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Figure 1. What Is the Cellular Reporter of Wnt Stimulation?
(A) Wnt stimulation induces accumulation of β-catenin. (B) Together with the Tcf/Lef transcription factor, β-catenin activates transcription of specific target genes. (C) We found that the absolute level of β-catenin is sensitive to nonspecific variation that may naturally occur in cells. (D) Interestingly, the fold-change in β-catenin induced by Wnt stimulation is buffered against variation in parameters. (E) Together with functional data from Xenopus embryos, we propose that fold-changes in β-catenin, and not absolute levels of β-catenin, are the output of Wnt signaling.
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Figure 2. Fold-Change in β-Catenin, Not Absolute Levels, Is Predicted to Be Insensitive to Variation in Cellular Parameters
(A) A kinetic model of the Wnt pathway (modified from Lee et al., 2003). (B and C) Parameter sensitivity analysis. The parameters measured in the Xenopus extracts are used as the “unperturbed setpoint†(the first column). From this setpoint, each parameter was increased by 5-fold, one at a time (corresponds to each column). For each parameter set, we simulated the accumulation of β-catenin induced by a step Wnt stimulation and recorded the level of β-catenin after Wnt stimulation (B) and fold-change in β-catenin induced by Wnt (C). (D and E) For example, when Axin1 synthesis rate is increased by 5-fold (D) and the system is then stimulated with Wnt, the Wnt-induced level of β-catenin is 30 nM, and the Wnt-induced fold-change in β-catenin is 6-fold. When β-catenin synthesis rate is increased by 5-fold (E) and the system is then stimulated with Wnt, the Wnt-induced level of β-catenin is 1200 nM, and the Wnt-induced fold-change in β-catenin is 9-fold. Please see Figure S1 for a more complete sensitivity analysis.
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Figure 3. There Is a Region of Parameters where the Wnt-Induced Fold-Change in β-Catenin Is Insensitive to Variation in Parameters (A and B) Analytical solutions for the level of β-catenin after Wnt stimulation (A) and the Wnt-induced fold-change in β-catenin (B). These surfaces were computed analytically using equations 29-30 derived in the Supplemental Data. The black dot denotes the Xenopus parameters (α = 66, γ = 1). (C) The light blue shading indicates the region where the fold-change in β-catenin varies by less than ± 10%. Suppose that the cells reside within this region (point a), and we gradually inhibit degradation (α), thereby moving from point a to b to c. At each point, we stimulate the cells with Wnt, shown in the right panel. The absolute levels of β-catenin vary immediately, but the fold-change in β-catenin is identical for points a and b, which reside within the blue-shaded region.
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Figure 4. The Wnt-Induced Fold-Change in β-Catenin Is Insensitive to Perturbations in the Degradation Machinery
In human RKO cells, most β-catenin is cytoplasmic due to absence of cadherin (Breen et al., 1993, Giannini et al., 2000). In each experiment, two to three biological replicates were examined, each analyzed using two to four dot blots. Unless noted, error bars are standard deviation (SD) across biological samples. (A) A typical Wnt stimulation in RKO cells (i.e., incubation with Wnt3A-conditioned media). Level of β-catenin was quantified using dot blot. (B) Redrawn from Figure 3B. This figure predicts how the Wnt-induced fold-change in β-catenin would respond to various perturbations, depicted by the arrows. (C) Definition of the quantities measured in the experiments. (D) Cells overexpressing Axin1 were stimulated with Wnt. Three independent experiments were performed. 95% CI = 1.96 × SD across experiments. (E and F) Cells were pretreated with different doses of lithium (3 hr) and then stimulated with Wnt in the presence of lithium. (E) Representative profiles of Wnt-induced β-catenin accumulation in the control and treated cells. (F) Results from four independent experiments. Please see Figure S4 for a detailed experimental protocol and S6 for similar results using BIO, another GSK3β inhibitor. (G) Cells overexpressing β-catenin were stimulated with Wnt. Two independent experiments were performed. 95% CI = 1.96 × SD across experiments. (H) Cells overexpressing β-catenin were pretreated with different doses of lithium (3 hr) and then stimulated with Wnt in the presence of lithium. Three independent experiments were performed.
