Epigenetic stability increases extensively during Drosophila follicle stem cell differentiation

Abstract

Stem and embryonic cells facilitate programming toward multiple daughter cell fates, whereas differentiated cells resist reprogramming and oncogenic transformation. How alterations in the chromatin-based machinery of epigenetic inheritance contribute to these differences remains poorly known. We observed random, heritable changes in GAL4/UAS transgene programming during Drosophila ovarian follicle stem cell differentiation and used them to measure the stage-specific epigenetic stability of gene programming. The frequency of GAL4/UAS reprogramming declines more than 100-fold over the nine divisions comprising this stem cell lineage. Stabilization acts in cis, suggesting that it is chromatin-based, and correlates with increased S phase length. Our results suggest that stem/early progenitor cells cannot accurately transmit nongenetic information to their progeny; full epigenetic competence is acquired only gradually during early differentiation. Modulating epigenetic inheritance may be a critical process controlling transitions between the pleuripotent and differentiated states.

Fig. 1.

GAL4/UAS programming is unstable downstream from follicle stem cells. (A) Epigenetic fidelity can be measured in a cell lineage as the frequency of heritable gene expression changes (dark green). (B) Propagation upon replication fork passage of genetic information (black lines), and epigenetic information such as macromolecular complexes (green, purple) bearing secondary modifications (star). (C). Follicle stem cell (FSC) lineage gives rise to multiple cell types, including stalk, main body, stretch, border, polar and posterior follicle cells, as illustrated on the Drosophila ovariole showing the germarium (left) and older developing follicles (right). Two FSCs (green triangles) produce daughters (green hexagons) that surround germ cells (blue) to form new follicles (stage 1). After nine mitotic divisions (stage 7), three endocycles are completed (stage 10), followed by terminal differentiation. (D) c768 GAL4/UAS-EGFP expression varies among the follicle cells on a stage 10B follicle. (Inset) Three independent mitotic clones (red) induced by a single heat shock show uniform clonal growth and lack of mixing. (Scale bar, 25 μm.)

Fig. 2.

GAL4/UAS variegation is not due to position-effect variegation or gene silencing (A) Constructs showing GAL4/UAS variegation (green triangles) are located at diverse euchromatic sites (triangles), whereas PZ07128 (red) showing PEV is inserted near heterochromatin. (B–F) Su(var)3–9 suppresses PEV (D vs. C, and Insets) but not GAL4/UAS variegation (F vs. E) as quantitated in C. (G–I) EGFP expression from a UAS construct containing one (G) or two (H) GAL4 binding sites. (I) Structure of UAS constructs. Genotypes are indicated. (Scale bar, 25 μm.)

Fig. 3.

GAL4/UAS-EGFP expression is epigenetically inherited. (A) Predicted relationship to cell lineage (red) if EGFP expression (green) is epigenetically inherited, as discussed in text. (B–F) EGFP expression (green or white) compared with lacZ-marked clones (red, yellow outlines; Insets). (B) A 22-cell clone located within a region of uniform low EGFP expression. (C) EGFP channel from B showing correspondence between EGFP expression and clonal boundaries (yellow). (D) An 18-cell clone located within a region of uniform high EGFP expression that contains a four-cell subregion of medium expression. (E) An 18-cell clone corresponding to a region of uniform medium EGFP expression. (F) A 29-cell clone of medium expression that contains a one-cell subregion of low expression. (Scale bar, 25 μm).

Fig. 4.

Epigenetic stability increases during development (A) Stage 10B follicle with clones highlighted (yellow lines) for counting the number and size of clones up to 32 cells, each corresponding to a single expression change (Table S2). (B) Graph showing the probability EGFP expression will change at the indicated division (Div#). Values represent total changes at a specific lineage division divided by the total number of cells generated at that division in the sample analyzed (SI Materials and Methods). (C) A stage 3 follicle imaged by two-photon confocal microscopy showing early lineage clones of EGFP expression. (D) Graph showing the change probabilities for early lineage divisions determined from early follicle data (Table S3). (E) Composite of B and D. (Scale bar, 25 μm)

Fig. 5.

Epigenetic instability acts in cis. (A) UAS-dsRED and UAS-EGFP expression in a stage 10B follicle driven by c768-GAL4. (Inset) Predicted EGFP and dsRED expression if trans-acting factors fluctuate (correlated expression, solid yellow border, yellow arrow) or if the cis-acting factors at the site of the individual UAS constructs vary (uncorrelated expression, dashed yellow border, dashed arrow). Both types of patterns are seen. Separate green (B) and red (C) channels are shown. (D) Epigenetic stability plot of cis-acting fluctuations. (Scale bar, 25 μm)

Fig. 6.

Epigenetic stability correlates with increasing S phase length. (A) EdU incorporation in an ovariole after 1 h. (B) EdU labeling frequency reveals that S phase length increases during follicle cell development, shown in terms of developmental stages (x axis) or FSC divisions (arrows). (C) Frequency with which epigenetic changes increase (blue) or decrease (purple) EGFP expression. (D) Epigenetic changes in clones of one, two, or four cells usually arise in one daughter (“single” blue) rather than in “twin spots” (green). (E) Changes in epigenetic stability (red triangle) during follicle development take place before extensive follicular patterning by intercellular signaling (bar). (F) Model of epigenetic inheritance at replication mediated by specific chromatin machinery (green box and crescents). (Scale bar, 25 μm)