Journal: Science Advances

Article Title: Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation

doi: 10.1126/sciadv.adn8963

Figure Lengend Snippet: ( A ) Total bone marrow cells in Nup358 fl/fl (control) and Nup358 fl/fl CreER T2 (Nup358 knockout) mice treated with tamoxifen. ( B ) Schematic illustration of hematopoiesis. HSCs, hematopoietic stem cells; MPPs, multipotent progenitors; LSK, Lin − Sca1 + cKit + cells; MyP, myeloid progenitors; CMP, common myeloid progenitors; MEP, megakaryocyte erythrocyte progenitors, GMP, granulocyte monocyte progenitors; LMPP, lymphoid-myeloid primed progenitors; CLP, common lymphoid progenitors; Meg, megakaryocytes; Er, erythrocytes; Mo, monocytes; Ne, neutrophils; DC, dendritic cells; B, B lymphocytes, T, T lymphocytes; NK, natural killer cells. ( C to H ) Flow cytometry analysis of bone marrow cells from control and Nup358 knockout mice. Mature immune cells (Lin + ) and hematopoietic progenitors (Lin − ) numbers (C), Lin + and Lin − percentage of viable cells (D). Number of MyP (E), LMPP and CLP (F). Number of monocytes (Mon), neutrophils (Neu), and macrophages (Mac) (G), NK cells, and B cells (H). ( I ) Number of CD3 + , CD4 + , and CD8 + T lymphocytes in spleen of control and Nup358 knockout mice. ( J ) Colony formation assay of bone marrow cells from control and Nup358 knockout mice in conditions promoting myeloid differentiation. ( K ) Representative images of macrophages differentiated in vitro from bone marrow cells of tamoxifen-treated Nup358 fl/fl and Nup358 fl/fl CreER T2 mice. Scale bars, 75 μm. ( L ) Representative images macrophages differentiated in vitro from bone marrow cells of Nup358 fl/fl and Nup358 fl/fl CreER T2 mice treated with tamoxifen after differentiation. Scale bars, 75 μm. ( M ) Viability of differentiated macrophages from (L). Data are means ± SD ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001 by either unpaired Student’s t test [(A), (E), and (M)] or multiple unpaired Student’s t test with Holm-Sidak method to correct for multiple comparisons [(C) and (D) and (F) to (I)]. Data are pooled from eight (A), six [(C) and (D)], three [(E) and (F)], two [(G) to (I)], and four (I) independent experiments with at least n = 3 mice of each genotype.

Article Snippet: To generate Nup358 fl/fl -CreER T2 mice, Nup358 fl/fl mice were crossed with R26-CreER T2 mice (The Jackson Laboratory).

Techniques: Knock-Out, Flow Cytometry, Colony Assay, In Vitro

Journal: Science Advances

Article Title: Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation

doi: 10.1126/sciadv.adn8963

Figure Lengend Snippet: ( A ) Schematic illustration of the bone marrow transplantation experiments. ( B ) Number of neutrophils (NE), monocytes (MO), and lymphocytes (LY) per milliliter in peripheral blood of CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358 fl/fl or Nup358 fl/fl -CreER T2 mice after 12 weeks of reconstitution and 5 days after tamoxifen administration. ( C ) Total bone marrow cells from CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358 fl/fl or Nup358 fl/fl -CreER T2 mice at 12 days after tamoxifen administration. ( D to G ) Flow cytometry analysis on bone marrow cells isolated from CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358 fl/fl or Nup358 fl/fl -CreER T2 mice after 12 weeks of reconstitution and 12 days after tamoxifen administration. Lin + and Lin − cells as number (D) or percentage of viable bone marrow cells (E), and HSC LT , HSC ST MPP2, MPP3, and MPP4 as number (F) or percentage of LSK cells (G) at 12 days after tamoxifen injection. ( H and I ) Flow cytometry analysis on splenocytes isolated from CD45.1 wild-type mice transplanted with bone marrow cells from either Nup358 fl/fl or Nup358 fl/fl -CreER T2 mice after 12 weeks of reconstitution and 12 days after tamoxifen administration. Total CD3 + T lymphocytes (H), CD4 + T lymphocytes, and CD8 + T lymphocytes (I). Data are means ± SD. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ***** P ≤ 0.00001 by unpaired Student’s t test [(C) and (H)] or multiple unpaired Student’s t test with Holm-Sidak method to correct for multiple comparisons [(D) to (G) and (I)].

