Temporal competition between differentiation programs determines cell fate choice

Abstract

Multipotent differentiation, where cells adopt one of several possible fates, occurs in diverse systems ranging from bacteria to mammals. This decision-making process is driven by multiple differentiation programs that operate simultaneously in the cell. How these programs interact to govern cell fate choice is poorly understood. To investigate this issue, we simultaneously measured activities of the competing sporulation and competence programs in single Bacillus subtilis cells. This approach revealed that these competing differentiation programs progress independently without cross-regulation before the decision point. Cells seem to arrive at a fate choice through differences in the relative timing between the two programs. To test this proposed dynamic mechanism, we altered the relative timing by engineering artificial cross-regulation between the sporulation and competence circuits. Results suggest a simple model that does not require a checkpoint or intricate cross-regulation before cellular decision-making. Rather, cell fate choice appears to be the outcome of a 'molecular race' between differentiation programs that compete in time, providing a simple dynamic mechanism for decision-making.

Figure 1

Competence initiation during the progression to spore formation. (A) Average fluorescence time trace of sporulating cells from 0A-IIR strain with P_spo0A-YFP (blue) and P_spoIIR-CFP (green). Individual traces (n=32) are normalized in amplitude and aligned in time with respect to P_spoIIR initiation (60% of peak activity) before averaging. The blue area corresponds to standard deviation. A representative filmstrip of sporulating microcolony of 0A-IIR strain is shown above. For strain definitions, see Supplementary Table S2. We note that the drop of fluorescence signal after the maximum is due to lysis of the mother cell during the spore formation process. (B) Sample quantitative traces illustrating how the activity of sporulation was determined at the initiation of competence. Specifically, the panel shows a single cell (0A-comG strain) undergoing a competence event followed by sporulation. Competence reporter P_comG activity is measured by CFP fluorescence intensity (shown in red) and P_spo0A activity is measured by YFP fluorescence intensity (in blue). The competence and sporulation events are highlighted by the cartoon and the filmstrip on top. The traces are aligned in time with respect to maximum P_spo0A activity at sporulation (set as zero time point). These data are utilized to determine P_spo0A activity (black circle highlighted by red arrow) at initiation of competence (defined by >30% P_comG activity relative to maximum P_comG amplitude at competence). For comparison, both time traces are normalized with respect to amplitude. (C) P_spo0A activity (black circles) at initiation of competence measured in single cells, n=67, from 0A-comG strain, as shown in (B). The filled circle highlighted by red arrow corresponds to the measurement shown in (B). The traces are aligned with respect to maximum P_spo0A activity at sporulation as described in (B). (D) Conditional probability of competence as a function of normalized P_spo0A activity. P_spo0A expression at onset of competence was measured in single cells from the 0A-comG strain as shown in (B) and (C). Conditional probability of competence is calculated as the number of competence initiations observed at specified values of P_spo0A expression normalized by the number of total surrounding cells with the specified P_spo0A amplitude (Supplementary Figure S1; Supplementary information, S2.4.3). Only data for P_spo0A activities between 20 and 80% are shown, because data <20% is within error and cells >80% are too close to spore formation ((A)). Error bars represent counting error.

Figure 2

Cross-regulation genes expression in single cells displays a cell fate-specific pattern only after cell fate choice. Panels (A) and (B) show mean P_abrB-YFP (A) and P_sinI-YFP (B) fluorescence measured in single cells of strains 0A-comG-abrB (n=10 competent and 40 spores), and 0A-comG-sinI (n=10 competent and 25 spores), respectively. The fluorescence measurements were taken from time points either 40 min before initiation of respective cell fate reporter (30% of maximum value for P_comG, 90% of maximum value for P_spo0A), labeled ‘pre-decision’, or 40 min after the maximum expression of respective cell fate reporter, labeled ‘execution’. Error bars represent standard error of the mean (s.e.m.). *P<0.001.

