Cell-Size Pleomorphism Drives Aberrant Clone Dispersal in Proliferating Epithelia

Abstract

As epithelial tissues develop, groups of cells related by descent tend to associate in clonal populations rather than dispersing within the cell layer. While this is frequently assumed to be a result of differential adhesion, precise mechanisms controlling clonal cohesiveness remain unknown. Here we employ computational simulations to modulate epithelial cell size in silico and show that junctions between small cells frequently collapse, resulting in clone-cell dispersal among larger neighbors. Consistent with similar dynamics in vivo, we further demonstrate that mosaic disruption of Drosophila Tor generates small cells and results in aberrant clone dispersal in developing wing disc epithelia. We propose a geometric basis for this phenomenon, supported in part by the observation that soap-foam cells exhibit similar size-dependent junctional rearrangements. Combined, these results establish a link between cell-size pleomorphism and the control of epithelial cell packing, with potential implications for understanding tumor cell dispersal in human disease.

Keywords: Epithelial Topology; Pleomorphism; TOR; cell growth; cell rearrangement; cell size; epithelial junctions.

Figure 1.. Small-cell clones tend to disperse in silico

(A) In the in silico model, cells are represented by polygons with straight sides (cyan) and parametrized by vertices (green). Their mechanical state is characterized by their surface areas A (magenta) and side lengths l (black). The depicted equation relates the potential energy W of the tissue to the sum of work done to acquire cell side lengths l_i and to deviate cell areas A_j from their preferred value a_j. Line-tension γ and area-compression k moduli remained constant in all simulations. During simulation, the vertices move so as to minimize the potential energy. (B) Implementing cell growth and division in the vertex model. Cells spend the majority of time in a rest phase. Subsequently, cells enter a phase of growth when their preferred area is increased linearly with time. Cells divide when their preferred area is double the rest phase preferred area. (C and D) Representative snapshots of mosaic tissue generated after ~8 rounds of cell division. Clonal cells (magenta) were derived from a single progenitor cell at the start of the simulation. The preferred clone cell area a_c was set at either 1 (C) or 0.2 (D), while the preferred area of non-clone cells a_nc was set at 1. All cells had the same division rate and featured random orientation of cleavage. Instances of dispersal are circled with dashed lines. (E) Clone cell dispersal after ~8 rounds of cell division over a range of preferred clone cell areas between 0.1 and 2, while that of non-clone cells was 1. Clone cell dispersal is the number of instances where non-clone neighbors intervene clonal cell population. For each preferred cell area, the simulation was repeated 200 times. (F) The temporal dynamics of clone cell dispersal over a range of preferred clone cell areas between 0.1 and 2. (G) Cell sidedness after ~8 rounds of cell division. Clonal cells at the non-clone interface are denoted with green, their immediate non-clone 1^o neighbors with brown and their 2^o neighbors with blue.

Figure 2.. Tor^ΔP clones are frequently interposed by larger neighboring cells

(A and B) FRT40A control (A, n = 29 wing discs) and Tor^ΔP (B, n = 62 wing discs) cell clones expressing GFP (magenta) and stained for Dlg (green) to visualize junctions in wing disc epithelia. Scale bars, 20 μm. (C and D) Skeletonized junctions with cell-sidedness inscribed within the clonal FRT40A control (C) and Tor^ΔP (D) cells. (E) The frequency distribution of relative area in FRT40A control (grey, n = 937 cells) and Tor^ΔP cells (red, n = 421 cells). (F) Cell dispersal in FRT40A control (n = 28 wing discs) and Tor^ΔP (n = 52 wing discs) clones. Clone cell dispersal is the number of instances wherein mutant cells (magenta) were intervened by exactly one non-mutant neighbor in the wing pouch. Unless otherwise specified, in this and every following box-plot, circles represent mean values from individual wing discs, the diamond box contains 25–75% percentiles of the data and the bar denotes the median. P-value, unpaired two-sample t-test. (G) The frequency distribution of sidedness in FRT40A control (grey, n = 937 cells) and Tor^ΔP cells (red, n = 421 cells).

Figure 3.. Tor^ΔP cells lose sides to their larger neighbors

(A) Illustration of a cell clone (dark green) and its non-clone 1⁰ and 2⁰ neighbors (brown and blue, respectively). (B and C) Cell sidedness of FRT40A control clones (B, n = 937 cells) and Tor^ΔP clones (c, n = 421 cells) along with their 1⁰ and 2⁰ neighbor cells. While Tor^ΔP cells tend to lose sides, their immediate 1⁰ neighbors tend to gain sides. P-values, one-way ANOVA.