Article Snippet: To generate Nup358 fl/fl -CreER T2 mice, Nup358 fl/fl mice were crossed with R26-CreER T2 mice (The Jackson Laboratory).

Techniques: Transplantation Assay, Flow Cytometry, Isolation, Injection

Journal: Science Advances

Article Title: Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation

doi: 10.1126/sciadv.adn8963

Figure Lengend Snippet: ( A ) Uniform Manifold Approximation and Projection plot of control and Nup358 knockout cells showing 15 clusters identified by dimensionality reduction and unsupervised clustering. ( B ) Distribution of control (blue) and Nup358 knockout (red) cells among clusters. ( C ) Violin plots representing the aggregated expression of genes associated with neutrophils, megakaryocytes/erythrocytes, and lymphocytes in the identified clusters. ( D ) Distribution of control and Nup358 knockout cells in unprimed and primed clusters. ( E ) Distribution of control and Nup358 knockout cells in neutrophil-primed (N-primed), megakaryocytes/erythrocyte-primed (ME-primed), and lymphocyte-primed (L-primed) clusters. ( F ) Heatmaps showing genes differentially expressed between control and Nup358 knockout LSK cells from clusters 4 and 9 (fold change > ±1.25; P < 0.05). ( G ) Volcano plot of single-cell transcriptome data showing the differentially expressed genes in Nup358 knockout cells from clusters 4 and 9. Genes are organized by log 2 fold change and –log 10 P value. Genes below the cutoff (dashed line, P < 0.05) are shown in gray. ( H ) Ten most significantly altered pathways in Nup358 knockout LSK cells from cluster 4 and cluster 9 ranked by P value. ( I ) Cell cycle analysis on MPP2 in control ( Nup358 fl/fl ) and Nup358 knockout ( Nup358 fl/fl CreER T2 ) by flow cytometry.

Article Snippet: To generate Nup358 fl/fl -CreER T2 mice, Nup358 fl/fl mice were crossed with R26-CreER T2 mice (The Jackson Laboratory).

Techniques: Knock-Out, Expressing, Cell Cycle Assay, Flow Cytometry

Journal: Science Advances

Article Title: Nucleoporin Nup358 drives the differentiation of myeloid-biased multipotent progenitors by modulating HDAC3 nuclear translocation

doi: 10.1126/sciadv.adn8963

Figure Lengend Snippet: ( A ) Schematic illustration of the in vitro myeloid differentiation of primary hematopoietic progenitors. ( B ) The total number of viable cells in cultures of Nup358 fl/fl and Nup358 fl/fl CreER T2 hematopoietic progenitors treated with tamoxifen in vitro was measured over time. ( C to F ) Flow cytometry analysis of hematopoietic progenitor cultures 96 hours after treatment with tamoxifen. Fold change relative to vehicle-treated cells. ( G to J ) Cell growth and viability of Nup358 fl/fl and Nup358 fl/fl CreER T2 hematopoietic progenitor cultures treated with either tamoxifen or vehicle for 96 hours and HDAC3 inhibitor RGFP966 or vehicle for 48 hours. ( K to M ) Flow cytometry analysis of hematopoietic progenitor cultures treated with either tamoxifen or vehicle for 96 hours and HDAC3 inhibitor RGFP966 or vehicle for 48 hours. Data are means ± SD * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, ***** P ≤ 0.00001 by unpaired Student’s t test (D) or multiple unpaired Student’s t test with Holm-Sidak method to correct for multiple comparisons [(C), (E), and (F)] or analysis of variance (ANOVA) with Turkey correction for multiple comparison [(K) to (M)]. Data in (B) to (F) are representative of two independent experiments with at least n = 3 biological samples of each genotype.

Article Snippet: To generate Nup358 fl/fl -CreER T2 mice, Nup358 fl/fl mice were crossed with R26-CreER T2 mice (The Jackson Laboratory).

