Integrated β-catenin, BMP, PTEN, and Notch signalling patterns the nephron
- The Roslin Institute, University of Edinburgh, United Kingdom
- MRC Institute of Genetics and Molecular Medicine, United Kingdom
- University of Edinburgh, United Kingdom
- Columbia University, United States
- Erasmus MC, University Medical Centre, Netherlands
- Cancer Research UK Beatson Institute, United Kingdom
- Maine Medical Center Research Institute, United States
- Beatston Institute for Cancer Research, United Kingdom
Figures
Figure 1 with 1 supplement
β-catenin activity levels form a reversed gradient along the axis of the nephron.
(A–B) TCF/Lef::H2B-GFP expression in nephrons: (A) late renal vesicle/early comma-shaped body nephron, (B) S-shaped body nephron, lines: white–nephron axis, purple–ureteric bud, green–distal …
Figure 1—figure supplement 1
β-catenin reporter and antibody data show different β-catenin activity levels along the axis of the nephron.
(A) TCF/Lef:H2B-GFP time-lapse data of a nephron developing through post-MET Pretubular Aggregate (PTA), Renal Vesicle (RV), Comma-shaped (CB), and S-shaped (SB) stages. These data are shown as …
Figure 2 with 5 supplements
Pharmacological modulation of β-catenin signalling alters proximal segment development.
(A) Time-lapse analysis of treated Wt1+/GFP kidneys—same as shown in Video 5. (B) Quantification of mean time taken for first glomeruli to mature to crescent-shaped stage where glomeruli are tightly …
Figure 2—figure supplement 1
β-catenin signalling is altered in response to pharmacological inhibitors.
(A–B) TCF/Lef:H2B-GFP kidneys displaying low levels of β-catenin signalling in IWR1 conditions whereas strong activation of β-catenin signalling is detected in CHIR conditions. Typical nephrons …
Figure 2—figure supplement 2
‘Just-right’ β-catenin signalling levels drive MET.
(A) Response of β-catenin target gene (Lef1), induction markers (Pax2 and Pax8), and epithelialisation marker (Cdh1) to different CHIR concentrations. Inserts showing all channels for kidneys …
Figure 2—figure supplement 3
Ectopic nephrons form from Six2 expressing progenitors.
Six2GFPCre with conditional RFP lineage tracing highlighting all nephron progenitors cell (GFP+) and all nephron lineages (RFP+). Kidneys cultured in CHIR show ectopic RFP+ structures forming from …
Figure 2—figure supplement 4
Co-inhibition experiments confirm specificity of pharmacological inhibitors.
(A) Co-inhibition of GSK-3β (CHIR) and Tankyrase (IWR1) or CBP (ICG001). ICG001 blocks TCF interacting with the CBP co-activator (Emami et al., 2004) downstream of (CHIR). CHIR effects are not …
Figure 2—figure supplement 5
The proximal cell-identity is promoted by decreased β-catenin signalling.
(A) qRT-PCR data for genes indicative of terminally differentiated glomerular cells. (B) Wt1+/GFP kidneys stained for Pax8 and Cdh1–arrowheads indicating structures positive for GFP. (C) Size of …
Figure 3 with 1 supplement
Pharmacological modulation of β-catenin signalling alters distal segment development.
(A) Lgr5-EGFP expression in treated nephrons with segmentation markers. (B) Time-lapse analysis of treated Lgr5+/EGFP-IRES-CreERT2 kidneys–arrowheads indicate developing nephrons, red-dashed line …
Figure 3—figure supplement 1
Lgr5 expression domain in heterozygous and homozygous Lgr5+/EGFP-IRES-CreERT2 kidneys.
Cultured Lgr5+/EGFP-IRES-CreERT2 kidneys stained for segmentation marker Jag1 and epithelial marker Cdh1. (A and C) Lgr5+/EGFP-IRES-CreERT2 heterozygous kidneys and kidneys and (B and D) Lgr5 EGFP-IR…
Figure 4 with 2 supplements
Shifts in positional identity by altered β-catenin activity.
(A) Model of predicted changes in segmentation if the gradient of β-catenin activity specifies positional identities in the nephron. Nephrons depicted as spheres representing renal vesicle stage. …
Figure 4—figure supplement 1
Gradual shifts in positional identity by gentle changes in β-catenin activity.
(A) Typical nephrons for their conditions as cultured at incremental CHIR dosages (0 µM, 0.50 µM, 0.75 µM, 1 µM, 1.25 µM, and 1.5 µM). Dashed lines indicate the axis and lengths of nephron segments. …
Figure 4—figure supplement 2
β-catenin activity dosage-dependent phenotypes in series of Apc and Ctnnb1 models.
Kidneys characterised using anti-Wt1, Jag1, Podxl, Lam, and Cdh1. (A) Ctnnb1Y654/E654 and Ctnnb1E654/E654, (B) Apc+/1572T Ctnnb1Y654/Y654 and Apc +/1572T Ctnnb1Y654/E654. Ctnnb1Y654 is the wild-type …
Figure 5 with 2 supplements
Modulating β-catenin activity shifts positional identities along the nephron without altering proliferation.
(A) Proliferation in TCF/Lef::H2B-GFP expressing nephrons treated with CHIR and IWR1. Nephron axis–dashed white line. Phosphorylated Histone 3 used as a marker for mitotic cells. (B) Quantification …
Figure 5—figure supplement 1
Modulating β-catenin activity shifts positional identities along the nephron regardless of proliferation levels.
