Critical role of WNK1 in MYC-dependent early mouse thymocyte development
- The Francis Crick Institute, United Kingdom
- Kings College London, United Kingdom
- University of Birmingham, United Kingdom
- Imperial College, United Kingdom
Figures
Figure 1 with 1 supplement
Essential role for WNK1 in thymocyte development.
(A) Flow cytometric analysis of thymocytes from Wnk1fl/+CD2-Cre and Wnk1fl/flCD2-Cre mice showing double negative (DN, Thy1.2+Lineage-) thymocytes subdivided into DN1 (CD44+CD25-), DN2+DN3 (CD25+), …
Figure 1—figure supplement 1
Expression of genes encoding WNK pathway proteins.
Mean ± SEM expression of genes in the WNK1 pathway in the indicated thymocyte subsets, re-analyzed from previously published data (Hu et al., 2013).
Figure 2
WNK1 is required for thymocytes to develop past the pre-TCR checkpoint.
(A) Left: flow cytometric analysis of Thy1.2+Lineage- thymocytes separated by expression of CD44 and CD25, and then DN3 (CD44-CD25+) cells separated into DN3a (small, FSClowCD28-) and DN3b (large, …
Figure 3
RNAseq of WNK1-deficient thymocytes reveals significant changes in five distinct processes.
(A) Thymi were harvested from Wnk1fl/+Rag1-/-CD2-Cre and Wnk1fl/flRag1-/-CD2-Cre mice that were either untreated (0 hr) or had been injected with anti-CD3ε antibody 3, 24 or 48 hr earlier. DN3+DN4 …
Figure 4 with 1 supplement
WNK1 deficiency does not perturb early pre-TCR-induced signaling.
(A) Analysis of RNAseq experiment described in Figure 3A, showing heatmap and hierarchical clustering of selected genes from the TCR signaling signature at 0 hr in Wnk1fl/+Rag1-/-CD2-Cre and Wnk1fl/f…
Figure 4—figure supplement 1
WNK1 deficiency does not perturb protein translation.
(A) Analysis of RNAseq experiment described in Figure 3A, showing heatmap and hierarchical clustering of selected genes from the translation signature at 0 hr. (B) Histograms showing levels of …
Figure 5
WNK1 regulates thymocyte migration and adhesion.
(A) Heatmap and hierarchical clustering of selected genes from the adhesion signature at 24 and 48 hr after injection of anti-CD3ε into Wnk1fl/+Rag1-/-CD2-Cre and Wnk1fl/flRag1-/-CD2-Cre mice from …
Figure 6 with 1 supplement
WNK1 controls entry into S-phase.
(A) Analysis of RNAseq experiment described in Figure 3A, showing heatmap and hierarchical clustering of selected genes from the cell-cycle signature at 24 hr after injection of anti-CD3ε. (B) …
Figure 6—figure supplement 1
Loss of WNK1 affects expression of genes associated with S-phase, but not late G1.
Analysis of RNAseq experiment described in Figure 3A. (A) Mean ± SEM expression of selected genes associated with progression from the early to late G1 phase of the cell cycle at 0 hr and 3 hr …
Figure 7 with 1 supplement
WNK1 is required for pre-TCR induced post-transcriptional regulation of MYC.
(A, B) Analysis of RNAseq experiment described in Figure 3A. (A) Table shows top six transcription factors whose known target genes were most enriched among all significant DEGs at 24 hr or among …
Figure 7—figure supplement 1
Expression of anti-apoptotic genes in the absence of WNK1.
(A) Analysis of RNAseq experiment described in Figure 3A, showing mean ± SEM expression of anti-apoptotic genes. No expression was detected of the Bcl2a1 genes. (B) Graph shows mean ± SEM% of DN2 …
Figure 8
OXSR1 and STK39 are required for pre-TCR induced proliferation and regulation of MYC.
(A–C) Mean ± SEM number of thymocytes at each developmental stage in Stk39+/+ and Stk39T243A/T243A mice (A) or in Rag1-/- radiation chimeras reconstituted with fetal liver cells from Oxsr1+/+ and Oxs…
Figure 9
Ectopic expression of MYC promotes development of WNK1-deficient thymocytes through the pre-TCR checkpoint.