Figure 4.. E-cadherin overexpression does not rescue Tor^ΔP clone dispersal

(A) Tor^ΔP clones (magenta, cells marked with asterisks) in the wing pouch expressing endogenous E-cadherin fused to tdTomato (n = 12 wing discs). The range of tdTomato fluorescent intensities is indicated by the calibration bar. Scale bar, 20 μm. (B) Fluorescence recovery of junctional E-cad^Tomato after photobleaching (FRAP) three regions of interest (red circles) simultaneously, at −0.5 s. Clonal Tor^ΔP cells (magenta) are marked with asterisks. The red circles (ROIs) are on junctions shared between nonclonal (N:N), clonal-nonclonal (C:N) or clonal (C:C) cells. Scale bar, 2 μm. (C) FRAP in N:N (grey, n = 10 ROIs, from 6 wing discs), C:N (blue, n = 6 ROIs, from 5 wing discs) and C:C (red, n = 8 ROIs, from 5 wing discs) junctions. The dots represent junctional E-cad^Tomato enrichment, determined by normalizing the mean pixel intensity of an ROI to that of the entire field of view. The average prebleach enrichment in each of the three junction types (at −1 s) and the corresponding postbleach recovery fits (≥ 0 s) are denoted by crosses and solid curves, respectively. (D) The immobile fraction of junctional E-cad^Tomato. F-value, one-way ANOVA. (E, F) FRT40A control (E, n = 15 wing discs) and Tor^ΔP (F, n = 13 wing discs) clones overexpressing E-cad and GFP (magenta, cells marked with asterisks). The range of E-cad immunostaining fluorescent intensities is indicated by the calibration bar. Scale bars, 20 μm. (G) Cell dispersal in FRT40A control and Tor^ΔP clones overexpressing E-cad. P-value, unpaired two-sample t-test.

Figure 5.. Apoptosis does not have a causal role in dispersing clonal Tor^ΔP cells

(A and B) FRT40A control (A, n = 5 wing discs) and Tor^ΔP (B, n = 6 wing discs) clones expressing GFP (magenta) and stained for cDcp1 (green) to mark apoptotic cells. Yellow dotted perimeter and circles indicate the wing disc pouch region and cDcp1 + cells, respectively. (C) Tor^ΔP clones that overexpress the cell-death repressor p35 and GFP (magenta). (D) Cell dispersal in FRT40A control (n = 14 wing discs) and Tor^ΔP (n = 14 wing discs) clones overexpressing the caspase inhibitor p35. P-value, unpaired two-sample t-test. Scale bars, 20 μm.

Figure 6.. Smaller cells can match side-lengths with their neighbors by decreasing sidedness

(A) The side-lengths of regular hexagons with areas 1 (brown) and 0.5 (red) are mismatched. (B) The side-lengths of regular heptagons with areas 1 and that of pentagons with 0.5 are comparable. (C) The equation in the inset relates the side-length l of a regular polygon to its area A. The value of the coefficient λ depends on polygon sidedness p. The side-lengths of regular polygons are plotted against area for indicated polygon classes. (D) Optimal tiling for regular polygons with mismatched areas. (E) The square root function fits relating side-lengths and areas of epithelial cells from the wing disc for cells in the indicated polygon categories. Tor^ΔP cells (n = 591 cells) and their 1⁰ neighbors (n = 558 cells) are represented by dotted and solid curves, respectively. In agreement with geometric considerations, relative cell side-lengths tend to negatively correlate with cell-sidedness for a given relative apical area for both Tor^ΔP clones and their 1⁰ neighbors. (F-H) The frequency distribution of relative cell perimeters (F), sidedness (G) and relative side-lengths (H) for Tor^ΔP clones (green, n = 591 cells) and their 1⁰ neighbors (brown, n = 558 cells).

Figure 7.. Reducing the size discrepancy between Tor^ΔP cells and their neighbors restores clone contiguity

(A) Tor^ΔP clones co-expressing a constitutively active version of S6 kinase (S6K^CA) and GFP (magenta) stained for Dlg (n = 26 wing discs). Scale bar, 20 μm. (B, C, and D) Normalized cell area (B), cell sidedness (C) and cell cycle duration (D) of FRT40A control (grey) or Tor^ΔP (red) clones with and without S6K^CA overexpression. P-values, one-way ANOVA. (E and F) Cell sidedness (E) and dispersal (F) of clonal cells plotted against their cell area. Each data point is the mean value from a wing disc containing clonal FRT40A control cells (grey, n = 937 cells), Tor^ΔP cells (red, n = 421 cells), FRT40A control cells with S6K^CA overexpression (orange, n = 621 cells), Tor^ΔP cells with S6K^CA overexpression (green, n = 451 cells) or Tor^ΔP cells with Tor overexpression (blue, n = 394 cells). Magenta triangles represent data from wing discs containing clonal Tor^ΔP cells surrounded by M^−/+ cells (n = 605 cells). The dotted line is a linear fit of the data.