m6A RNA methylation impacts fate choices during skin morphogenesis
- Howard Hughes Medical Institute, Robin Chemers Neustein Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, United States
- Bioinformatics Resource Center, The Rockefeller University, United States
- Electron Microscopy Resource Center, The Rockefeller University, United States
- Department of Pharmacology, Weill Cornell Medicine, Cornell University, United States
Figures
Figure 1 with 1 supplement
miCLIP and ribosomal profiling analyses of the mouse skin epithelial progenitors.
(A) Schematic depicting the major factors involved in regulating the cellular dynamics of m6A modification. (B) Schematic depicting embryonic development of mammalian epithelial skin progenitors. …
Figure 1—figure supplement 1
miCLIP experiment setup and correlation analysis.
(A) Hematoxylin and eosin (H and E) stained sagittal sections of wild-type P0 back skin before and after dispase treatment (scale bars: 50 µm). (B) Sorting strategy to isolate basal skin progenitors …
Figure 2 with 2 supplements
Krt14-Cre driven conditional Mettl3 knockout mice display severe defects in HF morphogenesis.
(A) Representative pictures of Krt14-Cre-/-, Rosa26-YFPfl/+ and Krt14-Cre+/-, Rosa26-YFPfl/+ littermate embryos demonstrating the onset of uniform YFP expression in the E14.5 skin epithelium (scale …
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Figure 2—source data 1
Quantification of m6A/A ratio through TLC signals in (C).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig2-data1-v2.xlsx
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Figure 2—source data 2
Quantification of HF density in (F).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Breeding strategy to generate the Mettl3 cKO animals and the control (Ctrl) littermates.
By mating the Mettl3+/fl, K14-Cre+/- and Mettl3fl/fl, Rosa26-YFPfl/fl breeders, we got offspring with 4 kinds of genotypes being born at Mendel’s ratio, among which Mettl3+/fl, Rosa26-YFP+/fl, …
Figure 2—figure supplement 2
Additional information on the Mettl3 cKO phenotypes.
(A) Representative pictures of P6 control and cKO littermates (scale bars: 1 cm). (B) Quantification of body weights of control and cKO littermates (error bars: standard deviation, for each …
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Figure 2—figure supplement 2—source data 1
Quantification of neonates' body weights in (B).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig2-figsupp2-data1-v2.xlsx
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Figure 2—figure supplement 2—source data 2
Quantification of TEWL in (C).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig2-figsupp2-data2-v2.xlsx
Figure 3 with 1 supplement
Loss of m6A results in diminished WNT signaling and signs of perturbed HF fate.
(A) Left panel: representative images from E17.5 sagittal sections immunolabeled for LEF1 and PCAD (scale bars: 50 µm). White solid lines denote skin surface and dashed lines denote dermal-epidermal …
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Figure 3—source data 1
Quantification of LEF1 immunofluorescence signals at E17.5 in (A).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig3-data1-v2.xlsx
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Figure 3—source data 2
Quantification of LEF1 immunofluorescence signals at P0 in (B).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Examination of METTL3 and YFP expression in the grafted skin.
Images of back skin engrafted to Nude (Nu/Nu) mice for 15 days immunolabeled for PCAD, METTL3 and YFP demonstrating in the grafted tissue METTL3 is still largely depleted in the cKO epithelium …
Figure 4 with 1 supplement
Perturbations in WNT-driven dermal papilla engulfment and in actin-mediated cellular polarity within Mettl3 cKO HFs.
(A) Ultrastructure of HF in control and Mettl3 cKO mice in P0 back skin. Matrix (Mx) cells engulf the dermal papilla (DP, colored in pink) in the control HF but often fail to do so in the cKO. …
Figure 4—figure supplement 1
Analysis of DNA fragmentation, apoptosis and cell division angles in HF morphogenesis.
