Posterior Distribution of τ, <i>g</i>, α, and ɛ

<div><p>Results are obtained using the model with context-dependent methylation rate and ignoring data from patient X.</p><p>(Row 1) Reports the posteriors of the parameters τ, the average depth of the genealogy of the stem cells (in years), and <i>g,</i> the length of cell differentiation in terms of number of cell generations. These two parameters control, together with <i>N,</i> the shape of the genealogy.</p><p>(Row 2) Reports the results for the parameters α and ɛ, responsible, together with ν, for the polymorphism given the genealogy: α is the relative amount of methylation/demethylation events in the <i>g</i> generations of cell differentiation compared with the number of events in a stem cell lineage up to the most recent common ancestor of the stem cell population; ɛ is the sequencing error rate.</p></div

Posterior Correlation between <i>N</i> and α

<p>The posterior of <i>N</i> subject to the constraint α = 0, 0.01, 0.02, or 0.03 (dotted lines) are compared with the posterior of <i>N</i> without those constraints (solid line).</p

A layer 2 FSC moves to EC region 2a.

Germarium expressing UAS-H2BRFP and ubi-GFP 16d after shift to 18C. Initial layer assignment of FSCs was determined by the relative strength of RFP expression. A layer 2 FSC (white arrow) moved to layer 3 at 45 min, and later moved anterior to region 2a. Images were collected every 9 min. Time is in hours:min. (MOV)</p

A layer 3 FSC moves to layer 1.

Germarium expressing UAS-H2BRFP and ubi-GFP 16d after shift to 18C. Initial layer assignment of FSCs was determined by the relative strength of RFP expression. A layer 3 FSC (purple arrow), initially anterior to a layer 2 FSC (red arrow) and a layer 1 FSC (cyan arrow) moved past the layer 2 FSC to layer 1. Images were collected every 5 min 20 sec. Time is in min:sec. (AVI)</p

FSCs move in both directions between layers 1 and 2.

A germarium expressing UAS-H2B-RFP and ubi-GFP 21d after shift to 18C. Initial layer assignment of FSCs was determined by the relative strength of RFP expression. A layer 1 FSC expressing ubi-GFP and very low H2B-RFP (blue arrow) moved to layer 2. A layer 2 FSC expressing H2B-RFP and ubi-GFP (white arrow) moved to layer 1. Images were collected every 5 min. Time is in hours:min (MOV)</p

FUCCI reporter responses to key cell cycle regulators and signals.

Examples of responses of FUCCI reporters to conditional expression of indicated transgenes, showing Fas3 (blue) staining to infer FSC identity and location, (A-H) GFP and RFP or (A’-H”) only RFP. All samples were also stained with DAPI to be able to count all cells, including those in S-phase (red arrows). All G1 (green arrows) and G2/M (yellow arrows) FSCs in the range of z-sections shown are indicated, as is the anterior Fas3 border (purple arrows). (A) Excess Stg increased G1 and decreased G2 FSC numbers. (B) Excess Stg together with CycE greatly increased S and decreased G2 FSC numbers. (C) Dominant-negative PI3K decreased G1 and increased G2 FSC numbers. (D) Excess JAK-STAT activity decreased G2 and increased S FSC numbers, but (E) principally increased G1 and decreased G2 FSC numbers together with excess Dacapo, Cdk2 inhibitor. (F) Decreased JAK-STAT activity increased G2 and decreased S FSC numbers. ECs were mainly in G1 in all samples but (D) and (E) illustrate conversion of some to G2 by excess JAK-STAT activity. Scale bar is 10μm.</p

Similar patterns of initial cell labeling for H2B-RFP and GFP driven by the same promoter.

(A-C) Flies with UAS-H2B-RFP, UAS-GFP, actin-GAL4 and tub-tsGAL80 were kept at 29C for 4d and then fixed to show initial expression levels before any chase period at 18C. Both H2B-RFP (red) and GFP (green) showed strong expression in ECs and layer 2 and 3 FSCs (blue and yellow arrows), weaker expression in layer 1 FSCs (white arrows) and even weaker expression in FCs (pink bracket). Three consecutive middle z slices were combined for (A) RFP and GFP together, (B) RFP alone, and (C) GFP alone. Scale bar, 20μm. (PDF)</p

FUCCI reporter and EdU labeling to score cell cycle phase of all FSCs and ECs.