(A) Bone marrow from Wnk1fl/+RCE or Wnk1fl/-RCE mice (CD45.1-CD45.2+) was infected in vitro with MIGR1 retroviral vectors expressing MYC, MYCT58A or no gene of interest (Empty); all vectors …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | Wnk1 | Ensembl | ENSMUSG00000045962 | |
| Gene (Mus musculus) | Oxsr1 | Ensembl | ENSMUSG00000036737 | |
| Gene (Mus musculus) | Stk39 | Ensembl | ENSMUSG00000027030 | |
| Gene (Mus musculus) | Slc12a2 | Ensembl | ENSMUSG00000024597 | |
| Gene (Mus musculus) | Myc | Ensembl | ENSMUSG00000022346 | |
| Genetic reagent (Mus musculus) | Wnk1tm1Clhu; Wnk1fl | Xie et al., 2009 doi:10.2353/ajpath.2009.090094 | RRID:MGI:4360972 | |
| Genetic reagent (Mus musculus) | Wnk1tm1.1Clhu; Wnk1- | Köchl et al., 2016 doi:10.1038/ni.3495 | RRID:MGI:6286269 | |
| Genetic reagent (Mus musculus) | Oxsr1tm1.1Ssy; Oxsr1fl | Lin et al., 2011 doi:10.1073/pnas.1107452108 | RRID:MGI:5297491 | |
| Genetic reagent (Mus musculus) | Tg(CD2-icre)4Kio; CD2Cre | de Boer et al., 2003 doi:10.1002/immu.200310005 | RRID:MGI:2449947 | |
| Genetic reagent (Mus musculus) | Wnk1tm1.1Tyb; Wnk1D368A | Köchl et al., 2016 doi:10.1038/ni.3495 | RRID:MGI:6286266 | |
| Genetic reagent (Mus musculus) | Gt(ROSA)26Sortm1(cre/ERT2)Thl; ROSA26CreERT2, RCE | de Luca et al., 2005 doi:10.1172/JCI24059 | RRID:MGI:3701992 | |
| Genetic reagent (Mus musculus) | Oxsr1tm1.1Arte; Oxsr1T185A | Rafiqi et al., 2010 doi:10.1002/emmm.200900058 | RRID:MGI:5462099 | |
| Genetic reagent (Mus musculus) | Stk39tm1.1Arte; Stk39T243A | Rafiqi et al., 2010 doi:10.1002/emmm.200900058 | RRID:MGI:5462098 | |
| Genetic reagent (Mus musculus) | Slc12a2tm1Ges; Slc12a2- | Flagella et al., 1999 doi:10.1074/jbc.274.38.26946 | RRID:MGI:1935144 | |
| Genetic reagent (Mus musculus) | Rag2tm1Fwa; Rag2- | Shinkai et al., 1992 doi:10.1016/0092-8674(92)90029 C | RRID:MGI:1858556 | |
| Genetic reagent (Mus musculus) | Rag1tm1Mom; Rag1- | Mombaerts et al., 1992 doi:10.1016/0092-8674(92)90030 G | RRID:MGI:1857241 | |
| Genetic reagent (Mus musculus) | C57BL/6J.129S4-Tg(Prm-cre)70Og | O'Gorman et al., 1997 doi:10.1073/pnas.94.26.14602 | RRID:MGI:2388049 | |
| Cell line (Mus musculus) | OP9-DL1 | Schmitt and Zúñiga-Pflücker, 2002 doi:10.1016/s1074-7613 (02)00474–0 | RRID:CVCL_B218 | |
| Cell line (Homo-sapiens) | Platinum E (Plat-E) | Morita et al., 2000 doi:10.1038/sj.gt.3301206 | RRID:CVCL_B488 | Retroviral packaging cell line |
| Recombinant DNA reagent | pMIGR1 (plasmid) | Pear et al., 1998 doi:10.1182/blood.V92.10.3780 | RRID:Addgene_27490 | Retroviral construct encoding GFP |
| Recombinant DNA reagent | pMIGR1-MYC (plasmid) | Luo et al., 2005 doi:10.1182/blood-2005-02-0734 | Retroviral construct encoding MYC and GFP | |
| Recombinant DNA reagent | pMIGR1-MYCT58A (plasmid) | This paper | Retroviral construct encoding MYC-T58A and GFP | |