(A) Quantification of TUNEL+ and cleaved Caspase-3+ cells in HFs at different developmental stages [placodes (Plcd), germs and pegs] at P0. Quantifications are from images of P0 sagittal sections …
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Figure 4—figure supplement 1—source data 1
Quantification of cell death events in HFs in (A).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig4-figsupp1-data1-v2.xlsx
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Figure 4—figure supplement 1—source data 2
Quantification of cell division angles in (B).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig4-figsupp1-data2-v2.xlsx
Figure 5 with 1 supplement
Single-cell transcriptomics of Mettl3 cKO compared to control skin epithelial lineages.
(A) YFP+ progenitors were FACS-enriched from E17 control and Mettl3 cKO whole back skins and subject to scRNA-seq as described in the Materials and methods. Shown are the data from unbiased …
Figure 5—figure supplement 1
Cell isolation for scRNA-seq analysis and cell identity verification.
(A) Images of E17.5 back skin sagittal sections before (whole skin, Wh) and after dispase treatment immunolabeled for PCAD demonstrating Mettl3 cKO affects the separation of developing HFs between …
Figure 6 with 2 supplements
Investigation of RNAs whose levels diminish upon METTL3 loss.
(A) scRNA-seq data from Figure 5A were binned according to four major classifications: Epi basal, Epi suprabasal, HF WNThi and HF WNTlo. Scatter plots of mRNAs in these cells were then analyzed …
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Figure 6—source data 1
Lef1 qPCR in (C).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-data1-v2.xlsx
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Figure 6—source data 2
Quantification of HES1 immunofluorescence signals in (D).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-data2-v2.xlsx
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Figure 6—source data 3
Quantification of EdU+ and BrdU+ cells in (E).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-data3-v2.xlsx
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Figure 6—source data 4
Quantification of cell division angles in (F).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-data4-v2.xlsx
Figure 6—figure supplement 1
Correlation between the levels of m6A modification and changes in steady-state RNA levels upon Mettl3 ablation.
Correlation analysis of the Z score (cKO/Ctrl) from scRNA-seq to different parameters of assessing m6A modification levels. The different parameters tested include m6A site number (site #), m6A site …
Figure 6—figure supplement 2
Additional analysis of epidermal perturbations upon Mettl3 cKO.
(A) Confocal images of E16.5 whole-mount back skin immunolabeled for PCAD, ECAD and YFP (scale bars: 20 µm). PCAD and ECAD expression was quantified in the basal progenitors of skin epithelium …
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Figure 6—figure supplement 2—source data 1
Quantification of PCAD, ECAD immunofluorescence signals in (A).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-figsupp2-data1-v2.xlsx
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Figure 6—figure supplement 2—source data 2
Quantification of EdU+ cells and the suprabasal/basal cell number ratio in (B).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-figsupp2-data2-v2.xlsx
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Figure 6—figure supplement 2—source data 3
Quantification of cell sizes by cytospin in (C).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-figsupp2-data3-v2.xlsx
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Figure 6—figure supplement 2—source data 4
Quantification of cell death events in epidermis in (F).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig6-figsupp2-data4-v2.xlsx
Figure 7 with 1 supplement
Investigation of RNAs upregulated upon METTL3 loss.
(A) Scatter plots of mRNAs in the indicated groups of cells (as in Figure 5A) based on expression changes (cKO/Ctrl) assessed by scRNA-seq Z score and correlated with the coding sequence m6A density …
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Figure 7—source data 1
qPCR of selected transcripts in (D).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig7-data1-v2.xlsx
Figure 7—figure supplement 1
Additional analysis of features affected by the upregulated genes upon Mettl3 cKO.
(A) ECDF plots of the Z score (cKO/Ctrl) from scRNA-seq demonstrate transcripts with higher levels of m6A modification (assessed by the coding sequence SN-uTPM per nt value from miCLIP) in wild-type …
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Figure 7—figure supplement 1—source data 1
Bmyc qPCR in (B).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig7-figsupp1-data1-v2.xlsx
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Figure 7—figure supplement 1—source data 2
Myc qPCR in (C).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig7-figsupp1-data2-v2.xlsx
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Figure 7—figure supplement 1—source data 3
Quantification of MYC immunofluorescence signals in (D).