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Figure 5. Phenotypic Outcome of Wnt Signaling in Xenopus Patterning Is Insensitive to Perturbations in the Degradation Machinery
(A) Model prediction. To facilitate comparison with experimental data, we plot the Wnt-induced fold-change in β-catenin against the basal level of β-catenin. (Please also see Figure S8 to help visualize this.) From the Xenopus setpoint, perturbing degradation (α, green) will vary β-catenin level without initially affecting the Wnt-induced fold-change in β-catenin. However, increasing synthesis (γ, red) will increase both β-catenin level as well as the Wnt-induced fold-change. (B) RNA was injected to all four cells at 4-cell stage. Lithium treatment was performed at 48-cell stage for 5 min. Dorsoanterior phenotype was scored using the DAI index (Kao and Elinson, 1988). Cadherin-free pool of β-catenin was quantified using western blot on stage 8, around the onset of target gene expression. (C) Lithium treatment. (D) Plotting median DAI versus measured level of β-catenin for lithium treatment, GBP RNA injection, and Axin1 RNA injection. (E and F) β-catenin injection. (F) Plotting median DAI versus measured level of β-catenin for β-catenin RNA injection. Note: Phenotypic scoring, β-catenin quantitation, and RT-PCR (in Figure 6) were performed in sibling embryos. SD of β-catenin is from two to four western blots. For more raw data and controls, see Table S1. Sixty percent of the DAI 7/8 embryos developed as Janus twins (Kao and Elinson, 1988). Image in (B) is reprinted from Kao and Elinson (1988).
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Figure 6. Transcriptional Outcome of Wnt Signaling Is Insensitive to Variations in the Degradation Parameters
(A and B) Quantitative RT-PCR analysis of two direct target genes of the Wnt pathway, siamois and Xnr3, in embryos treated with lithium (A) and injected with β-catenin RNA (B). RT-PCR was performed at stage 10. SD = 2–3 measurements. Cadherin-free pool of β-catenin was quantified at stage 8–9 using quantitative western blot. SD = 2–4 blots. (C) Embryos were injected with TopFlash and RL-TK constructs at 4-cell stage and then treated with lithium. Luciferase activity was assayed at stage 10. (D) RKO cells stably expressing BAR reporter and Renilla luciferase (Major et al., 2007) were treated with lithium. β-catenin and luciferase profiles were measured in the same experiment.
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Figure S1. Other Features of β-Catenin Accumulation Are Sensitive to
Parameters
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Figure S2. The Insensitivity to Parameters Can Still Be Seen when the Total Pool of β-Catenin (the Measurable Quantity) Is Examined Instead.
Same as Figure S1, except that the total pool of β-catenin is plotted here, as opposed to free, un-phosphorylated β-catenin in Figure S1. The total pool of
β-catenin is the quantity that can be measured using Western blot.
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Figure S3. Sensitivity to Ligand Concentration
As a control, the basal level of β-catenin is not affected by variations in δ (~ligand stimulation). The Wnt-induced level and fold-change in β-catenin is sensitive to
δ. The higher γ (~higher Wnt concentration), the higher the Wnt-induced level of
βcatenin, and consequently the fold-change in β-catenin. γ=1 is used in these calculations.
