Cell-Cell Contact Area Affects Notch Signaling and Notch-Dependent Patterning

Abstract

During development, cells undergo dramatic changes in their morphology. By affecting contact geometry, these morphological changes could influence cellular communication. However, it has remained unclear whether and how signaling depends on contact geometry. This question is particularly relevant for Notch signaling, which coordinates neighboring cell fates through direct cell-cell signaling. Using micropatterning with a receptor trans-endocytosis assay, we show that signaling between pairs of cells correlates with their contact area. This relationship extends across contact diameters ranging from micrometers to tens of micrometers. Mathematical modeling predicts that dependence of signaling on contact area can bias cellular differentiation in Notch-mediated lateral inhibition processes, such that smaller cells are more likely to differentiate into signal-producing cells. Consistent with this prediction, analysis of developing chick inner ear revealed that ligand-producing hair cell precursors have smaller apical footprints than non-hair cells. Together, these results highlight the influence of cell morphology on fate determination processes.

Keywords: Notch signaling; cell morphology; cell-cell contact; inner ear; lateral inhibition; live cell imaging.

Figure 1. The live-cell Notch trans-endocytosis (TEC) assay allows dynamic tracking of N1-Dll1 interaction.

(A) A schematic of the Notch TEC assay. In this assay a signal sending cell expressing Dll1-mCherry (gray-red, top) under a doxycycline inducible promoter is co-cultured with a signal receiving cell expressing N1G4-citrine (gray-green, bottom). The N1G4-citrine has a citrine (green) inserted in the extracellular domain of N1 (NECD) and Gal4 replacing its intracellular domain. Upon Interaction between Dll1-mCherry and the N1G4-citrine the extracellular domain of N1G4-citrine trans-endocytoses into the Dll1-mCherry cell. (B) A schematic of a co-culture experiment. N1G4-Citrine cells (green) are co-cultured with Dll1-mCherry cells (white/red). At the beginning of the experiment Dll1-mCherry is induced by doxycycline. Upon induction of Dll1-mCherry, trans-endocytosed vesicles (yellow) appear in signal sending cells. (C) A filmstrip showing a co-culture experiment as described in (B). Here, Dll1-mCherry (red) cells are co-cultured with N1G4-ctirine cells (green in the top row, gray in the bottom row) (see also Movie S1). The bottom row shows only the N1G4-citrine. Dll1-mCherry cells (red) were pre-induced with 100 ng/mL of doxycycline 3 hr prior to the first frame (t = 0). TEC is observed as vesicles containing both N1G4-citrine and Dll1-mCherry within the Dll1-mCherry cells (yellow in the top row, arrows in the bottom row). Scale-bar 10µm. See also associated Figure S1.

Figure 2. The two-cell TEC assay allows measuring the dependence of TEC on contact width.

(A) A schematic of the device used for the two-cell assay. (B) A schematic of a two-cell TEC assay in a pair of cells with small contact width (~2.5 µm). (C) A bright field image of the cell pair used in (D). Scale-bar as indicated (D) A filmstrip showing a Dll1-mCherry cell (red) and a N1G4-citrine cell (green in top row, gray in bottom row) interacting in a two-cell microwell (see movieS2 in supplementary). Bottom row shows only the N1G4-citrine fluorescence. Dll1-mCherry was pre-induced with 100 ng/ml doxycycline one hour prior to the first frame of the movie (t=0). Arrows indicate TEC events. (E) A schematic of a two-cell TEC assay on a pair of cells with large contact width. In this experiment, cells broke out of the microwells and formed a large contact (~25 µm length). (F) A bright field image of the cell pair used in (G). Scale-bar as indicated. (G) A filmstrip showing TEC in a pair of cells with a large contact width (see movie S3 in supplementary). Experimental procedure and labeling is the same as shown in (D). (H-I) Quantitative analysis of the filmstrips in (D) and (G), respectively. The levels of TEC (green, left axis) and Dll1-mCherry fluorescence (red, right axis) are plotted as a function of time. Grayed area in (F) indicate cell division period, where cells are partially out of the image plane. (J) Comparison of the normalized TEC (nTEC) between small and large contact widths (i.e. between (H, black) and (I, blue)). nTEC is defined as the level of TEC divided by total Dll1-mCherry level. See also associated Fig. S2.

Figure 3. nTEC is correlated with contact width.

A plot showing the mean nTEC level between 90-150 min after the movie started, as a function of the mean contact width between 0-150 min (in both cases the mean is taken over time). Data shown is from 30 two-cell assay experiments (as in Fig. 2D,G, circles) and one data point from a free co-culture experiment (square). The solid line is a linear fit to the data points. This data shows that nTEC levels correlate with the width of the contact between the cells (n=31, ρ=0.83, p-value<10^-8 calculated using Pearson correlation). See also associated Fig. S3.

Figure 4. Dependence of Notch signaling on contact area can bias lateral inhibition patterning.

(A) A schematic of a model for lateral inhibition that takes into account dependence of signaling on contact area. The model assumes that Notch signaling in each cell depends on the number of Notch-Delta pairs formed (red and green, respectively). Notch signaling in each cell activates a repressor (R) that inhibits Delta production. Finally, Delta is distributed evenly on cell boundaries. (B) A simulation of the lateral inhibition model shown in (A) on a disordered cell lattice. Cells that express high levels of Delta are red and cells that express low level of Delta are green. The simulation was performed as described in the STAR methods (Eqs. 15-17). Parameters used: β_N = 3.9, β_D = 3.9, β_R = 194.1, m = 3, l = 3, ${\text{k}}_{\text{t}}^{-1}$ = 0.2 (C) A histogram showing the distribution of cell perimeters for high-Delta cells (red, n=827) and low Delta cells (green, n=1974) collected from simulations on 20 random lattices. (D) Analysis of the dynamics of the simulation in (B) showing a correlation between repressor level (R in (A)) and cell perimeter, at the fate determination point, τ_fate (n=140, ρ=0.59, p-value<10^-13 calculated using Pearson correlation). The solid line is a linear fit to the data points. Inset: a plot of repressor levels for each cell as a function of time. The fate determination point, τ_fate (dashed line), is defined as the latest time where there is no significant difference in the Delta levels of the prospective high and low Delta cells. (E) An image from the distal region of the chick basilar papilla taken at E6 (from (Goodyear and Richardson, 1997)). Samples were stained using both anti-cingulin (membrane/tight junction marker) and anti-HCA (Hair cell marker). Scale-bar 10μm. (F) A histogram of cell perimeters in (E) showing that cell with hair cell (HC) marker (red, n=62) have smaller apical perimeters on average than cells without HC marker (green, n=732). See Fig. S4J-L and methods for description of analysis. In (C) and (F) the middle bar and error bars denote the median and interquartile range, respectively. Three stars denote p-value<0.001 determined by Wilcoxon rank sum test. See also associated Fig. S4.