- https://cdn.elifesciences.org/articles/56980/elife-56980-fig7-figsupp1-data3-v2.xlsx
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information | |
|---|---|---|---|---|---|
| Genetic reagent (M. musculus) | C57BL/6J | Jackson lab | Stock #: 000664 | ||
| Genetic reagent (M. musculus) | Mettl3flox | PMID:31412241 J. Brüning (Max Planck Institute for Metabolism Research and Policlinic for Endocrinology, Diabetes and Preventive Medicine) | |||
| Genetic reagent (M. musculus) | Krt14-Cre | PMID:10411913 E. Fuchs (Rockefeller University) | MGI:1926500 | ||
| Genetic reagent (M. musculus) | Rosa26-YFPflox | Jackson lab | Stock #: 006148 | ||
| Genetic reagent (M. musculus) | Gli-LacZ | PMID:12361967 A. Joyner (MSKCC) | MGI:2449767 | ||
| Genetic reagent (M. musculus) | Nude (Nu/Nu) | Charles River Laboratories | Strain code: 088 | ||
| Antibody | Rat anti-BrdU | Abcam | Cat. #: ab6326 RRID:AB_305426 | IMF (1:200) | |
| Antibody | Rat anti-CD104 | BD Pharmingen | Cat. #: 553745 RRID: AB_395027 | IMF (1:500) | |
| Antibody | Armenian hamster anti-CD29 | BioLegend | Cat. #: 102201 RRID:AB_312878 | IMF (1:500) | |
| Antibody | Rat anti-CD49f | BioLegend | Cat. #: 313602 RRID:AB_345296 | IMF (1:1000) | |
| Antibody | Rabbit anti-Cleaved Caspase-3 | R&D Systems | Cat. #: AF835 RRID:AB_2243952 | IMF (1:200) | |
| Antibody | Rabbit anti-ECAD | Cell Signaling | Cat. #: 3195 RRID:AB_2291471 | IMF (1:500) | |
| Antibody | Rabbit anti-FLG | E. Fuchs (Rockefeller University) | IMF (1:2000) | ||
| Antibody | Rabbit anti-HES1 | E. Fuchs (Rockefeller University) | IMF (1:200) | ||
| Antibody | Rabbit anti-iNV | BioLegend | Cat. #: 924401 RRID:AB_2565452 | IMF (1:2000) | |
| Antibody | Guinea pig anti-K5 | E. Fuchs (Rockefeller University) | IMF (1:500) | ||
| Antibody | Rabbit anti-K10 | Covance | Cat. #: PRB-159P-100 RRID:AB_291580 | IMF (1:1000) Flow (1:500) | |
| Antibody | Guinea pig anti-LEF1 | E. Fuchs (Rockefeller University) | IMF (1:5000) | ||
| Antibody | Rabbit anti-LHX2 | E. Fuchs (Rockefeller University) | IMF (1:5000) | ||
| Antibody | Rabbit anti-LOR | E. Fuchs (Rockefeller University) | IMF (1:4000) | ||
| Antibody | Rabbit anti-METTL3 | Abcam | Cat. #: ab195352 RRID:AB_2721254 | IMF (1:100-1:500) | |
| Antibody | Rabbit anti-MYC | Abcam | Cat. #: ab32072 RRID:AB_731658 | IMF (1:100) | |
| Antibody | Goat anti-PCAD | R&D Systems | Cat. #: AF761 RRID:AB_355581 | IMF (1:300-1:600) | |
| Antibody | Rabbit anti-SOX9 | Millipore | Cat. #: AB5535 RRID:AB_2239761 | IMF (1:1000) | |
| Antibody | Rabbit anti-Survivin | Cell Signalling | Cat. #: 2808 RRID:AB_2063948 | IMF (1:500) | |
| Antibody | Chicken anti-GFP/YFP | Abcam | Cat. #: ab13970 RRID:AB_300798 | IMF (1:1000-1:1200) | |
| Antibody | anti-CD29 PECy7 | Thermo Fisher Scientific | Cat. #: 25-0291-82 RRID: AB_1234962 | FACS (1:1000) | |
| Antibody | anti-CD31 APC | Thermo Fisher Scientific | Cat. #: 17-0311-82 RRID:AB_657735 | FACS (1:1000) | |
| Antibody | anti CD45-APC | Thermo Fisher Scientific | Cat. #: 17-0451-83 RRID:AB_469393 | FACS (1:1000) | |