| Antibody | anti-B220-biotin (RA3-6B2) (Rat monoclonal) | Invitrogen | Cat# 13-0452-86; RRID:AB_466451 | FACS (1:200) |
| Antibody | anti-B220-FITC (RA3-6B2) (Rat monoclonal) | Biolegend | Cat# 103206; RRID:AB_312991 | FACS (1:200) |
| Antibody | anti-active Caspase 3-PE (C92-605) (Rabbit monoclonal) | BD Pharmingen | Cat# 550821; RRID:AB_393906 | FACS (1:50) |
| Antibody | anti-CD3ε-biotin (2C11) (Armenian hamster monoclonal) | Biolegend | Cat# 100304; RRID:AB_312669 | FACS (1:200) |
| Antibody | anti-CD3ε-FITC (2C11) (Armenian hamster monoclonal) | eBioscience | Cat# 11-0031-85; RRID:AB_464883 | FACS (1:200) |
| Antibody | anti-CD3ε purified, endotoxin low (2C11) (Armenian hamster monoclonal) | Tonbo | Cat# 40–0031 M001; RRID:AB_464883 | IV injection |
| Antibody | anti-CD4-biotin (RM4-4) (Rat monoclonal) | eBioscience | Cat# 13-0043-85; RRID:AB_466334 | FACS (1:200) |
| Antibody | anti-CD4-FITC (GK1.5) (Rat monoclonal) | eBioscience | Cat# 11-0041-86; RRID:AB_464894 | FACS (1:200) |
| Antibody | anti-CD4-BV421 (RM4-5) (Rat monoclonal) | Biolegend | Cat# 100544; RRID:AB_11219790 | FACS (1:200) |
| Antibody | anti-CD4-PerCP (RM4-5) (Rat monoclonal) | Biolegend | Cat# 100538; RRID:AB_893325 | FACS (1:200) |
| Antibody | anti-CD4-APC-CY7 (GK1.5) (Rat monoclonal) | BD Pharmingen | Cat# 552051; RRID:AB_394331 | FACS (1:200) |
| Antibody | anti-CD8-biotin (53–6.7) (Rat monoclonal) | Biolegend | Cat# 100704; RRID:AB_312743 | FACS (1:200) |
| Antibody | anti-CD8-FITC (53–6.7) (Rat monoclonal) | Biolegend | Cat# 100706; RRID:AB_312745 | FACS (1:200) |
| Antibody | anti-CD8-BV421 (53–6.7) (Rat monoclonal) | Biolegend | Cat# 100738; RRID:AB_11204079 | FACS (1:200) |
| Antibody | anti-CD8-APC (53–6.7) (Rat monoclonal) | eBioscience | Cat# 17-0081-83; RRID:AB_469336 | FACS (1:200) |
| Antibody | anti-CD8-PE-CY7 (53–6.7) (Rat monoclonal) | Biolegend | Cat# 100722; RRID:AB_312761 | FACS (1:200) |
| Antibody | anti-CD11b-biotin (M1/70) (Rat monoclonal) | Biolegend | Cat# 101204; RRID:AB_312787 | FACS (1:200) |
| Antibody | anti-CD11b-FITC (M1/70) (Rat monoclonal) | Biolegend | Cat# 101206; RRID:AB_312789 | FACS (1:200) |
| Antibody | anti-CD11c-biotin (N418) (Armenian Hamster monoclonal) | Biolegend | Cat# 117304; RRID:AB_313773 | FACS (1:200) |
| Antibody | anti-CD11c-FITC (HL3) (Armenian Hamster monoclonal) | BD Pharmingen | Cat# 553801; RRID:AB_395060 | FACS (1:200) |
| Antibody | anti-CD19-biotin (1D3) (Rat monoclonal) | BD Pharmingen | Cat# 553784; RRID:AB_395048 | FACS (1:200) |
| Antibody | anti-CD19-FITC (MB19-1) (Mouse monoclonal) | eBioscience | Cat# 11-0191-85; RRID:AB_464966 | FACS (1:200) |
| Antibody | anti-CD25-eF450 (PC-61.5) (Rat monoclonal) | eBioscience | Cat# 48-0251-82; RRID:AB_10671550 | FACS (1:200) |
| Antibody | anti-CD25-FITC (7D4) (Rat monoclonal) | BD Pharmingen | Cat# 553072; RRID:AB_394604 | FACS (1:200) |