Techniques: In Vitro, Flow Cytometry, Comparison

Journal: Science Advances

Article Title: N 6 -methyladenosine modification–tuned lipid metabolism controls skin immune homeostasis via regulating neutrophil chemotaxis

doi: 10.1126/sciadv.adp5332

Figure Lengend Snippet: ( A ) Representative images of m 6 A immunofluorescence staining in the lesional skin of patients with psoriasis or healthy control. ( B and C ) RT-qPCR of the indicated genes (B) and immunochemistry staining of indicated proteins (C) in the lesional skin of patients with psoriasis or the healthy skin. Scale bar, 100 μm. ( D ) Representative images of m 6 A and METTL3 immunofluorescence staining in the lesional skin of patients with AD or healthy control. ( E and F ) Representative images of m 6 A (E) and METTL3 (F) immunofluorescence staining in the dorsal skin from WT mice topically treated with IMQ for 6 days consecutively. ( G and H ) RT-qPCR analysis of Mettl3 mRNA (G) and immunoblot of METTL3 (H) in the epidermis from WT mice topically treated with IMQ. ( I ) Immunoblot of METTL3 in the dorsal skin treated with IMQ for 6 days consequently or in the recovery skin (on day 30). ( J and K ) Representative images of m 6 A (J) and METTL3 (K) immunofluorescence staining in the dorsal skin from WT mice topically treated with MC903 for 8 days consecutively. ( L and M ) RT-qPCR analysis of Mettl3 mRNA (L) and immunoblot of METTL3 (M) in the epidermis from WT mice topically treated with MC903. ( N ) Immunoblot of METTL3 in ear skin treated with MC903 for 12 days consequently or in the recovery skin (on day 30). Scale bar, 50 μm [(A), (D), (E), (F), (J) and (K)]; scale bar, 100 μm (C). Pso, psoriasis; KRT14, keratin 14; IMQ, imiquimod; AD, atopic dermatitis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. * P < 0.05; ** P < 0.01; *** P < 0.001. The P values were analyzed by unpaired, two-tailed Student’s t test [(B), (G), and (L)]. Data are presented as means ± SEM.

Article Snippet: The KRT14-CreER T2 transgenic mice (catalog no. NM-KI-190024) were obtained from Shanghai Model Organisms Center Inc. Mettl3 fl/fl mice were crossed with the KRT14-CreER T2 transgenic mice to generate KRT14-CreER T2 ; Mettl3 fl/fl mice.

Techniques: Immunofluorescence, Staining, Control, Quantitative RT-PCR, Western Blot, Two Tailed Test

Journal: Science Advances

Article Title: N 6 -methyladenosine modification–tuned lipid metabolism controls skin immune homeostasis via regulating neutrophil chemotaxis

doi: 10.1126/sciadv.adp5332

Figure Lengend Snippet: ( A and B ) Mouse construct (A) and schematic diagram (B) for CreER-inducible Mettl3 conditional ablation specific in keratinocytes and harvest time in (C) and (D). ( C and D ) Immunofluorescence staining of METTL3 (C) or m 6 A (D) in the dorsal skin from Mettl3 cKO (KRT14-CreER T2 ; Mettl3 fl/fl ) or WT ( Mettl3 fl/fl ) mice. Scale bars, 50 μm. ( E ) Schematic diagram for experimental design for inducible keratinocyte-specific ablation of Mettl3 observation in (F) to (N). ( F ) Incidence of skin lesions in the tail or palm of Mettl3 cKO and WT mice over time ( n = 20). ( G and H ) Dermoscopic images, H&E staining of the tail (G) or palm (H) skin lesions, and their quantitation of acanthosis and dermal cellular infiltrates on the lesional skin of Mettl3 cKO and WT mice skin in the indicated locations. Scale bars, 100 μm. ( I ) Immunofluorescence staining of loricrin in lesional skins of Mettl3 cKO mice or WT mice skin in the indicated locations. Scale bars, 50 μm. Dashed line indicates the border between the epidermis and dermis. ( J and K ) Top 10 GO terms (J) and KEGG pathways (K) enriched in DEGs identified by RNA-seq between the spontaneously inflamed tail skin of Mettl3 cKO mice and the normal tail skin of WT mice. ( L ) Heatmap showing expression of selected genes [Fragments Per Kilobase of transcript per Million mapped reads (FPKM)] involved in immune responses in WT and Mettl3 cKO skin. ( M ) GSEA of genes in Mettl3 cKO skin relative to control skin with DEGs in the human psoriatic skin (left) or AD skin (right) (GSE121212) enriched. TAM, tamoxifen; AD, atopic dermatitis; ECM, extracellular matrix; NES, normalized enrichment score. * P < 0.05; ** P < 0.01; *** P < 0.001. The P values were analyzed by unpaired, two-tailed Student’s t test [(G) and (H)]. Data are presented as means ± SEM.

