Cell segregation and border sharpening by Eph receptor–ephrin-mediated heterotypic repulsion

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.

Actomyosin regulation by Eph receptor signaling couples boundary cell formation to border sharpness

“…By generating a series of point and deletion mutants of epha4, we find that forward signaling is essential for boundary marker expression, with a strong input of kinase-dependent signaling and lesser input of PDZ binding domain dependent signaling. These findings are consistent with studies of the regulation of cell repulsion and cortical tension by Eph receptor signaling (Canty et al, 2017;Fagotto et al, 2013;O'Neill et al, 2016;Rohani et al, 2011;Rohani et al, 2014;Taylor et al, 2017). Cell repulsion and tension are regulated by increased Rho activity, which leads to myosin light chain phosphorylation and actomyosin contraction at borders where Eph receptor activation is occurring (Fagotto et al, 2013;Rohani et al, 2014).…”

“…We find that for r3 and r5 they form on one side of each interface, in the epha4-expressing cells, and this is because they are induced by forward and not by reverse signaling. This finding is consistent with evidence that although reverse signaling can trigger cell repulsion, forward signaling leads to much stronger cell repulsion and actomyosin contraction and thus has a dominant role in cell segregation and border sharpening (Canty et al, 2017;Fagotto et al, 2013;O'Neill et al, 2016;Rohani et al, 2011;Rohani et al, 2014;Taylor et al, 2017;Wu et al, 2019). However, rfng expression is also detected in some cells adjacent to r3 or r5 that are not expressing epha4, in particular at the r5/r6 border.…”

“…Multiple kinase-dependent pathways have been found to link Eph receptor forward signaling to Rho activation (Jorgensen et al, 2009;Kania and Klein, 2016;Pasquale, 2008). Eph kinase-independent signaling can also lead to cell repulsion and segregation (Taylor et al, 2017) and can activate Rho, for example through binding of Dishevelled to the PDZ domain binding motif of Eph receptors (Tanaka et al, 2003). Such kinase-independent signaling leads to a less sustained cell repulsion response than occurs when Eph kinase function is intact (Taylor et al, 2017).…”

“…Eph kinase-independent signaling can also lead to cell repulsion and segregation (Taylor et al, 2017) and can activate Rho, for example through binding of Dishevelled to the PDZ domain binding motif of Eph receptors (Tanaka et al, 2003). Such kinase-independent signaling leads to a less sustained cell repulsion response than occurs when Eph kinase function is intact (Taylor et al, 2017). Taken together, these findings reveal some functional overlap between kinase-and PDZBD-dependent signaling, with a greater role of Eph kinaseactivated pathways both in cell segregation and boundary cell induction.…”

“…Eph receptors and ephrins that have a high affinity for each other are expressed in complementary domains in many tissues (Cayuso et al, 2015;Gale et al, 1996;Rohani et al, 2014), such that activation of forward and reverse signaling occurs at the interface. Eph receptor and ephrin signaling can drive cell segregation and border sharpening through multiple mechanisms that likely depend upon whether the tissue is epithelial or mesenchymal: by decreasing cell-cell adhesion (Fagotto et al, 2013;Solanas et al, 2011), by increasing cortical tension (Calzolari et al, 2014;Canty et al, 2017), or by triggering cell repulsion (Poliakov et al, 2008;Rohani et al, 2011;Taylor et al, 2017;Wu et al, 2019). In addition, Eph-ephrin signaling has been found to regulate cell differentiation in a number of tissues (reviewed by (Laussu et al, 2014;Wilkinson, 2014)).…”