Early anteroposterior regionalisation of human neural crest is shaped by a pro-mesodermal factor
- Centre for Stem Cell Biology, School of Biosciences, University of Sheffield, United Kingdom
- Neuroscience Institute, The University of Sheffield, Western Bank, United Kingdom
- Computational and Data Science Laboratory, High Performance Computing and Networking Institute, National Research Council of Italy, Italy
- Laboratory for Human Organogenesis, RIKEN Center for Biosystems Dynamics Research, 2-2-3 Minatojima-minamimachi, Chuo-ku, Japan
- CeMM Research Center for Molecular Medicine, Austrian Academy of Sciences, Austria
- Molecular Biotechnology Center, Department of Molecular Biotechnology and Health Sciences, University of Torino, Italy
- Institute of Artificial Intelligence, Center for Medical Statistics, Informatics, and Intelligent Systems, Medical University of Vienna, Austria
- St. Anna Children's Cancer Research Institute, Austria
- Laboratory of Molecular Cell Biology and Development, Department of Animal Development and Physiology, Graduate School of Bio-studies, Kyoto University, Japan
- University of Cassino and Southern Lazio, Italy
Figures
Figure 1
Brachyury expression marks neural crest (NC)-fated axial progenitors.
Top: schematics showing location and orientation of immunostaining data in in embryonic day (E) 8.75 (Theiler stage [TS]13) and E9.0 (TS14) embryos. (A) Confocal sections of wholemount …
Figure 2 with 1 supplement
Effect of TBXT reduction on human pembryonic stem cell (hES)-derived neuromesodermal progenitors (NMPs).
(A) Differentiation/tetracycline (Tet) treatment scheme. (B) Immunofluorescence analysis of the expression of TBXT in shRNA hEScell-derived NMPs in the presence and absence of Tet. Scale bar = 100 …
Figure 2—figure supplement 1
Effect of Tet treatment on control B2M shRNA hESC-NMPs.
(A) Immunofluorescence analysis of the expression of TBXT in control B2M shRNA hESC-derived NMPs in the presence and absence of Tet. Scale bar = 100 μm. (B) qPCR expression analysis of indicated HOX …
Figure 3 with 1 supplement
TBXT depletion impairs posterior axial identity acquisition by neural crestNC.
(A) Differentiation/tetracycline (Tet) treatment scheme. (B) qPCR expression analysis of indicated HOX genes in control vs Tet-treated NMP-derived trunk NC cells. Error bars represent SD (n=4–6). *p<…
Figure 3—figure supplement 1
TBXT depletion does not influence the number of neurectodermal cells in trunk NC cultures.
(A) Representative flow cytometry (FACS) plots showing the presence of SOX1-positive cells in trunk NC cultures generated from NMPs in the presence and absence of tetracycline (Tet) following …
Figure 4
TBXT-driven programming of a posterior neural crest (NC) axial identity is an early event.
(A) Differentiation/tetracycline (Tet) treatment scheme. The temporal Tet treatment regimens employed were: no Tet control (− −); Tet treatment between days 3 and 7 (− +); Tet treatment between days …
Figure 5 with 2 supplements
Early programming of a posterior axial identity in neuromesodermal progenitor (NMP)-derived neural crest(NC) cells is primarily WNT-dependent.
(A) Scheme of treatments during the differentiation of human pluripotent stem cells (hPSCs) toward NMPs. (B) qPCR expression analysis of representative WNT-FGF targets in NMP cultures treated with …
Figure 5—figure supplement 1
Influence of WNT and BMP signalling on trunk NC specification.
(A) Immunofluorescence analysis of the expression of HOXC9, SOX1, and SOX10 in trunk NC cultures treated with the indicated combinations of WNT-BMP agonists/antagonists. Scale bar = 100 µm. (B) …
Figure 5—figure supplement 2
WNT signalling dynamics during posterior NC emergence.
(A) Fluorescence analysis of R26-WntVis mouse embryos at embryonic day (E) 8.75 (Theiler stage [TS]13) and E9.0 (TS14) demonstrating EGFP reporter responsiveness to WNT signalling. (B) Sagittal …
Figure 6
Posterior axial identity acquisition by neuromesodermal progenitor (NMP)-derived, WNT-FGF-induced pre-neural spinal cord cells is TBXT-independent.
(A, D) Differentiation/treatment schemes associated with different time windows of TBXT knockdown during spinal cord differentiation from NMPs. (B–C, E–F) qPCR expression analysis of indicated HOX …
Figure 7 with 1 supplement
Posterior axial identity acquisition by NMP-derived pre-neural spinal cord progenitors is FGF-dependent.