Article Snippet: The KRT14-CreER T2 transgenic mice (catalog no. NM-KI-190024) were obtained from Shanghai Model Organisms Center Inc. Mettl3 fl/fl mice were crossed with the KRT14-CreER T2 transgenic mice to generate KRT14-CreER T2 ; Mettl3 fl/fl mice.

Techniques: Construct, Immunofluorescence, Staining, Quantitation Assay, RNA Sequencing Assay, Expressing, Control, Two Tailed Test

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet: (A) Expansion of CD44 hi CD4 cells in the spleen and peripheral lymph nodes with I-A b :2W1S 55–68 commensal Ca-2W1S specificity after intestinal colonization, and their expression of lineage-defining markers including FOXP3 (Tregs), RORγt (Th17), TBET (Th1), IRF4 (Th2), and PD-1/CXCR5/BCL6 (Tfh). (B) Single-cell gene expression profiling of commensal Ca-2W1S I-A b :2W1S 55–68 CD44 hi CD4 cells in the spleen and peripheral lymph nodes based on expression of lineage-defining markers (RORC for Th17, IRF4 for Th2, CXCR5 for Tfh, and FOXP3 for Treg), with the remaining unaccounted lineage-negative cells unified by KLF2 expression. (C) Relative expression of each lineage-defining marker and KLF2 by cells in each cluster identified in (B). (D) KLF2 expression by CD44 hi CD4 cells in the spleen and peripheral lymph nodes with I-A b :2W1S 55–68 surrogate commensal specificity in Ca-2W1S-colonized KLF2(GFP) reporter mice compared with tetramer-negative cells of irrelevant bulk specificity. (E) KLF2 expression by CD44 hi CD4 cells isolated from each tissue with I-A b :CBir1 464–472 specificity in KLF2(GFP) reporter mice compared with tetramer-negative cells of irrelevant bulk specificity. SLO, secondary lymphoid organ; mLN, mesenteric lymph node; PP, Peyer patch; SI-LP, small intestine lamina propria; LI-LP, large intestine lamina propria; SPF, specific pathogen free; ABX, drinking water supplementation with five antibiotics (ampicillin, gentamicin, metronidazole, neomycin, vancomycin) to eliminate commensal bacteria. Each point indicates the data from an individual mouse, and these results represent data from at least five independently colonized mice and two independent experiments each with similar results. *p < 0.05, **p < 0.01, ****p < 0.001, using paired t test; ns, not significant. See also , , and and .

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Expressing, Gene Expression, Marker, Isolation, Bacteria

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet: (A) Rectal prolapse incidence in CD4 Cre KLF2 f/f (filled red) compared with co-housed KLF2 f/f littermate controls (black), or for CD4 Cre KLF2 f/f mice administered antibiotics (ampicillin, gentamicin, metronidazole, neomycin, vancomycin) in the drinking water beginning at age 40 days (open red). (B) Colonic histology and inflammation scoring for CD4 Cre KLF2 f/f mice that did not develop rectal prolapse through 150 days of age (red) compared with co-housed KLF2 f/f littermate control mice (black), with some mice in each group administered antibiotics as described in (A) (open circles). (C) Fecal levels of each cytokine for each group of mice described in (B). (D) Fecal Lipocalin2 levels for each group of mice described in (B). (E) Percent RORγt- and FOXP3-expressing mLN CD4 cells for each group of mice described in (B). Each point indicates the data from an individual mouse, combined from at least two independent experiments each with similar results. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001, using one-way ANOVA; bar, mean ± SEM. See also .

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Control, Expressing

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet: (A) Phenotypic comparison of FOXP3+ and FOXP3-negative mLN CD4 cells in CD4 Cre KLF2 f/f (red) compared with Cre-negative littermate control mice (black). (B) Representative plots showing GITR and CD25 expression by mLN CD4 cells in CD4 Cre KLF2 f/f or Cre-negative littermate controls, and FOXP3 expression by GITR+ CD25+ (green) compared with GITR-negative CD25-negative (purple) cells from each group of mice. (C) CFSE dilution by responder CD4 splenocytes afteranti-CD3/CD28 stimulation (96 h) and co-culture with purified GITR+ CD25+ mLN CD4 cells from CD4 Cre KLF2 f/f (red) compared with GITR+ CD25+ mLN CD4 cells from Cre-negative control mice (black) each at a 1:1 ratio, or responder cells alone (gray). Each point indicates the data from an individual mouse, combined from at least two independent experiments each with similar results. ****p < 0.001, using one-way ANOVA; bar, mean ± SEM.

