An orderly retreat: Dedifferentiation is a regulated process

Abstract

Differentiation is a highly regulated process whereby cells become specialized to perform specific functions and lose the ability to perform others. In contrast, the question of whether dedifferentiation is a genetically determined process, or merely an unregulated loss of the differentiated state, has not been resolved. We show here that dedifferentiation in the social amoeba Dictyostelium discoideum relies on a sequence of events that is independent of the original developmental state and involves the coordinated expression of a specific set of genes. A defect in one of these genes, the histidine kinase dhkA, alters the kinetics of dedifferentiation and uncouples the progression of dedifferentiation events. These observations establish dedifferentiation as a genetically determined process and suggest the existence of a developmental checkpoint that ensures a return path to the undifferentiated state.

Fig. 1.

Dedifferentiation markers. Cells were developed on filters to the indicated stage [Agg, aggregation (♦); Finger (▪); Mex, Mexican hat (▴)], dissociated, incubated in HL5, and sampled as indicated.(A) Developmental morphology. Aggregates appear after 10 hr, fingers after 13 hr, and Mexican hats after 16 hr of development. (B) Proliferation. Dedifferentiating cells were counted and the data were plotted relative to the initial density. Data are means and standard deviation of four experiments. (C) Nuclear DNA synthesis. Dedifferentiating cells were labeled with BrdUrd as indicated. Nuclear DNA was dot-blotted and BrdUrd incorporation was detected (photographs). Labeling intensity was plotted relative to the terminal level in each experiment (graphs). (D) Erasure. Dedifferentiating cells were starved on filters and reaggregation was determined microscopically (right y axis). Erasure is the fraction (percent, left y axis) of the time required for vegetative cells to aggregate (10 hr).

Fig. 2.

Transcriptional profiles of dedifferentiation. RNA samples were collected from dedifferentiating cells at the indicated times (hr). Color charts (Agg, dedifferentiation from aggregates; Finger, from fingers; Mex, from Mexican hats; Development, normal development) represent the expression pattern of 2,066 developmentally regulated genes (rows) (18). The gene order is identical in all charts. Vertical lines delineate three transcriptional phases (I, II, and III). Colors indicate lower than average (blue), average (gray), and higher than average (yellow) expression as described (18). Scale: log₂ of the ratio between sample and standard.

Fig. 3.

Dedifferentiation-specific gene expression. Data were collected as in Fig. 2. Time (hr) is indicated above and the developmental stage below each chart (Agg, dedifferentiation from aggregates; Finger, from fingers; Mex, from Mexican hats; Development, normal development). (A) The 259 genes up-regulated in phase II. The gene order is identical in all charts (the genes are represented by 272 targets). (B) The 122 genes coregulated during dedifferentiation but not during normal development.

Fig. 4.

Annotation of dedifferentiation genes. Genes from Fig. 3 were GO-annotated and the “biological process” annotation of significantly enriched groups is shown (P values are indicated below). The GO tree levels of the “biological process” annotation are shown as numbers inside bars (ranging from 2 to 9). The table on the right indicates the number of genes in each group (List), genes with that annotation on the entire array (Total), the P value, and the annotation. Bar lengths represent the fold enrichment (scale, x axis). Indented bars are subgroups of bars immediately above, as indicated by the branching pattern. Bar colors represent the group annotation at GO level 2: white, physiological process; black, cellular process. (A) Genes regulated during phase II of dedifferentiation and during development as shown in Fig. 3A (P < 0.1). (B) Genes regulated during phase II of dedifferentiation but not during development as shown in Fig. 3B (P < 0.15).

Fig. 5.

Dedifferentiation of dhkA^- cells. Wild-type and mutant aggregate (WT, solid diamonds; dhkA^-, hatched diamonds) and finger cells (WT, solid squares; dhkA^-, hatched squares) were dedifferentiated and sampled as indicated. (A) Growth. Cells were counted and the data are plotted relative to the initial density. Data are means and standard deviation of three or four experiments. The WT data are from Fig. 1 A.(B) Viability. Plating efficiency of vegetative and dissociated finger cells was measured. Viability is the percentage of microscopically visible cells that formed plaques. Results are means and standard deviations of three experiments. WT, gray bars; dhkA^-, hatched bars. (C) Nuclear DNA synthesis. Cells were labeled with BrdUrd; incorporation into nuclear DNA was determined and quantified as in Fig. 1 (photographs). WT, gray bars; dhkA^-, hatched bars. (D) Erasure. Dedifferentiating cells were starved on filters and reaggregation time was monitored microscopically. Erasure is the fraction (percent, left x axis) of the time required for vegetative cells to aggregate (10 hr).