Figure 3

Cells identified in the DA state suggest lack of cross-regulation before cell fate decision. (A) Filmstrip of a representative competent cell visualized by competence reporter P_comG-CFP (red) (see also Supplementary Figure S6A). (B) Filmstrip of a representative DA cell expressing the same reporter (P_comG-CFP) as shown in (A) (see also Supplementary Figure S6B). (C) Sample time traces illustrating how the activity of sporulation was determined specifically in DA cells. The panel shows a single DA cell (0A-comG strain), in which P_comG activity is measured by CFP fluorescence intensity (shown in red) and P_spo0A activity is measured by YFP fluorescence intensity (in blue). The traces are aligned in time with respect to maximum P_spo0A activity at sporulation (set as zero time point). There data are utilized to determine P_spo0A activity (red circle) at initiation of P_comG expression in DA cells (defined by >30% P_comG activity relative to maximum observed P_comG amplitude), similarly to Figure 1B. For comparison, both time traces are normalized with respect to amplitude. (D) P_spo0A activity measured in single DA cells (red circles), n=67. The data were combined from measurements of 0A-comG, [0A-comG]^0A−K, [0A-comG]^IIG−K strains (see Supplementary Figure S8 and the manuscript below). The traces are aligned with respect to maximum P_spo0A activity at sporulation as described in (C). P_spo0A activity at the onset of competence (black circles) determined in Figure 1C is shown for comparison. For strain definitions, see Supplementary Table S2. (E) Average time traces of single DA cells measured in strains containing pairwise combinations of each of the indicated sporulation reporters together with P_comG-CFP; strains are as follows: 0A-comG (n=15), IIE-comG (n=7), IIG-comG (n=13), IIR-comG (n=4). All traces are normalized in amplitude and aligned in time with respect to P_comG activity (30% of peak activity). See also Supplementary Figure S8A. (F) Left: The design of the NoE strain. An E. coli protease tag ssrA was translationally fused to SpoIIE protein, and a specific E. coli protease factor SspB that targets this tag for degradation was expressed from P_comG promoter. Simultaneous activation of both differentiation programs, a characteristic of DA state, in engineered NoE cells would result in targeted degradation of SpoIIE. For details, see Supplementary information, S2.3. Right: Competent and DA cell frequencies observed in a strain lacking the NoE modification (‘No protease’) compared with the NoE strain. Black bars show competent cells fraction, while red bars indicate DA cells fraction. Both strains have enhanced background DA frequency for easier analysis as a result of ectopic ComK expression from P_spoIIG promoter (see Figure 5 and Supplementary Table S1). Error bars represent standard error of the mean (s.e.m.).

Figure 4

Temporal competition between differentiation programs determines cell fate. (A) Filmstrip showing a typical competent cell from the strain expressing P_comG-CFP (red) and ComGA-YFP protein fusion (green). Time between P_comG initiation (>30% maximum) and ComGA localization to cell poles (11.7 h) is indicated as t_comp. (B) Filmstrip of a representative DA cell visualized by P_comG-CFP (red) and SpoIIE-YFP (orange). Time between P_comG initiation (>30% maximum) and SpoIIE localization to asymmetric septum (>40% maximum, 5.3 h) is indicated as t_DA. (C) Histogram of time between P_comG initiation defined as in (A) (set as zero time point) and either ComGA localization to cell poles in competent cells (t_comp, n=27), or SpoIIE localization to asymmetric septum in DA cells (t_DA, n=32) measured in single cells as described in (A) and (B). (D) Diagram of the ‘molecular race’ hypothesis. Competence (top panel) occurs if ComK expression begins early enough to give sufficient time for ComGA localization (green dot) before SpoIIE (yellow line). However, functional competence cannot develop if ComK is expressed within the critical time window (termed ‘Decision’ and shown in pink) close to SpoIIE localization, resulting in DA state where sporulation takes over (middle panel). Sporulation is shown in the bottom panel for reference.

Figure 5

Perturbation of timing between sporulation and competence changes cell fate outcome. (A) Engineered cross-regulatory links between sporulation and competence circuits designed to test the ‘molecular race’ hypothesis. Top panel, comK is ectopically expressed either from the early stage of sporulation (P_spo0A promoter) before the decision time window discussed in Figure 4D (‘Decision’, pink area), the later stage close in time to decision (P_spoIIG promoter) or past the decision window (P_spoIIR promoter). Middle panel, two independent copies of comK gene are ectopically expressed from the indicated promoters (P_spo0A and P_spoIIG). Bottom panel, yneA is ectopically expressed together with comK from either early (P_spo0A) or later (P_spoIIG) stage of sporulation. (B) Competence and DA fractions measured in the strains expressing yneA and/or comK from early and late stage-specific sporulation promoters as described in (A). All strains are also expressing P_comG-CFP to report ComK activity. Each dot indicates the labeled specific strain. ‘Native’ denotes the PY79 B. subtilis strain expressing only the P_comG reporter. ‘Before’ and ‘During’ expression corresponds to ectopic expression from P_spo0A and P_spoIIG promoters, respectively, as shown in (A). Red arrows indicate comK expression. Green arrows point to strains expressing two copies of P_spo0A-comK (‘2 × before’) and P_spoIIG-comK (‘2 × during’) denoted ‘2 × P_spo0A-comK’ and ‘2 × P_spoIIG-comK’, respectively. Blue arrows show yneA expression from either P_spo0A or P_spoIIG promoter, termed ‘before+delay’ and ‘during+delay’, respectively. Detailed statistics for all strains can be found in Supplementary Table S1. Error bars represent standard deviation.