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Comparison, Control, Expressing, Co-Culture Assay, Purification, Negative Control

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet: (A) CFSE dilution by responder CD4 splenocytes after anti-CD3/CD28 stimulation (96 h) and co-culture with purified KLF2+ CD44 hi CD4 splenocytes at each ratio (red), anti-CD3/CD28-stimulated responder cells alone (gray filled), or responder cells without stimulation (gray line). (B) CFSE dilution by responder CD4 splenocytes after anti-CD3/CD28 stimulation (96 h) and co-culture with purified FOXP3-negative KLF2+ CD44 hi (red) compared with FOXP3+ (blue) CD4 splenocytes each at a 1:1 ratio, or anti-CD3/CD28-stimulated responder cells alone (gray). (C) Percent FOXP3+ among purified FOXP3-negative KLF2+ CD44 hi (red) compared with FOXP3+ (blue) CD4 cells after anti-CD3/CD28 stimulation, and co-culture with responder cells as described in (B). (D) CFSE dilution by responder CD4 splenocytes after anti-CD3/CD28 stimulation (96 h) and co-culture with each subset of FOXP3-negative cells including KLF2+ CD44 hi (red), KLF2-negative CD44 hi (green), and KLF2+ CD44 lo (orange) cells, or FOXP3+ cells including KLF2+ (blue dotted) compared with KLF2-negative (blue line) cells at each ratio compared with anti-CD3/CD28-stimulated responder cells alone (gray). (E) Fecal Lipocalin2 levels day 20 after transfer of CD45RB hi cells (5 × 10 4 cells; CD45.1 congenic) into Rag1−/− recipient mice (gray), or Rag1−/− mice co-transferred CD45RB hi cells with purified KLF2+ CD44 hi (FOXP3-negative) (5 × 10 5 cells; red) or FOXP3+ cells (5 × 10 5 cells; blue). (F) BrdU incorporation by mLN CD45RB hi CD45.1 donor cells for the mice described in (E). (G) FOXP3 expression by mLN KLF2+ CD44 hi (FOXP3-negative) (red) or FOXP3+ cells (blue) (CD45.1-negative, CD45.2-positive) donor CD4 cells for the mice described in (E). Each point indicates the data from an individual mouse, combined from at least two independent experiments each with similar results. *p< 0.05, **p < 0.01, ***p < 0.005 using one-way ANOVA for multiple groups or unpaired t test for two groups; bar, mean ± SEM; dotted line, limit of detection. See also .

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Co-Culture Assay, Purification, BrdU Incorporation Assay, Expressing

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet: (A) CFSE dilution by responder CD4 splenocytes from BG2 TCR transgenic mice without stimulation (black), after β-gal peptide stimulation (gray), or β-gal peptide stimulation plus co-culture (96 h) with purified FOXP3-negative KLF2+ CD44 hi cells (red) at a 1:1 ratio. (B) Differential gene expression by KLF2+ CD44 hi (FOXP3-negative) cells upon anti-CD3/CD28 stimulation (48 h) compared with no simulation controls (y axis), and differential gene expression by KLF2+ CD44 hi (FOXP3-negative) compared with KLF2-negative CD44 hi (FOXP3-negative) cells after stimulation (x axis); with genes significantly different (p < 0.05) between KLF2+ CD44 hi cells after stimulation compared with no simulation controls highlighted in blue, genes significantly different between KLF2+ CD44 hi (FOXP3-negative) compared with KLF2-negative CD44 hi (FOXP3-negative) cells after stimulation highlighted in green, and those significantly different in both parameters highlighted in red. (C) IL-10 concentration in culture supernatants of purified KLF2+ compared with KLF2-negative CD44 hi FOXP3-negative cells after anti-CD3/CD28 stimulation as described in (B). (D) CFSE dilution by responder CD4 cells after anti-CD3/CD28 stimulation (96 h) and co-culture with purified KLF2+ CD44 hi FOXP3-negative CD4 cells at a 1:1 ratio with medium supplemented with neutralizing antibodies against each cytokine or cell-associated molecule with statistics compared with no-blockade (isotype treatment) controls. (E) Representative plots and composite data showing IL-10 (CD90.1) expression by FOXP3-negative splenic CD4 cells from triple reporter (10BiT, IL-10[CD90.1];KLF2[GFP]; FOXP3[RFP]) mice after anti-CD3 stimulation, and expression of each Tr1-associated marker by IL-10-producing KLF2+ compared with KLF2-negative cells. (F) Fecal Lipocalin2 levels in CD4 Cre KLF2 f/f recipient mice day 20 after transfer of KLF2+ CD44 hi (FOXP3-negative) cells from WT (10 6 cells; blue) or Il10 −/− donors (10 6 cells; red) compared with no transfer controls (black). (G) Percent RORγt+ or IL-17A+ after PMA-ionomycin stimulation by mLN CD4 cells for each group of mice described in (F). Each point indicates the data from an individual mouse, combined from at least two independent experiments each with similar results. *p < 0.05, **p < 0.01, ***p < 0.005, ****p < 0.001 using one-way ANOVA for multiple groups or paired t test for two groups; bar, mean ± SEM. See also and .

