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. 2018 Nov 13;7:e37851. doi: 10.7554/eLife.37851

Figure 4. A trans-acting step, occurring in parallel with the cis-acting step, provides an additional input for Bcl11b activation.

(A) Candidate models for Bcl11b activation from the DN2 stage, involving a single cis-acting switch (top left), sequential trans-, then cis-acting switches (bottom left), and parallel, independent trans- and cis- acting switches (right). (B) Plots show best fits of different models to the time evolution of Bcl11b allelic activation states, observed by timelapse imaging (Figure 2). Gray-shaded area indicates time delay for detection of indicated allelic state as a result of the time required for stable fluorescence protein accumulation. Best fit rate constants indicated in legend. (C) Bar charts show reduced chi-squared values for each model fit, that is the normalized sum-squared fit errors over all time points and allelic states, divided by the degrees of freedom (d.f.) (see Materials and methods). Both sequential and parallel trans-cis models fit the data significantly better than the cis-only model (F-test, F = 12.2, p=0.0052, sequential vs. cis-only model; F = 8.13, p=0.021, parallel vs. cis-only model). (D) Three possible classes of Bcl11b activation states observable from clonal lineage data. Lineage trees and transition diagrams show examples of simulated lineages that fall into the indicated classes. (E) Pie charts show expected distribution of allelic activation states predicted for clonal lineages of non-expressing progenitors in either the sequential (left) or the parallel (right) trans-cis model, obtained from N = 30,000 simulations, using parameters derived from bulk fitting (see Appendix). (F) Pie chart (left) shows observed distribution of activation states observed across an entire imaging time course. Colored scatterplots (right) show Bcl11b-mCh versus Bcl11b-YFP levels of single-cell lineages, falling into the indicated categories. Clones were scored according to observable fluorescence across an entire developmental trajectory, from 0 hr (cyan) to 120 hr (purple). The observed frequency of clones with ‘single mono-allelic’ expression of Bcl11b (7/9 = 77%) is significantly different than that predicted for the sequential trans-cis Model (20.1%, **- p<0.001, χ2 = 14.9, d.f. = 1), but not significantly different from that predicted for the parallel trans-cis Model (63.9%, χ2 = 0.27, d..f = 1, n.s.). Results are representative of three independent experiments. See Figure 4—figure supplement 2 for data for independent replicate experiments.

Figure 4—source data 1. Quantitative analysis of timelapse imaging data used to test three minimal models.
Figure 4—source data 1B shows the mean population fractions and 95% confidence intervals of mono- and bi-allelic expressing cells as observed by timelapse imaging. Figure 4B was plotted from these data points. Figure 4—source data 1C gives the reduced chi-squared values calculated for each model and is represented in Figure 4C. Figure 4—source data 1E, plotted as pie charts in Figure 4E, shows expected fractions of each class of Bcl11b activation state from 30,000 Monte-Carlo simulations for both sequential and parallel trans-cis models. Figure 4—source data 1F shows number of single cell lineages scored for each class of Bcl11b activation state in each observed experiment (3 independent experiments). Both sequential and parallel trans-cis models predict different frequencies of activation states.
elife-37851-fig4-data1.xlsx (13.3KB, xlsx)
DOI: 10.7554/eLife.37851.020

Figure 4.

Figure 4—figure supplement 1. Least-squares fitting of 2D histograms of Bcl11b expression levels.

Figure 4—figure supplement 1.

Two-dimensional plots show experimental (right) and best-fit (left) heat maps of Bcl11b levels at the indicated time points. Data are taken from 5 hr time windows centered on the indicated time points. White horizontal and vertical bars (right) provide a visual guide for discerning Bcl11b activation levels, while red arrows (t = 7.5 hr) show emergence of the two mono-allelic expressing populations, together with emergence of bi-allelic expressing population.
Figure 4—figure supplement 2. Clones show mono-allelic expression from a single predominant allele during Bcl11b activation.

Figure 4—figure supplement 2.

(A) Table shows observed numbers of clones with indicated allelic activation patterns, showing data from three independent experiments. Simulations of clonal lineages from sequential or parallel trans-cis activation models are shown (N = 30,000 simulations). See Figure 4 and main text for model description and allelic pattern definition. (B) Live images show cells from two clonal lineages showing a single mono-allelic pattern of activation, with mono-allelic expression of either the red allele only (Clone I), or the yellow allele only (Clone II). Images shown are from Experiment 3, whereas images and data in Figure 4 are shown from Experiment 1.