Journal: Nature

Article Title: Human neural tube morphogenesis in vitro by geometric constraints.

doi: 10.1038/s41586-021-04026-9

Figure Lengend Snippet: Fig. 2 | Formation of a neural and non-neural ectoderm bilayer. a, Schematic of in vitro neural tube. b–p, Immunostained vertical (b–e) and horizontal (f–p) sections through the stem cell-derived neural tube (time indicated in hours post-BMP exposure). d, Single cells were visualized by co-culture of CAAX-mCherry cells and non-fluorescent cells. F-ACT, F-actin. e, Fibronectin (FN1) is shown in relation to neural tissue (NCAD). f, Horizontal section through an approximately 1 mm elongated neural tube culture on a rectangular micropattern of 500 µm width. g, h, Anterior neural markers PAX6 and OTX2 at 96 h. i, Apical marker PKCζ and primary cilia marker ARL13B at 120 h. j–l, Neural crest markers SOX10 (96 h), PAX7 (72 h), FOXD3 (96 h). m, n, Non-neural ectoderm markers TFAP2α and KRT8 at 96 h. o, p, Focal adhesion marker phosphorylated focal adhesion kinase (pFAK) and FN1 at 96 h. q, Uniform manifold approximation and projection (UMAP) plot showing the main cell populations defined by scRNA-seq. r, Top differentially expressed genes. Scale bars 25 μm (b–e), 50 μm (f), 10 μm (g–p).

Article Snippet: Materials & experimental systems n/a Involved in the study Antibodies Eukaryotic cell lines Palaeontology and archaeology Animals and other organisms Human research participants Clinical data Dual use research of concern Methods n/a Involved in the study ChIP-seq Flow cytometry MRI-based neuroimaging Antibodies Antibodies used SOX2-Rb (Cell Signaling mAB3579), OCT4-Ms (Milipore MAB4401), NANOG Goat R&D AF1997, N-Cadherin Ms Cell Signaling mAb #14215, N-Cadherin Ms Sigma C2542, E-Cadherin Rb Cell Signaling mAb #3195, OTX2 Goat Neuromics GT15095 PAX6 Rb Biolegend 11511 (Covance # PRB-278P), PAX6 Ms BD Biosciences 561462, TFAP2A Ms DSHB 3B5 Keratin8 Ms DSHB TROMA-I, Sox10 Gt R&D Systems AF2864, Fibronectin Rb Abcam Ab2413, Collagen IV Gt Biorad 134001, SOX17 Gt R&D Systems AF-1924, Brachyury Goat ThermoFisher PA5-46984, CDX2 Ms Abcam Ab157524, ZO1 Rb Abcam ab216880, PROM1 Rb Abcam ab216323, PKCζ Ms Santa Cruz Biotech Sc-17781, ARL13B Rb Proteintech 17711-1-AP, GBX2 Gt Abcam ab109726, EN1 Rb ThermoFisher PA5-14149, FOXD3 Gt R&D Systems AF2819ת P75 Ms BD Biosciences 560877, pMLC2 Rb Cell Signaling 3671, FOXA2 Gt R&D Systems AF2400, NKX2.1 Rb Abcam ab76013, NKX2.1 Ms ThermoFisher MA5-13691, Tubulin Ms Cell Signaling mAb 12152S, Shroom3 Rb Sigma Aldrich HPA047784-25UL, Secondary Alexa 405 Dk anti Gt abcam ab175664, Secondary Alexa 568 Dk anti Rb abcam ab175470, Secondary Alexa 647 Dk anti Ms abcam ab160107.

Techniques: In Vitro, Derivative Assay, Co-Culture Assay, Marker

Journal: bioRxiv

Article Title: Foxd3 controls heterochromatin-mediated silencing of repeat elements in mouse embryonic stem cells and represses the 2-cell transcription program

doi: 10.1101/2021.05.01.442081

Figure Lengend Snippet: : MA plot showing analysis of up and down regulated genes and repeats in total RNA-Seq preparations of control and Foxd3 KO cells. Significantly dysregulated genes are shown in blue and repeats are shown in yellow. The most highly upregulated repeat is marked in red. : Validation of select upregulated repeats in Foxd3 KO cells by RT-qPCR. The data is plotted as average fold change relative to control, after normalization to Gapdh . Error bars indicate SEM (N=3 biological replicates). Asterisks indicate statistically significant differences when compared to control (*: p<0.05, paired t-test). : Immunofluorescence analysis of the MERVL-gag protein in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells exhibiting the depicted phenotype is indicated. Scale bar represents 10 μm, n=150. : Venn diagram representing the overlap between genes upregulated in Foxd3 KO cells (yellow) and in MERVL: Tomato+ cells (representing 2CLCs, blue) ( Macfarlan et al , 2012 ). : Immunofluorescence analysis of 2CLC markers ZSCAN4 and DPPA3 in control and Foxd3 KO cells. Nuclei are labelled with DAPI. The percentage of counted cells exhibiting the depicted phenotype is indicated. Scale bar represents 10 μm, n=150. : Heat map depicting the comparative gene expression profiles of the top 100 upregulated genes in Foxd3 KO cells. The columns indicate expression in Foxd3 KO cells, 2-cell stage embryos and MERVL:Tomato+ cells.