| Antibody | anti-CD25-PE (3C7) (Rat monoclonal) | BD Pharmingen | Cat# 553075; RRID:AB_394605 | FACS (1:200) |
| Antibody | anti-CD28-PE-CY7 (E18) (Mouse monoclonal) | Biolegend | Cat# 122014; RRID:AB_604079 | FACS (1:200) |
| Antibody | anti-CD44-PE (IM7) (Rat monoclonal) | eBiosciene | Cat# 12-0441-83; RRID:AB_465665 | FACS (1:200) |
| Antibody | anti-CD44-APC (IM7) (Rat monoclonal) | eBioscience | Cat# 17-0441-83; RRID:AB_469391 | FACS (1:200) |
| Antibody | anti-CD44-APC-eF780 (IM7) (Rat monoclonal) | eBioscience | Cat# 47-0441-82; RRID:AB_1272244 | FACS (1:200) |
| Antibody | CD45.1-PerCP-Cy5.5 (A20) (Mouse monoclonal) | eBioscience | Cat# 45-0453-82; RRID:AB_1107003 | FACS (1:200) |
| Antibody | CD45.2-APC-eF780 (104) (Mouse monoclonal) | eBioscience | Cat# 47-0454-82; RRID:AB_1272175 | FACS (1:200) |
| Antibody | anti-CXCR4-PE (2B11) | BD Pharmingen | Cat# 12-9991-82; RRID:AB_891391 | FACS (1:100) |
| Antibody | anti-DX5-biotin (Rat monoclonal) | eBioscience | Cat# 13-5971-85; RRID:AB_466826 | FACS (1:200) |
| Antibody | anti-DX5-FITC (Rat monoclonal) | Biolegend | Cat# 108906; RRID:AB_313413 | FACS (1:200) |
| Antibody | anti-GR-1-biotin (RB6-8C5) (Rat monoclonal) | Biolegend | Cat# 108404; RRID:AB_313369 | FACS (1:200) |
| Antibody | anti-LFA-1-PE (M17/4) (Rat monoclonal) | BD Bioscience | Cat# 12-0111-82; RRID:AB_465544 | FACS (1:200) |
| Antibody | anti-mouse IgG1-biotin (A85-1) (Rat monoclonal) | BD Pharmingen | 553441; RRID:AB_394861 | FACS (1:100) |
| Antibody | anti-MYC-AF647 (Y69) (Rabbit monoclonal) | Abcam | Cat# ab190560; RRID:AB_2876372 | FACS (1:400) |
| Antibody | anti-NK1.1-biotin (PK136) (Mouse monoclonal) | eBioscience | Cat# 13-5941-85; RRID:AB_466805 | FACS (1:200) |
| Antibody | anti-NK1.1-FITC (PK136) (Mouse monoclonal) | Biolegend | Cat# 108706; RRID:AB_313393 | FACS (1:200) |
| Antibody | anti-pS325-OXSR1/pS383-STK39 (sheep polyclonal) | MRC-PPU | Cat# S670B; RRID:AB_2876373 | IB (1 µg/ml) |
| Antibody | anti-PTCRA (2F5) (Mouse monoclonal) | BD Pharmingen | Cat# 552407; RRID:AB_394381 | FACS (1:100) |
| Antibody | anti-pS235/pS236-S6-AF488 (D57.2.2E) (Rabbit monoclonal) | Cell Signaling | Cat# 5317; RRID:AB_10694920 | FACS (1:50) |
| Antibody | anti-TCRβ-bio (H57-597) (Armenian Hamster monoclonal) | eBioscience | Cat# 13-5961-85; RRID:AB_466820 | FACS (1:200) |
| Antibody | anti-TCRβ-FITC (H57-597) (Armenian Hamster monoclonal) | Biolegend | Cat# 109206; RRID:AB_313429 | FACS (1:200) |
| Antibody | anti-TCRβ-APC (H57-597) (Armenian Hamster monoclonal) | Tonbo | Cat# 20–5961 U100, RRID:AB_2621612 | FACS (1:200) |
| Antibody | anti-TCRγδ-biotin (UC7-13D5) (Armenian Hamster monoclonal) | eBioscience | Cat# 13-5811-82; RRID:AB_466684 | FACS (1:200) |
| Antibody | anti-TCRγδ-FITC (UC7-13D5) (Armenian Hamster monoclonal) | Biolegend | Cat# 107504; RRID:AB_313313 | FACS (1:200) |
