isci.2022.103943

Figure Lengend Snippet: Endothelial-progenitor-cells-specific deletion of Ccm3 results in progressive formation of lesions throughout the brain Procr CreERT2−IRES-tdTomato/+/ Ccm3 f/f mice received a single dose of tamoxifen at P1 and were analyzed at the indicated times. (A and B) Representative photographs of whole brains (A) and tiling of brain sections (B) stained for PECAM1, showing lesion burden and distribution. Black arrowheads, lesions. Scale bars: 1000 μm. (C) Quantification of total lesioned area. Data are means ± SE. Each dot represents an animal; p < 0.0001 among groups (ANOVA); ∗p < 0.05, ∗∗p < 0.01 (Tukey’s post hoc tests). (D) Quantification of lesion numbers. Data are means ± SE. Each dot represents an animal; p < 0.001 among groups (Kruskal-Wallis tests); ∗p < 0.05, ∗∗p < 0.01 (Dunn’s post hoc tests). (E) Quantification of distribution of lesion area between cerebellum and cerebrum. Data are means ± SE. Each dot represents an animal; p < 0.001 among groups (ANOVA) ∗p < 0.00 1 (Tukey’s post hoc tests). (F) Quantification of distribution of lesion numbers between cerebellum and cerebrum. Data are means ± SE. Each dot represents an animal; p < 0.001 among groups (Kruskal-Wallis tests) ∗p < 0.05, ∗∗p < 0.01 (Dunn’s post hoc tests).

Article Snippet: The Cdh5 (PAC)-Cre-ER T2 / Ccm3 f/f mice in which Ccm3 f/f mice with exons 4–5 of the Ccm3 gene flanked by loxP sites (Taconic Artemis GmbH) were bred with Cdh5 (PAC)-Cre-ER T2 mice to obtain endothelial-specific and tamoxifen-inducible loss of function of the Ccm3 gene, as previously described ( ).

Techniques: Staining

Journal: iScience

Article Title: A murine model of cerebral cavernous malformations with acute hemorrhage

doi: 10.1016/j.isci.2022.103943

Figure Lengend Snippet: The spleen shows hematopoietic disorders Representative images of the spleen from 3-month-old wild-type (WT) and Ccm3 EPCKO (KO) mice. Four WT and nine KO mice were analyzed. (A) Representative H&E staining. (B) Quantification of white and red pulp areas. Data are means ± SE. Each dot represents an animal. ∗p < 0.001 (Student’s t-tests). (C) Representative immunostaining for different cell populations. Scale bar: 50 μm (A); 100 μm (C).

Article Snippet: The Cdh5 (PAC)-Cre-ER T2 / Ccm3 f/f mice in which Ccm3 f/f mice with exons 4–5 of the Ccm3 gene flanked by loxP sites (Taconic Artemis GmbH) were bred with Cdh5 (PAC)-Cre-ER T2 mice to obtain endothelial-specific and tamoxifen-inducible loss of function of the Ccm3 gene, as previously described ( ).

Techniques: Staining, Immunostaining

Journal: iScience

Article Title: A murine model of cerebral cavernous malformations with acute hemorrhage

doi: 10.1016/j.isci.2022.103943

Figure Lengend Snippet: The spleen shows increased vascular density (A and B) Representative images of the spleen stained for the endothelial markers PECAM1 (A), VE-Cadherin (endothelial cells) and Endomucin (sinusoids) (B). Scale bar: 50 μm. (C) Quantification of vascular density and mean vessel diameter. Data are means ± SE. Each dot represents an animal; ∗p < 0.005 (Mann-Whitney tests). Four WT and nine KO 3-month-old mice were analyzed.

Article Snippet: The Cdh5 (PAC)-Cre-ER T2 / Ccm3 f/f mice in which Ccm3 f/f mice with exons 4–5 of the Ccm3 gene flanked by loxP sites (Taconic Artemis GmbH) were bred with Cdh5 (PAC)-Cre-ER T2 mice to obtain endothelial-specific and tamoxifen-inducible loss of function of the Ccm3 gene, as previously described ( ).

Techniques: Staining, MANN-WHITNEY

Journal: iScience

Article Title: A murine model of cerebral cavernous malformations with acute hemorrhage

doi: 10.1016/j.isci.2022.103943

Figure Lengend Snippet: Propranolol administration did not ameliorate the pathological phenotype (A–C) Procr CreERT2−IRES-td-Tomato/+/ Ccm3 f/f were treated with 333 mg/L of propranolol dissolved in the drinking water from one to three months of age. Quantification of (B) total lesioned area and (C) number of lesions in the brain. (D) Quantification of erythrocytes extravasation expressed as percentage of Ter119-positive area outside the vascular area. (E) Quantification of chronic hemorrhage expressed as percentage of mice with at least one field positive for Perls' Prussian stain. (F) Quantification of leukocytes extravasation expressed as percentage of CD45-positive area on nonvascular area. For all the plots, data are means ± SE. Each dot represents an animal; p > 0.05 (Student’s t test).

Article Snippet: The Cdh5 (PAC)-Cre-ER T2 / Ccm3 f/f mice in which Ccm3 f/f mice with exons 4–5 of the Ccm3 gene flanked by loxP sites (Taconic Artemis GmbH) were bred with Cdh5 (PAC)-Cre-ER T2 mice to obtain endothelial-specific and tamoxifen-inducible loss of function of the Ccm3 gene, as previously described ( ).

Techniques: Staining

Journal: iScience

Article Title: A murine model of cerebral cavernous malformations with acute hemorrhage

doi: 10.1016/j.isci.2022.103943

Figure Lengend Snippet:

Article Snippet: The Cdh5 (PAC)-Cre-ER T2 / Ccm3 f/f mice in which Ccm3 f/f mice with exons 4–5 of the Ccm3 gene flanked by loxP sites (Taconic Artemis GmbH) were bred with Cdh5 (PAC)-Cre-ER T2 mice to obtain endothelial-specific and tamoxifen-inducible loss of function of the Ccm3 gene, as previously described ( ).

Techniques: Plasmid Preparation, Recombinant, Software