Abstract
Eph receptor and ephrin signalling has a major role in cell segregation and border formation, and may act through regulation of cell adhesion, repulsion or tension. To elucidate roles of cell repulsion and adhesion, we combined experiments in cell culture assays with quantitations of cell behaviour which are used in computer simulations. Cells expressing EphB2, or kinase-inactive EphB2 (kiEphB2), segregate and form a sharp border with ephrinB1-expressing cells, and this is disrupted by knockdown of N-cadherin. Measurements of contact inhibition of locomotion reveal that EphB2-, kiEphB2- and ephrinB1-expressing cells have strong heterotypic and weak homotypic repulsion. EphB2 cells have a transient increase in migration after heterotypic activation, which underlies a shift in the EphB2–ephrinB1 border but is not required for segregation or border sharpening. Simulations with the measured values of cell behaviour reveal that heterotypic repulsion can account for cell segregation and border sharpening, and is more efficient than decreased heterotypic adhesion. By suppressing homotypic repulsion, N-cadherin creates a sufficient difference between heterotypic and homotypic repulsion, and enables homotypic cohesion, both of which are required to sharpen borders.
Highlights
The establishment of organized tissues during development requires that adjacent cell populations with distinct tissue or regional identity do not intermingle with each other
We found that kinase-inactive EphB2 (kiEphB2) and ephrinB1 cells segregate, but rather than forming islands, kiEphB2 cells are interconnected and have a greater cluster size than EphB2 cells
To further test the behaviour of kiEphB2 cells, we carried out segregation assays in which EphB2, kiEphB2 and ephrinB1 cells are mixed. kiEphB2 cells selectively segregate to the interface with ephrinB1 cells, with EphB2 cells preferentially located at the centre of EphB2/kiEphB2 cell clusters: kiEphB2 cells comprise 44% (560/1267) of the total EphB2 þ kiEphB2 cells, but 84% (118/140) of the cells at the interface with ephrinB1 cells
Summary
The establishment of organized tissues during development requires that adjacent cell populations with distinct tissue or regional identity do not intermingle with each other. This is reflected by the formation of sharp borders between tissues or regional domains, despite the proliferation and intercalation of cells that can cause intermingling. Insights into mechanisms that prevent intermingling have come from experiments in which cells from different tissues are mixed in vitro, which for many cell types leads to their segregation. Such segregation occurs during development, on a more local scale, at borders that initially are fuzzy and sharpen. Differential adhesion or tension is generated by differences
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