| Antibody | anti-TCRγδ-PE (GL3) (Armenian Hamster monoclonal) | eBioscience | Cat# 12-5711-82; RRID:AB_465934 | FACS (1:200) |
| Antibody | anti-Thy1.2-BV605 (53–2.1) (Rat monoclonal) | Biolegend | Cat# 140317; RRID:AB_11203724 | FACS (1:500) |
| Antibody | anti-Thy1.2-FITC (53–2.1) (Rat monoclonal) | Biolegend | Cat# 140304; RRID:AB_10642812 | FACS (1:500) |
| Antibody | anti-Thy1.2-APC (53–2.1) (Rat monoclonal) | eBioscience | Cat# 17-0902-83; RRID:AB_469423 | FACS (1:500) |
| Peptide, recombinant protein | Streptavidin-PE | Biolegend | Cat #: 405204 | FACS (1:200) |
| Peptide, recombinant protein | Streptavidin-PerCP | Biolegend | Cat #: 405213 | FACS (1:200) |
| Peptide, recombinant protein | mouse CXCL12 | R and D systems | Cat # 460-SD-010/CF | (500 ng/ml) |
| Peptide, recombinant protein | mouse ICAM1-Fc | R and D systems | Cat # 796-IC-050 | Transwell assay (500 ng/ml); Adhesion Assay (25 µg/ml) |
| Commercial assay or kit | Foxp3/Transcription factor staining buffer set | eBioScience | Cat # 00-5523-00 | |
| Chemical compound, drug | Tamoxifen | Millipore-Sigma | Cat # T5648 | |
| Software, algorithm | Graphpad Prism 8 | Graphpad | RRID:SCR_002798 | |
| Software, algorithm | Flowjo 10 | Flowjo | RRID:SCR_008520 | |
| Other | 7AAD | Merck | Cat # SML1633 | |
| Other | FxCycle Violet Stain | Thermo Fisher | Cat # F10347 | (1:1000) |
| Other | LIVE/DEAD NearIR | Thermo Fisher | Cat # L10119 | (1:500) |
| Other | Zombie Aqua | Biolegend | Cat # 423102 | (1:500) |
Additional files
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Supplementary file 1
RNAseq analysis of control and WNK1-deficient DN3 thymocytes following injection of anti-CD3ε.
Analysis of RNAseq experiment described in Figure 3A, showing expression of all genes measured in TPM at all time points.
- https://cdn.elifesciences.org/articles/56934/elife-56934-supp1-v2.xlsx
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Supplementary file 2
Differential gene expression in control and WNK1-deficient DN3 thymocytes following injection of anti-CD3ε.
Analysis of RNAseq experiment described in Figure 3A, showing differential gene expression analysis with DESeq2, taking into account all genes.
- https://cdn.elifesciences.org/articles/56934/elife-56934-supp2-v2.xlsx
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Supplementary file 3
Significant differential gene expression in control and WNK1-deficient DN3 thymocytes following injection of anti-CD3ε.
Analysis of RNAseq experiment described in Figure 3A, showing differential gene expression analysis with DESeq2 as in Supplementary file 2 but showing only genes that were statistically significantly differentially expressed (padj ≤0.05) and had an average expression value of TPM >3 over all conditions.
- https://cdn.elifesciences.org/articles/56934/elife-56934-supp3-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/56934/elife-56934-transrepform-v2.docx