Article Snippet: Wild type full-length Foxd3 cDNA was obtained from origene (Catalog number MR222218) and amplified using Foxd3 specific primers to be subcloned into the pGEX-6P1 plasmid and verified by sequencing.

Techniques: RNA Sequencing, Control, Biomarker Discovery, Quantitative RT-PCR, Immunofluorescence, Gene Expression, Expressing

Journal: bioRxiv

Article Title: Foxd3 controls heterochromatin-mediated silencing of repeat elements in mouse embryonic stem cells and represses the 2-cell transcription program

doi: 10.1101/2021.05.01.442081

Figure Lengend Snippet: : Transcription factor binding sites with Transfac IDs predicted by PROMO in repeat element consensus sequences. : Consensus sequences of MSR, MERVL-LTR, MERVL-int with FOXD3 binding site highlighted (Blue boxes). The underlined sequences indicate statistically significant FOXD3 binding sites. : Electrophoretic mobility shift assay (EMSA) with increasing concentration (0, 15nM, 30nM, 60nM, 1.25μM and 2.5μM) of GST-FOXD3 and fixed concentration (50nM) of 5’-Cy5 labelled dsDNA oligonucleotides (35 bp each) from Sox15 promoter (positive control), MSR, MERVL-LTR, MERVL-int, L1MdA and Hprt promoter (negative control). : ChIP-qPCR enrichment of FOXD3 in mESCs using primers specific for MERVL, MSR, L1MdA sequences. Sox15 promoter and Actb promoter primers are used as positive and negative control respectively. Data is represented as percentage of input and the average of 3 biological replicates is plotted. Error bar indicates standard error of the mean (SEM). Asterisks indicate statistically significant differences compared to no antibody control levels (*: p< 0.05, paired t-test).

Article Snippet: Wild type full-length Foxd3 cDNA was obtained from origene (Catalog number MR222218) and amplified using Foxd3 specific primers to be subcloned into the pGEX-6P1 plasmid and verified by sequencing.

Techniques: Binding Assay, Electrophoretic Mobility Shift Assay, Concentration Assay, Positive Control, Negative Control, ChIP-qPCR, Control

Journal: bioRxiv

Article Title: Foxd3 controls heterochromatin-mediated silencing of repeat elements in mouse embryonic stem cells and represses the 2-cell transcription program

doi: 10.1101/2021.05.01.442081

Figure Lengend Snippet: : Electrophoretic mobility shift assay (EMSA) with increasing concentrations (0, 30nM, 60nM, 1.25μM, 2.5μM and 5μM) of recombinant GST-FOXD3, DNA binding mutants GST-FOXD3 M1 and GST-FOXD3 M2 and fixed concentration (50nM) of 5’-Cy5 labelled dsDNA oligonucleotides (35 bp each) representing MERVL and MSR. : RT-qPCR analysis of MERVL, MSR and L1MdA in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh . The average data for 2 biological replicates is plotted, error bars represent standard deviation. Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test). : Immunofluorescence analysis of MERVL-gag protein in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Nuclei are labelled with DAPI. The percentage of counted cells exhibiting the depicted phenotype is indicated. Scale bar represents 10μm n=150. : RT-qPCR analysis of, 2C genes Zscan4c and Dppa3 in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Data is represented as fold change relative to respective untreated cells after normalizing to Gapdh . The average data for 2 biological replicates is plotted, error bars represent standard deviation. Asterisks indicate statistically significant differences. (**: p< 0.005, *: p<0.05 paired t-test). : Immunofluorescence analysis of the ZSCAN4 protein in Foxd3 cKO cells, Foxd3 cKO cells over expressing full length FOXD3 or FOXD3 M1 without and with 4-OHT. Nuclei are labelled with DAPI. The percentage of counted cells exhibiting the depicted phenotype is indicated. Scale bar represents 10μm n=150.