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Figure S4. Experimental Protocol of the Inhibitor Treatment
RKO cells were pre-treated with different doses of LiCl (0-80mM) for 3h (in growth media). After that, the cells were transferred into control media or Wnt3A-conditioned media (WCM) + 0-80mM lithium. Cells were harvested usually at
24h, lysed, and analyzed using dot blot. β-catenin (-Wnt) = level of β-catenin in cells incubated in control media + 0-80mM lithium. This normally changed little
from 0h onward (i.e., the effects of lithium equilibrated rapidly). β-catenin (+Wnt) = level of β-catenin in cells incubated in WCM + 0-80mM lithium after the new plateau in β-catenin was reached. The time to reach the new Wnt-induced plateau varied from ~3h (in untreated cells) to ~20-24h (in cells treated with the highest dose of lithium). It was most efficient to harvest all cells at the same time and it gave the same result as when the cells were harvested as soon as
β-catenin reached the new plateau. Wnt-induced fold-change in β-catenin = β-cat (+Wnt) / Basal β-cat (-Wnt) Control media: Using DMEM+serum or L-cell-conditioned media gave the same results. L cell line is the parental line for the L-Wnt3A cell line. Pre-treatment period: 3h or 6h pre-treatment gave the same result. The same protocol was used
for BIO treatment.
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Figure S5. The Basal Level of β-Catenin Increased in a Dose-Dependent Manner
with Lithium Treatment
RKO cells treated with different doses of lithium showed a dose-dependent increase in the level of β-catenin. Level of βcatenin was quantified using dot blot. The measurements reported here were taken after 24h incubation, but the same
results were obtained from measurements at various time points (6h, 12h, 24h).
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Figure S6. The Wnt-Induced Fold-Change in β-Catenin Is Insensitive to Moderate
Doses of BIO (GSK3 β Inhibitor)
Cells were pre-incubated with BIO for 3 hours, followed by Wnt treatment in the presence of BIO for 6-24 hours. The level of β-catenin was quantified using dot blot. As a negative control, treatment with DMSO or MeBIO gave no significant changes in the level of β-catenin.
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Figure S7. More on Inhibiting GSK3 β in Cells Overexpressing β-Catenin
These measurements were performed in the same experiment, side by side. For each data point, 2 biological replicates were examined; SD is the std. dev. from 3 independent dot blots. In the main text, these data are included in Figures 4EG. Here, they are presented side by side to show more directly that:
1) Cells overexpressing β-catenin responded more to Wnt stimulation (i.e., gave a higher fold-change).
2) Cells overexpressing
β-catenin were less buffered when treated with lithium (i.e., fold-change decreased more readily).
More repeat experiments are shown in the main text.
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Figure S8. Step-by-Step Details on How Figure 5A Was Generated
To illustrate how Figure 5A was generated, let us decrease α from the Xenopus setpoint. As depicted by the green arrow in Figure A, the basal level of β-catenin will increase whereas the Wnt-induced fold-change in β-catenin will remain
constant initially. As shown in Figure B, we then plot independently β-catenin level and fold-change as a function of α. Figure C merges the two plots in Figure B. For each value of α, we plot fold-change against basal level.
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Figure S9. More Protocol Details on Experiments in the Xenopus laevis Embryos
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Figure S10. Separating Cadherin-Bound Pool of βCatenin
(A) To separate the cadherin-bound pool of β-catenin, a standard protocol was followed (Guger and Gumbiner, 2000; McCrea et al., 1991). 100 uL of embryo lysates (10 embryos) were rotated at 4oC for 1h with 100uL Concanavalin A–Sepharose 4B slurry (Sigma C9017). Supernatant and beads were separated by centrifugation (three 5min spin at 16,000g).
(B) The supernatant showed trace bands with monoclonal anti-cadherin 6B6
(Hybridoma Bank, 1:500). The 6B6 detects C-/U-/EP-cadherin, the cadherins expressed in early embryos (Heasman et al., 1994). The lower bands (<100kDa) are likely to be cadherin degradation products because they disappear with RIPA-extraction.
(C) Calibrating the volume of ConA to use. Equal volumes were loaded for each lane. Ratio does not necessarily correspond to actual membrane/cytoplasmic ratio of β-catenin in the embryo.
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Figure S11. The Phenotypic Buffering in Response to â¤200 mM Lithium Treatment Is Highly Reproducible
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Figure S12. Quantitation of β-Catenin at Earlier Stages Does Not Alter Conclusions
At all stages measured, β-catenin mRNA injection has lower effect on β-catenin
protein level, and yet gives more severe phenotypes.
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Figure S13. Transcription Is Not Saturated
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