Figure 3. Molecular regulation of dedifferentiation.

(A) Testing the importance of transcription factors expressed early in dedifferentiation. Expression of bzpS and mybD during dedifferentiation in liquid medium alongside their developmental profiles (left). Right: PCA of transcriptome changes during dedifferentiation of wild type and bzpS and mybD mutants. See Table 1 for details of additional mutants. (B) Disrupted gene expression during dedifferentiation in forG^- and rasS^- mutants. Northern blots of PCNA, HspE and Rpl15 expression during dedifferentiation in wild-type, forG- and rasS- cells, with RNA loading indicated by 26S rRNA. PCNA blots representative of three experiments. (C) PCA of transcriptome changes during the dedifferentiation of forG- and wild-type cells. PCA carried out on the mean read counts of two biological replicates. (D) Onset of fluid uptake during dedifferentiation of wild-type (AX2) cells in liquid medium, measured as a fraction of the fluid uptake by undifferentiated cells. Data are the mean and SD of four replicates, except for 30 hr, with three replicates. (E) Rapid changes in phosphorylation of nutrient response markers during dedifferentiation in liquid culture. Phospho-western blotting of AMPKα and 4E-BP1 phosphorylation. UD = undifferentiated cell sample. Equal amounts of protein loaded, with histone H3 used as a standard (three replicates).

Figure 3—figure supplement 1. Analysis of candidate regulators of dedifferentiation.

(A) DhkA mRNA expression is strongly repressed during dedifferentiation. Expression of dhkA during dedifferentiation in bacteria and liquid medium alongside its developmental profile. (B) Schematic showing genomic location of mapped RNAseq reads at the PCNA locus. In the representative example shown from AX2 cells in liquid medium, read count within the PCNA gene is ~250, read count in upstream intergenic region is ~5 reads. (C) Northern blot showing size of transcripts detected in PCNA doublet. Samples are from wild-type (DH1) cells at 0, 1, 3, 5, 7 and 9 hr of dedifferentiation. (D) and E) Clonal recovery success of differentiating forG^- and rasS^- cells. Clonal recovery of single cells dedifferentiating on bacteria is shown for WT and mutant cells, alongside clonal recovery of undifferentiated WT cells. (D) ForG^-: four experiments, using at least 150 input cells, undifferentiated data from 3 days, at least 150 input cells. (E) For rasS- cells. Data show three repeats of at least 150 input cells. For D and E, paired data indicated by lines joining the points.