Article Snippet: Wild type full-length Foxd3 cDNA was obtained from origene (Catalog number MR222218) and amplified using Foxd3 specific primers to be subcloned into the pGEX-6P1 plasmid and verified by sequencing.

Techniques: Electrophoretic Mobility Shift Assay, Recombinant, Binding Assay, Concentration Assay, Quantitative RT-PCR, Expressing, Standard Deviation, Immunofluorescence

Journal: bioRxiv

Article Title: Foxd3 controls heterochromatin-mediated silencing of repeat elements in mouse embryonic stem cells and represses the 2-cell transcription program

doi: 10.1101/2021.05.01.442081

Figure Lengend Snippet: : STRING analysis to predict putative interaction between FOXD3, SUV39H1, SUV39H2, SETDB1 and SETDB2. FOXD3 was significantly predicted to interact with SUV39H1 and SETDB1 (p value < 0.01). : GFP immunoprecipitation analysis using GFP-SUV39H1 or GFP-SUV39H2 expressing Suv39h dn mESCs. The top panel represents immunoblot using GFP antibody and the bottom panel represents immunoblot using FOXD3 antibody. : MERVL-int expression in WT, Suv39h dn , Foxd3 cKO control and Foxd3 KO mESCs as determined by RNA-seq. The browser track shows representative MERVL-int repeat elements. 2 regions encompassing 7000-8000bp from chromosome 6 and chromosome 8 are depicted. : ChIP-qPCR analysis of endogenous SUV39H1 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input from 3 biological replicates. Error bar indicates SEM (*: p<0.05 paired t-test). : ChIP-qPCR analysis revealing H3K9me3 enrichment over MERVL, MSR and L1MdA in control and Foxd3 KO cells. Actb promoter is used as negative control. Data is represented as average percentage input of 3 biological replicates. Error bar indicates SEM (**: p<.005, *: p<0.05 paired t-test). : FOXD3 represses MERVL in mouse ES cells: FOXD3 (green) binds to MERVL-LTR and recruits SUV39H1 (yellow) to the LTR. SUV39H1 facilitates repression of MERVL by establishing H3K9me3 (orange hexagons). Foxd3 depletion disrupts the recruitment of SUV39H1 and leads to a reduction in H3K9me3 over MERVL. This in addition to an increase in H3K4me3 (green hexagons) on the region results in the activation of MERVL and a concomitant increase in 2CLC specific genes, shifting the balance between mESCs (Green and purple) and 2CLCs (Brown and blue). Foxd3 thus helps maintain ES cells in a pluripotent state by regulating MERVL.

Article Snippet: Wild type full-length Foxd3 cDNA was obtained from origene (Catalog number MR222218) and amplified using Foxd3 specific primers to be subcloned into the pGEX-6P1 plasmid and verified by sequencing.

Techniques: Immunoprecipitation, Expressing, Western Blot, Control, RNA Sequencing, ChIP-qPCR, Negative Control, Activation Assay

Journal: Clinical Epigenetics

Article Title: BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

doi: 10.1186/1868-7083-5-2

Figure Lengend Snippet: Pathways enriched for BRAF mutation-associated promoter methylation after exclusion of ES cell H3K27me 3 binding promoter regions

Article Snippet: For FOXB1 , FOXB2 , and FOXD3 , 2 μl of 125× diluted cDNA was amplified in a mix containing 1× iQ supermix (#170-8862, Bio-Rad) and 1× TaqMan assay (#Hs00247213_s1, #Hs02386300_s1, Hs00255287_s1, Hs00230962_m1, Applied Biosystems).

Techniques: Mutagenesis, Methylation, Binding Assay

Journal: Clinical Epigenetics

Article Title: BRAF mutation-specific promoter methylation of FOX genes in colorectal cancer

doi: 10.1186/1868-7083-5-2

Figure Lengend Snippet: Relation between methylation and expression. Tumor/normal expression ratios ( y axis) were plotted against the average DNA methylation measured by bisulfite sequence analysis for FOXB2 ( A ) and FOXF1 ( B ), and the methylation log2 ratio measured by CpG array for FOXD3 ( C ). BRAF mutant samples are shown in red, wildtype samples in black.

Article Snippet: For FOXB1 , FOXB2 , and FOXD3 , 2 μl of 125× diluted cDNA was amplified in a mix containing 1× iQ supermix (#170-8862, Bio-Rad) and 1× TaqMan assay (#Hs00247213_s1, #Hs02386300_s1, Hs00255287_s1, Hs00230962_m1, Applied Biosystems).

Techniques: Methylation, Expressing, DNA Methylation Assay, Sequencing, Mutagenesis