Programmed cell-to-cell variability in Ras activity triggers emergent behaviors during mammary epithelial morphogenesis

Abstract

Variability in signaling pathway activation between neighboring epithelial cells can arise from local differences in the microenvironment, noisy gene expression, or acquired genetic changes. To investigate the consequences of this cell-to-cell variability in signaling pathway activation on coordinated multicellular processes such as morphogenesis, we use DNA-programmed assembly to construct three-dimensional MCF10A microtissues that are mosaic for low-level expression of activated H-Ras. We find two emergent behaviors in mosaic microtissues: cells with activated H-Ras are basally extruded or lead motile multicellular protrusions that direct the collective motility of their wild-type neighbors. Remarkably, these behaviors are not observed in homogeneous microtissues in which all cells express the activated Ras protein, indicating that heterogeneity in Ras activity, rather than the total amount of Ras activity, is critical for these processes. Our results directly demonstrate that cell-to-cell variability in pathway activation within local populations of epithelial cells can drive emergent behaviors during epithelial morphogenesis.

Figure 1. Programming the Assembly of Mosaic Epithelial Aggregates of Defined Size, Composition, and Initial Cell-to-Cell Connectivity

(A) Scheme for the programmed assembly of mosaic epithelial microtissues. (B) Single H2B-GFP-expressing MCF10A cells assembled 1:50 with H2B-RFP-expressing MCF10A cells. (C) Aggregates after purification by FACS. (D) MCF10A aggregates as shown in (C) condensed into rounded microtissues after 8.5 hr culture in lrECM. (E) Time series from Movie S1 showing motion of fluorescently labeled nuclei during condensation of an aggregate (insets) into a microtissue. Scale bars, 10 μm. See also Figure S1.

Figure 2. Onset of Polarity, Growth Arrest, and Lumen Formation in MCF10A Cell Aggregates

(A) Representative confocal immunofluorescence images of MCF10A aggregates stained for α₆-integrin and β-catenin after 0, 12, and 24 hr in 3D culture, and representative immunofluorescence images of similarly sized microtissues grown from single cells for comparison. A schematic illustrating the correct localization of α₆-integrin and β-catenin for MCF10A microtissues undergoing morphogenesis is shown to the right. (B) Quantification of the onset of polarity in assembled aggregates of MCF10A^WT cells. Values are means with error bars representing the SDs of the mean of 60 observations from at least two independent replicates. (C) Representative confocal immunofluorescence images of assembled MCF10A aggregates indicating correct localization of basement membrane component laminin-5 and cytoskeletal component F-actin after 24 hr in 3D culture. (D) Representative confocal immunofluorescence images of assembled MCF10A aggregates stained for cleaved caspase-3 and Ki-67 after 1, 4, and 16 days in culture (left). Schematic of MCF10A microtissue proliferation, growth arrest, and lumen formation (right). (E) Quantification of growth arrest and apoptotic lumen formation in microtissues grown from MCF10A aggregates. Data are expressed as the mean, and error bars represent the SD of the mean from at least two independent experiments (n = 60). (F) MDCK aggregates and similarly sized cysts grown from single cells stained for basolateral marker β-catenin and apical marker gp135 after 6 or 7 days. Scale bars, 10 μm. See also Figure S2.

Figure 3. Assembled MCF10A^Ras Cells Undergo Morphogenesis Similar to MCF10A WT Cells

(A) Western blots for Ras, phospho-Akt, phospho-Erk, and E-cadherin (E-cad) for MCF10A^WT and MCF10A^Ras cells. (B) Phase-contrast images of MCF10A^WT, MCF10A^Ras, and pBabe-puro Ras^V12-transduced MCF10A cells cultured for 23 days in lrECM. Inset shows the pBabe-puro Ras^V12-transduced cells after 5 days of culture in lrECM. (C) Representative confocal immunofluorescence images of homogeneous MCF10A^Ras cell aggregates stained for polarity and adherens junction proteins after culture in lrECM for the indicated times. These images should be compared to representative immunofluorescence images of similarly sized microtissues grown from single cells (right) and MCF10A^WT-staining patterns as shown in Figures 2A and S2A. (D) Representative confocal immunofluorescent images of homogeneous MCF10A^Ras aggregates stained for cleaved caspase-3 and Ki-67 after 16 or more days in culture. Scale bars, 20 μm. See also Figure S2.

Figure 4. Emergent Behaviors in Microtis-sues Heterogeneous for Signaling Downstream of H-Ras

(A) Representative time-lapse images from Movie S4 showing normal and emergent phenotypes in the mosaic MCF10A^Ras/MCF10A^WT microtissues. (B) Quantification of normal, basal cell extrusion, and motile multicellular protrusion frequency in homogeneous and heterogeneous microtissues. Data are expressed as the mean of at least 700 observations from four independent experiments, and error bars represent the SD of the mean. (C) Sensitivity of the frequency of emergent phenotypes to the addition of high-activity DNase immediately after assembly. (D) Sensitivity of cell extrusion and (E) motile multicellular protrusions to inhibition of PI3K by LY294002 and MEK by PD325901. Values are the averages of at least 400 total events from three independent experiments, and error bars show the SD of the means. Scale bar, 20 μm. ***p < 0.001; ns, not significant (one-way ANOVA and Tukey’s test). See also Figure S3.

Figure 5. Quantitative Analysis of Cell Motility during Emergent Behaviors

(A) A total of 30 superimposed 24 hr trajectories for cells expressing H2B-GFP fusions growing in homogeneous and heterogeneous MCF10A^Ras/MCF10A^WT microtissues of the indicated composition. (B) Average maximum distance traveled and (C) speed of the H2B-GFP-expressing cell under the conditions in (A). (D) Average distance traveled as a function of time for H2B-GFP-expressing cell in either homogeneous or heterogeneous microtissues. Average distances for H2B-GFP-expressing cells in heterogeneous microtissues are broken down into normal, motile multicellular protrusion, and basal extrusion phenotypes. (E) Trajectories of H2B-GFP-expressing MCF10A^Ras cells and the centroid of the surrounding WT microtissue. Microtissue trajectories (center) are subtracted from MCF10A^Ras trajectories (left) to produce the residual trajectories (right). A representative MCF10A^Ras cell (green), associated microtissue (red), and residual trajectory (hatched green and red) are highlighted. Trajectory of a hypermotile cell leaves a large residual (dashed orange lines). For (B) and (C), values are expressed as the mean with SD of 525 observations for three replicate experiments. For (A), (D), and (E), data are shown from a single experiment that is representative of three replicates. Trajectories are bounded by a 100 μm radius. See also Figure S4.