(A) CHIR and IWR1 alter segmentation similarly with or without MTX. Dashed-white line indicates nephron axis. (B–C) Inhibiting proliferation does not block the formation of a GFP-gradient in TCF/Lef:…
Figure 5—figure supplement 2
Modulating β-catenin activity shifts positional identities along the nephron regardless of apoptosis levels.
(A) Treated kidneys stained with TUNEL-assay to detect apoptotic cells. (B) Treated kidneys (live) stained with Annexin V assay to detect apoptotic cells. 6-CF marks proximal tubules and PT and as …
Figure 6 with 2 supplements
The β-catenin activity gradient is modified by changes to BMP and PI3K signalling.
(A) BRE-LacZ pSMAD reporter shows strong labelling in medial segment; co-stained for Wt1, Jag1, Cdh1. (B) pSMAD1/5/8 specific antibody stain. Lines and labelling in A–B indicate different segments. …
Figure 6—figure supplement 1
Rescue/reversals experiments for kidneys treated with 20 µM Ly294002 or 4 µM LDN193189.
Kidneys were cultured for 96 hr in the inhibitors or for 48 hr in inhibitor followed by another 48 hr in control medium. Kidneys were stained for Wt1, Jag1, and Cdh1 to display nephron formation and …
Figure 6—figure supplement 2
Changes to BMP and PI3K signalling alters nephron segmentation and β-catenin signalling.
(A) Data from time-lapse captured TCF/Lef:H2B-GFP nephrons treated with LDN-193189 or Ly294002 or as controls. The percentage frequency is plotted against pixel intensity values. The GFP intensity …
Figure 7 with 2 supplements
Altered β-catenin activity rescues the loss of Notch.
(A) Kidneys treated with DAPT and DAPT/IWR1 and stained for LTL, β-laminin, Cdh1, and Podxl–arrowheads indicate LTL-positive nephrons, inserts show magnified nephrons with Podxl staining for …
Figure 7—figure supplement 1
Rescue/reversals experiments for kidneys treated with 2 µM DAPT.
Kidneys were cultured for 96 hr in DAPT or for 48 hr in DAPT followed by another 48 hr in control medium. Kidneys were stained for Wt1, Jag1, and Cdh1 to display nephron formation and overall …
Figure 7—figure supplement 2
Altered β-catenin activity or PI3K signalling rescues the loss of Notch.
Data from kidneys treated with 2 µM DAPT and in combination with 20 µM Ly294002 or 2 µM IWR1. (A–B) TCF/Lef:H2B-GFP nephrons stained for WT1, JAG1, and CDH1. WT1 and JAG1 stains are not optimally …
Figure 8
Model for molecular pathways interacting to control the patterning of the nephron.
β-catenin activity is necessary to determine a distal cell identity but must be excluded from the proximal nephron. BMP/PTEN/PI3K antagonises β-catenin activity in the medial segment and positively …
Videos
Video 1
3D reconstructions of nephrons.
3D reconstruction of renal vesicle (top), S-shaped body (middle), and more mature nephron (bottom). Nephrons are positive for TCF/Lef::H2B-GFP, Jag1-red, Cdh1-blue (left panel) and the TCF/Lef::H2B-G…
Video 2
Time-lapse capture of nephron.
The nephron is the same as shown in Figure 1—figure supplement 1A (from a TCF/Lef::H2B-GFP reporter kidney) is shown during the earliest stages of nephron development. Segments and stages are …
Video 3
Time-lapse capture of Six2GFPCre/Rosa26tdRFP kidneys.
Kidneys cultured in control medium and CHIR medium. Timing and conditions as shown in videos.
Video 4
Time-lapse capture of Pax8Cre YFPlox-stop kidneys.
Kidneys cultured in control medium, IWR1 and CHIR medium, or ICG001 and CHIR medium. Timing and conditions as shown in videos.
Video 5
Time-lapse capture of Wt1+/GFP kidneys.
Kidneys cultured in control conditions or treated with IWR1 or CHIR. Timing, conditions, and scale as specified.
Video 6
Time-lapse capture of Lgr5+/EGFP-IRES-CreERT2 kidneys.
Kidneys cultured in control conditions or treated with IWR1 or CHIR. Timing, conditions, and scale as specified.
Video 7
Time-lapse capture of Lgr5+/EGFP-IRES-CreERT2 and Lgr5 EGFP-IRES-CreERT2/EGFP-IRES-CreERT2 kidneys.
Kidneys cultured in control conditions over 6 days. Timing and scale as specified.
Video 8
Time-lapse capture TCF/Lef::H2B-GFP kidneys.
Kidneys cultured in control medium, 4 µM LDN-193189, 20 µM Ly294003, 1.5 µM CHIR, 4 µM LDN-193189, and 1.5 µM CHIR, or 20 µM Ly294003 and 1.5 µM CHIR. Timing and scales are as specified.
Video 9
Time-lapse capture TCF/Lef::H2B-GFP kidneys.
Kidneys cultured in control medium, 4 µM LDN-193189, 20 µM Ly294003, 1.5 µM CHIR, 4 µM LDN-193189, and 1.5 µM CHIR, or 20 µM Ly294003 and 1.5 µM CHIR. Timing and scales are as specified.
Video 10
Time-lapse capture of TCF/Lef::H2B-GFP kidneys.
Kidneys in this video were cultured in control medium or 20 µM Ly294003. Timing and scales are as specified.
Video 11
Time-lapse capture of TCF/Lef::H2B-GFP kidneys.
Kidneys in this video were cultured in 2 µM DAPT or 20 µM Ly294003 with 2 µM DAPT. Timing and scales are as specified.
Additional files
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Supplementary file 1
Primers and UPL probes are used in qRT-PCR analysis.
- https://doi.org/10.7554/eLife.04000.037
