Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: ( A ) Left: Representative in silico 3D transcriptomic analysis showing co-expression of FUT1 with pluripotent markers (NANOG, SOX2, and OCT3/4) on E6.0 in sections #351–370 from a sequential series of sections. Right: 2D corn plots showing FUT1 and pluripotent gene expression on E6.0. Abbreviations represent A, anterior ; P, posterior; L, left; R, right; Pr, proximal; D, distal. ( B ) 2D corn plots showing the expression of ectoderm markers, OTX2, PAX6, ZIC1, SOX2, SOX1, HOXC8, PHOX2b, OLIG2, PAX7, HOXB9, and DBX1 on E7.5 embryos. ( C ) 2D corn plots showing the expression of DE markers, HHEX, FOXA2, SOX17, and FOXA1 on E7.5. ( D ) 2D corn plots showing the spatiotemporal expression mesoderm genes, BRY (T gene) and MIXL1 on E7.0–E7.5; HOPX, NKX2.5, ISL1, HAND1, IRX3, MESP2, FOXC1, FOXF1, and CDX2 on E7.5. 2D and 3D gene expression analysis was obtained by using the database of mouse gastrulation on E5.5–E7.5 ( http://egastrulation.sibcb.ac.cn ).

Article Snippet: HHEX , Hematopoietically-expressed homeobox , HHEX , Hs01074519_m1.

Techniques: In Silico, Expressing, Gene Expression

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: ( A ) Up at left: Quantification of pluripotent gene expression as measured by qPCR. Up at right: Quantification of the percentage of hESCs and hESCs-derived DE on day 8 showing the expression of OCT3/4 in histograms, bar chart, and MFI relative to hESCs. Bottom: Quantification of the percentage of hESCs and hESCs-derived DE on day 8 showing the expression of NANOG in histograms, bar chart, and MFI relative to hESCs. n = 4 technical replicates. ( B ) Up and bottom at left: Quantification of DE-specific markers, FOXA2 and SOX17 as measured by qPCR after hESC differentiation into DE using protocol 1, and FOXA2, SOX17, and HHEX as measured by qPCR after hESC differentiation into DE using protocol 2 ( n = 3 technical replicates). Bottom at right: Quantification of the percentage of hESCs and hESCs-derived DE on day 8 showing the expression of α-FP in histograms and bar chart and MFI relative to hESCs. ( C ) Up at left: Schematic illustration of hESC differentiation into NPCs via bFGF and RA signaling and BMP inhibition over 6 d. Up at right: Representative immunofluorescent cultures illustrate day 6 NPCs expressing OTX2 (green) and PAX6 (red). Nuclei are stained with DAPI (blue). Scale bars represent 100 μm. Bottom: qRT-PCR and flow cytometry analyses of OCT3/4, PAX6, SOX1. The expression of FUT1 and α-fucose in iPSCs during pluripotency and differentiation is identical to that of hESCs ( D ) Quantification of the percent of positive cells expressing both the FT1 and OCT3/4 proteins and MFI in hESCs, a pool of hiPSCs and a pool of human fibroblasts. Pooled sample, n = 3 cell lines. n = 3 technical replicates. ( E ) Quantification of the percent of positive cells expressing FT1 protein and α-fucose residues and MFI in hESCs, a pool of hiPSCs, and a pool of human fibroblasts. Pooled sample, n = 3 cell lines. n = 3 technical replicates. Data information: In qPCR ( A , B ), data are presented as means ± SD. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001. In FACS ( A – E ), data are presented as means ± SD. Ordinary One-way ANOVA ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: HHEX , Hematopoietically-expressed homeobox , HHEX , Hs01074519_m1.

Techniques: Gene Expression, Derivative Assay, Expressing, Inhibition, Staining, Quantitative RT-PCR, Flow Cytometry, Two Tailed Test