(A) Scheme of treatments during the differentiation of hPSC-derived NMPs toward early spinal cord progenitors. (B–C) qPCR expression analysis of indicated HOX genes (B) and representative NMP/early …
Figure 7—figure supplement 1
Posterior axial identity acquisition by NMP-derived pre-neural spinal cord progenitors is FGF-dependent.
Immunofluorescence analysis of CDX2 and HOXC9 protein expression in NMP-derived early pre-neural spinal cord progenitor cultures treated with the indicated combinations of WNT-FGF …
Figure 8 with 2 supplements
TBXT controls HOX gene expression and WNT signalling in NMPs by influencing chromatin accessibility.
(A) Representative transcription factor-binding motifs enriched in TBXT binding sites. (B) Graph showing the percentages of differentially expressed genes (padj<0.05, log2FC>|1|) following TBXT …
Figure 8—figure supplement 1
Effect of TBXT binding on chromatin accessibility.
(A) Average density plot of tag distributions across peak regions corresponding to the neuromesodermal progenitor (NMP), human embryonic stem cells (hES), and input samples. (B) Genomic distribution …
Figure 8—figure supplement 2
Chromatin accessibility dynamics during NMP differentiation toward trunk NC and pre-neural spinal cord progenitors.
Correspondence between TBXT binding (ChIP) in human pluripotent stem cell (hPSC)-derived NMPs and chromatin accessibility changes (ATAC-seq) in NMPs (D3) and NMP-derived early spinal cord (D7 SC) …
Figure 9
Model.
Proposed model for the transcriptional and signalling control of posterior axial identity/HOX gene expression in hPSC derived NMPs and their derivatives.
Additional files
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Supplementary file 1
Significantly up- and downregulated transcripts in Tet-treated, TBXT-depleted hESC-derived NMPs.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp1-v2.xlsx
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Supplementary file 2
List of GO terms and corresponding gene lists enriched in Tet-treated, TBXT depleted hESC-derived NMPs.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp2-v2.xlsx
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Supplementary file 3
List of all genomic regions (Intervals) with peak p-value below the applied threshold bound by TBXT in hESC-derived NMPs and undifferentiated hESCs.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp3-v2.xlsx
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Supplementary file 4
List of GO terms and corresponding gene lists associated with TBXT binding sites in hPSC-derived NMPs.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp4-v2.xlsx
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Supplementary file 5
List of known HOMER database motifs enriched in TBXT binding sites in hESC-derived NMPs.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp5-v2.xlsx
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Supplementary file 6
List of TBXT target genes which are differentially expressed following Tet treatment (padj<0.05 log2FC>|0.5|) and Gene Ontology Biological Processes enrichment analysis.
Genes are listed in relation to the genomic position (in relation to TSS) of TBXT binding within their proximity. Blue highlight denotes downregulation while red represents upregulation in expression.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp6-v2.xlsx
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Supplementary file 7
List of ATAC-seq peaks associated with gain or loss of chromatin accessibility following TBXT depletion in hESC-derived NMPs.
Gene Ontology Biological Processes enrichment analysis, list of HOX genes as well as other genes (padj<0.05 log2FC>|1|) affected by TBXT depletion and are associated with changes in chromatin accessibility are also included.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp7-v2.xlsx
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Supplementary file 8
List of transcription factor DNA binding motifs enriched in ATAC-seq sites associated with chromatin accessibility gain, chromatin accessibility loss or both.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp8-v2.xlsx
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Supplementary file 9
ATAC-seq analysis of trunk NC cells generated from hPSCs via NMPs in the presence of Tet.
Includes: (i) Summary: Summary of detected peak numbers; (ii) all_peaks: List of all the peaks with P-value <0.05 (gained open, gained close, no change) with the relative annotations; (iii) Accessibility Loss padj <0.05: Significant Gained close peaks (pvalue adjusted (padj) <0.05); (iv) HOX genes: Peaks in HOX gene regions, yellow colour highlights the significant peaks (padj <0.05); (v) Enrichment-Accessibility Loss: Enrichment of Significant Gained close peaks (pvalue adjusted <0.05); (vi) Accessibility gain padj <0.05: Significant Gained close peaks (pvalue adjusted (padj) <0.05).
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp9-v2.xlsx
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Supplementary file 10
List of primers used.
- https://cdn.elifesciences.org/articles/74263/elife-74263-supp10-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/74263/elife-74263-transrepform1-v2.docx