(A) Cartoon representation of a germarium based on [18]. Cap cells (CCs) at the anterior (left) contact germline stem cells (not highlighted), which produce cystoblast daughters that mature into 16-cell germline cysts (white) as they progress posteriorly. Quiescent Escort cells (ECs) extend processes around germline cysts and support their differentiation. Follicle Stem Cells (FSCs) occupy three A/P rings (3, 2, 1) around the germarial circumference and immediately anterior to strong Fas3 expression (red) on the surface of all early Follicle Cells (FCs). FCs proliferate to form a monolayer epithelium, including specialized terminal Polar cells (PCs), which secrete the Upd ligand responsible for generating a JAK-STAT pathway gradient (green). Wnt pathway (red) and Hh pathway (blue) gradients have opposite polarity and are generated by ligands produced in CCs and ECs. (B) Percentage of cells in the designated locations in G1 (green), S (red) and G2/M (yellow), deduced from FUCCI reporters in wild-type germaria. SEM is shown from scoring 28 germaria (574 r1 ECS, 638 r2a ECs, 205 layer 3 FSCs, 323 layer 2 FSCs, 490 layer 1 FSCs). (C-H) The same C587>FUCCI germarium after EdU (white) labeling is shown using (C, E-H) several superimposed z-sections to capture roughly half of all ECs and FSCs present or (D) a single z-section to illustrate how cell locations are scored (see also Materials and Methods and S1 Fig). Cells expressing GFP-only (green arrows), RFP-only (red arrows) or both (yellow arrows) in (C) are clarified by images of the same set of z-sections showing only (E, F) GFP or only (G, H) RFP channels, with (E, F) or without (F, H) the EdU channel (Fas3 is present in all). All three FSCs in S-phase (red arrows; white EdU label) express neither GFP nor RFP. Many FCs, posterior to FSCs (further right), and a germline cyst (large clustered nuclei; orange arrow) have (white) EdU label. The anterior border of strong Fas3 staining is indicated by small purple arrows at the edge of the germarium. Sample are scored by examining each z-section, as shown in (D). Here, the Fas3 border has been outlined (blue dotted line), immediately adjacent to the nuclei of a G1 (green) layer 1 FSC at the top, a G2 (GFP plus RFP- clear only if GFP channel omitted) layer 1 FSC towards the bottom, and an S-phase layer 1 FSC (faint white from EdU because most of the nucleus is in an adjacent section) at the bottom. An S-phase layer 2 cell lies one more cell anterior (bottom), while a G2 (yellow) layer 3 FSC is two cells from layer 1 (top). Scale bar is 10μm. Raw data are in S1 Data (tab Fig 1B).</p

Live FUCCI reporter imaging shows cell cycle transitions and phase durations.

(A, B) Time-stamped frames from live imaging of (A) control and (B) C587>Hop germaria. (A) A yellow G2 cell (white arrow) changes to M-phase morphology at 20min, producing two G1 daughters (white arrows) with only GFP (no RFP) signal at 30min and at 40min. One daughter (red arrow) has lost GFP, indicating S-phase (red arrow) at 50min. The second daughter lost GFP, entering S-phase at 60min. The highlighted cell and its daughters are scored as layer 1 FSCs because they are at the posterior margin of strong C587-GAL4 expression driving the FUCCI reporter transgenes. See S1 Movie. (B) A yellow G2 cell (white arrow) changes to M-phase morphology at 45min, and then produced two G1 (green) daughters (white arrows) by 1h. The highlighted cells are two diameters away from the posterior edge of C587-GAL4 expression, indicating that they are layer 3 FSCs. Cell cycle transitions for layer 3 FSCs were observed only for cells with increased JAK-STAT activity, consistent with slow cycling of anterior FSCs resulting partly from insufficient JAK-STAT activity. See S2 Movie. Scale bar is 10μm. (C) Summary of calculated average duration of G1 (green), S (red), G2 (yellow) and M (white) phases of the cell cycle from the sum of all live imaging assays for control (left), C587>Hop (middle) and C587>CycE (right) germaria. Total average cell cycle length is shown in the center of each circle. For each genotype, the ratio of the whole cell cycle length for layer 2 relative to layer 1 FSCs is shown. The lengths of cell cycles as a percentage of controls are shown in red for excess JAK and CycE. Raw data are in S1 Data (tabs Figs 4, 4-WT and 4-UAS-CycE and 4-UAS-Hop).</p

Posteriors of <i>N</i> and <i>g</i> in Models with Short Niche Succession Time

<p>Three models that assume a niche succession time of about one year (τ = 1) are compared: same model as before but with τ = 1 (plain line); τ = 1 and high level of methylation/demethylation during cell differentiation (α > 0.1, dashed line); τ = 1, α > 0.1, without imposing a star-like genealogy for differentiation lineages (0 < <i>g</i> < 10, dotted line).</p