| Antibody | anti-CD49f PE | Thermo Fisher Scientific | Cat. #: 12-0495-81 RRID:AB_891478 | FACS (1:1000) | |
| Antibody | anti-CD49f PECy7 | Thermo Fisher Scientific | Cat. #: 25-0495-82 RRID:AB_10804881 | FACS (1:1000) | |
| Antibody | anti CD117-APC | Thermo Fisher Scientific | Cat. #: 17-1172-81 RRID:AB_469432 | FACS (1:1000) | |
| Antibody | anti CD140a-APC | Thermo Fisher Scientific | Cat. #: 17-1401-81 RRID:AB_529482 | FACS (1:500) | |
| Antibody | Mouse anti-rabbit IgG PE-Cy7 | Santa Cruz | Cat. #: sc-516721 | Flow (1:300) | |
| Chemical compound, drug | Alexa Fluor 647 Phalloidin | Thermo Fisher Scientific | Cat. #: A22287 RRID:AB_2620155 | IMF (1:50) | |
| Commercial assay or kit | In situ cell death detection kit, TMR red | Sigma-Aldrich | Cat. #: 12156792910 | ||
| Commercial assay or kit | Click-iT EdU cell proliferation kit for imaging, Alexa Fluor 647 dye | Thermo Fisher Scientific | Cat. #: C10340 | ||
| Commercial assay or kit | LIVE/DEADfixable Aqua dead cell stain kit, for 405 nm excitation | Thermo Fisher Scientific | Cat. #: L34957 | ||
| Commercial assay or kit | Fixation/ permeabilization solution kit | BD Biosciences | Cat. #: 554714 | ||
| Commercial assay or kit | Dynabeads mRNA purification kit | Thermo Fisher Scientific | Cat. #: 61006 | ||
| Commercial assay or kit | Direct-zol RNA miniprep kit | Zymo Research | Cat. #: R2051 | ||
| Commercial assay or kit | RQ1 RNase-free DNase | Promega | Cat. #: M6101 | ||
| Commercial assay or kit | High-capacity cDNA reverse transcription kit | Thermo Fisher Scientific | Cat. #: 4368814 | ||
| Commercial assay or kit | Power SYBR Green PCR Master Mix | Thermo Fisher Scientific | Cat. #: 4367660 | ||
| Commercial assay or kit | Chromium single cell 3' reagent kit | 10x Genomics | Cat. #: PN-120267 | ||
| Chemical compound, drug | TRI Reagent LS | Sigma-Aldrich | Cat. #: T3934 | ||
| Software, algorithm | Zen | Zeiss | https://www.zeiss.com/microscopy/int/products/microscope-software/zen.html | ||
| Software, algorithm | Fiji ImageJ | ImageJ | https://imagej.net/Fiji | ||
| Software, algorithm | Andor IQ3 | Oxford Instruments | https://andor.oxinst.com/products/iq-live-cell-imaging-software/ | ||
| Software, algorithm | GraphPad Prism 8.2 | GraphPad | https://www.graphpad.com/ | ||
| Software, algorithm | BD FACSDiva | BD Biosciences | https://www.bdbiosciences.com/en-us/instruments/research-instruments/research-software/flow-cytometry-acquisition/facsdiva-software | ||
| Software, algorithm | FlowJo | FlowJo, LLC | https://www.flowjo.com/solutions/flowjo | Version 10 | |
| Software, algorithm | R | CRAN | https://cran.r-project.org | R Version 3.6.3 Bioconductor Version 3.10 | |
| Software, algorithm | Flexbar | PMID:24832523 | https://github.com/seqan/flexbar | Version 2.5 | |
| Software, algorithm | pyCRAC | S. Granneman (SynthSys) | http://sandergranneman.bio.ed.ac.uk/pycrac-software | Version 1.1.3 | |
| Software, algorithm | Novoalign | Novocraft | http://www.novocraft.com/products/novoalign/ | Version 3.04.06 | |