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Transgenic Assay, Co-Culture Assay, Purification, Gene Expression, Concentration Assay, Expressing, Marker

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet: (A) Percent CD4 cells among CD45 + leukocytes in the peripheral blood and their expression of CD62L for CD4 CreER(T2) KLF2 f/f mice (red) or Cre-negative littermate controls (black) after initiating tamoxifen supplemented chow, compared with isogenic C57BL/6 control mice after initiating daily administration of the S1PR1 modulator FTY720 (20 μg IP; blue). (B) Fecal Lipocalin2 levels after initiating tamoxifen supplemented chow to CD4 CreER(T2) KLF2-floxed (red) or Cre-negative control (black) mice at age 6–8 weeks, with some mice in each group administered antibiotics (ampicillin, gentamicin, metronidazole, neomycin, vancomycin) in the drinking water (open), compared with mice day 20 after daily administration of the S1PR1 modulator FTY720 (blue). (C) Percent RORγt+ mLN CD4 cells for each group of mice described in (A) 25 days after initiating tamoxifen supplemented chow with or without additional drinking water antibiotic supplementation, compared with mice day 25 after daily administration of the S1PR1 modulator FTY720. (D) Colonic histologic inflammation scoring for the mice described in (C). (E) Percent RORγt+ mLN CD4 cells with I-A b :2W1S 55–68 specificity in Ca-2W1S colonized mice or I-A b :CBir1 464–472 specificity in specific pathogen-free CD4 CreER(T2) KLF2 f/f mice (red) or Cre-negative littermate controls (black) 20 days after initiating tamoxifen-supplemented chow. Each point indicates the data from an individual mouse, combined from at least two independent experiments each with similar results. *p < 0.05, **p < 0.01, ****p < 0.001, using one-way ANOVA or unpaired t test for two groups; bar, mean ± SEM.

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Expressing, Control, Negative Control

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet: (A) Percent KLF2+ or FOXP3+ among CD4 cells spanning multiple clusters (including Treg and Tfh clusters) in inflamed intestinal biopsy specimens from individuals with Crohn’s disease compared with healthy controls pooled from three separate datasets. (B) Percent KLF2+ or FOXP3+ among cells in individual CD4 cell clusters (non-Treg CD4 cells, non-Tfh CD4 cells, Treg cells, Tfh cells) for the datasets described in (A). (C) Gene expression comparison for KLF2+ CD4 cells from Crohn’s disease compared with controls, showing genes enriched (positive fold change) or repressed (negative fold change) in Crohn’s disease. Data from each individual are shown as a single data point color coded by dataset with differences between groups analyzed using the Mann-Whitney U test or one-way ANOVA. **p < 0.01, ***p < 0.005; bar, mean ± SEM. See also , , and and and .

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Gene Expression, Comparison, MANN-WHITNEY

Journal: Cell reports

Article Title: Kruppel-like factor 2+ CD4 T cells avert microbiota-induced intestinal inflammation

doi: 10.1016/j.celrep.2023.113323

Figure Lengend Snippet:

Article Snippet: Commercially available C57BL/6, CD45.1 (002014), CD90.1 (000406), FOXP3 RFP (008374), Rag1 −/− (002216), Il10 −/− (002251), CD4 Cre (017336) and CD4 CreER(T2) (022356) each on the C57BL/6 background were purchased from The Jackson Laboratory, intercrossed and maintained for at least 5 generations at Cincinnati Children’s Hospital.

Techniques: Virus, Derivative Assay, Recombinant, Cell Stimulation, Enzyme-linked Immunosorbent Assay, Staining, Software

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