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: ( A ) Left: Representative histograms showing counts and MFI of FUT1 positive hESCs (FUT1 + ECs) expressing Globo-H compared to WT hESCs. Center: Representative images showing hESC nuclei (blue) and Globo-H expression (green). Right: Representative histograms showing counts and MFI of FUT1 + ECs expressing SSEA-5 compared to hESCs. n = 3 biological replicates. ( B ) Left: Pluripotent NANOG, SOX2, and OCT3/4 mRNA expression levels in WT hESCs, control hESCs (control) and FUT1 + ECs, three passages after mock and FUT1 transfection, respectively. n = 3 clones for each clone n = 2 technical replicates. Right top: Representative bright-field and endogenous fluorescence protein (green) images of control colony morphology after one passage in culture, on day 3. Right bottom: Representative bright-field and endogenous fluorescence protein (green) images of FUT1 + EC colony morphology after one passage in culture, on day 3. n = 2 technical replicates. ( C ) mRNA expression levels of pluripotent OCT3/4 and mesoderm-specific markers, MESP1 and BRY, in WT hESCs, control, and FUT1 + ECs, on day 0 and after differentiation into LM for 1 d. n = 3 clones for each clone n = 2 technical replicates. ( D ) Top: mRNA expression levels of mesoderm-specific markers, FOXF1, IRX3, and HAND1 in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into LM for 2 d. n = 3 clones for each clone n = 2 technical replicates. Bottom: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing HAND1 protein and quantification of the percent of positive cells for HAND1 in WT hESCs and FUT1 + ECs after LM differentiation for 3 d. n = 3 technical replicates. ( E ) Top: mRNA expression levels of mesoderm-specific markers, NKX2.5, ISL1, and HOPX in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into LM for 3 d. n = 3 clones for each clone n = 2 technical replicates. Bottom: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing HOPX protein and quantification of the percent of positive cells for HOPX in WT hESCs and FUT1 + ECs after differentiation into LM for 3 d. Pools of FUT1 + ECs were used for FACS. n = 3 technical replicates. ( F ) Top Left: Schematic showing hESC differentiation into DE over three days by using the protocol of (Loh et al, ). Top right: mRNA expression levels of endoderm-specific markers, HHEX, FOXA2, and SOX17 in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into DE for 3 d, as measured by qPCR. n = 2 clones for each clone n = 3 technical replicates. Bottom: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing SOX17 protein and quantification of the fraction of positive cells for SOX17 in WT hESCs and FUT1 + ECs after differentiation into DE for 3 d. n = 3 technical replicates. ( G ) Left: mRNA expression levels of Ectoderm-specific marker, PAX6 in WT hESCs, control, and FUT1 + ECs on day 0 and after differentiation into NPCs for 3 d, as measured by qPCR. n = 2 clones for each clone n = 3 technical replicates. Center: Representative histograms showing relative counts of WT hESCs and FUT1 + ECs expressing PAX6 protein of the fraction of positive cells for PAX6 in WT hESCs and FUT1 + ECs after differentiation into NPCs for 3 d. n = 3 technical replicates. Right: Quantification of positive cells for PAX6 in WT hESCs and FUT1 + ECs after differentiation into NPCs for 3 d. n = 2 clones for each clone n = 2 technical replicates. ( H ) Left: Representative histograms showing relative counts of control and FUT1 + ECs expressing cTnT protein after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates. Left at center: Quantification of the fraction of positive cells for cTnT in control and FUT1 + ECs after differentiation into CMs for 20 d. Left at right: MFI of control and FUT1 + ECs expressing cTnT after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates, Right at left: Representative histograms showing relative counts of control and FUT1 + ECs expressing Myosin protein after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates. Right at center: Quantification of the fraction of positive cells for Myosin in control and FUT1 + ECs after differentiation into CMs for 20 d. Right at right: MFI of control and FUT1 + ECs expressing Myosin after differentiation into CMs for 20 d. n = 2 clones for each clone n = 3 technical replicates. ( I ) Left: Quantification of cardiac marker, TNNT2 mRNA expression in control and FUT1 + ECs on day 0 and after CM differentiation for 20 d, as measured by qPCR. Right: Quantification of cardiac marker, ACTC1 mRNA expression in control and FUT1 + ECs on day 0 and after CM differentiation for 20 d, as measured by qPCR. The housekeeping gene GAPDH, was used for normalization. n = 2 clones for each clone n = 3 technical replicates. More than n = 7 clones of control hESCs and FUT1 + ECs were generated for overexpression experiments; mRNA expression of n = 3 clones was measured by qPCR. Pools and n = 2 clones of control hESCs and FUT1 + ECs originating from WA09-transfected hESCs after sorting were used for imaging and FACS. n = 2 clones of control and FUT1 + ECs were differentiated into CMs and measured by qPCR and FACS. Data presented are relative to the values of day 0 WT hESCs. Data information: In ( A , B ), scale bars represent 100 μm. In ( A – F ), data are presented as means ± SDs. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 or non-significant (NS). In ( G ), data are presented as means ± SDs. Ordinary one-way ANOVA ** P < 0.01 or non-significant (NS), and for PAX 6, Two-tailed Student’s t -test * p < 0.05. In ( H ), data are presented as means ± SDs. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, and for Myosin, Ordinary one-way ANOVA ** p < 0.01, **** p < 0.0001. In ( I ), Data are presented as means ± SEM. Two-tailed Student’s t-tests * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001.

Article Snippet: HHEX , Hematopoietically-expressed homeobox , HHEX , Hs01074519_m1.

Techniques: Expressing, Control, Transfection, Clone Assay, Fluorescence, Marker, Generated, Over Expression, Imaging, Two Tailed Test

Journal: EMBO Reports

Article Title: Terminal α1,2-fucosylation of glycosphingolipids by FUT1 is a key regulator in early cell-fate decisions

doi: 10.1038/s44319-024-00243-1

Figure Lengend Snippet: Reagents and tools table

Article Snippet: HHEX , Hematopoietically-expressed homeobox , HHEX , Hs01074519_m1.

Techniques: Immunohistochemistry-Frozen IF, Western Blot, Recombinant, Staining, Plasmid Preparation, Sequencing, Control, Transfection, Over Expression, Software, TaqMan Assay, Gene Expression, Binding Assay, Marker