| Software, algorithm | CIMS | C. Zhang (Columbia University) | https://zhanglab.c2b2.columbia.edu/index.php/CTK_Documentation | ||
| Software, algorithm | Bedtools | PMID:20110278 A. Quinlan (University of Utah) | https://github.com/arq5x/bedtools2.git | Version 2.25.0 | |
| Software, algorithm | Samtools | PMID:19505943 H. Li (Harvard University) | http://samtools.sourceforge.net | Version 1.2 | |
| Software, algorithm | Cell Ranger | 10x Genomics | https://support.10xgenomics.com/single-cell-gene-expression/software/overview/welcome | Version 2.1.1 | |
| Software, algorithm | Seurat | PMID:29608179 R. Satija (New York University) | https://satijalab.org/seurat/ | Version 2.3.4 | |
| Software, algorithm | Monocle | PMID:28114287 C. Trapnell (University of Washington) | http://cole-trapnell-lab.github.io/monocle-release/ | Version 2.10.1 | |
| Software, algorithm | MAST | PMID:26653891 A. McDavid (University of Rochester Medical Center) | https://www.bioconductor.org/packages/release/bioc/html/MAST.html | Version 1.8.2 | |
Additional files
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Supplementary file 1
Summary of all identified m6A sites through miCLIP.
- https://cdn.elifesciences.org/articles/56980/elife-56980-supp1-v2.xlsx
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Supplementary file 2
Quantification of m6A levels based on the sum of normalized-to-input uTPM value of m6A along coding sequence (CDS SN-uTPM) and GSEA.
First sheet: Rank of mRNAs based on coding sequence SN-uTPM. Second sheet: GSEA of mRNAs weighted on coding sequence SN-uTPM. The gene sets with p values <0.25 are shown. Third sheet: GSEA of mRNAs with top 20% coding sequence SN-uTPM and top 20% translation efficiency. The gene sets with p values <0.10 are shown.
- https://cdn.elifesciences.org/articles/56980/elife-56980-supp2-v2.xlsx
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Supplementary file 3
Differential gene expression analysis through scRNA-seq.
The extent of differential gene expression assessed by Z score (reflecting the extent of differential expression) and false discovery rate (FDR) was calculated between groups of Ctrl and cKO cells with the same identity, as indicated by sheet names in the file.
- https://cdn.elifesciences.org/articles/56980/elife-56980-supp3-v2.xlsx
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Supplementary file 4
Different parameters used to assess m6A modification levels.
- https://cdn.elifesciences.org/articles/56980/elife-56980-supp4-v2.xlsx
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Supplementary file 5
GSEA of transcripts with Z score (cKO/Ctrl)>1.96, FDR <0.05 in scRNA-seq and m6A coding sequence SN-uTPM per nt among the top 20%.
The gene sets with p values <0.05 are shown.
- https://cdn.elifesciences.org/articles/56980/elife-56980-supp5-v2.xlsx
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Supplementary file 6
Sequences of genotyping and qPCR primers used in this study.
- https://cdn.elifesciences.org/articles/56980/elife-56980-supp6-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56980/elife-56980-transrepform-v2.pdf
