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

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.

A mechanical model of cell segregation driven by differential adhesion.

From simulations that begin with a random mix of two cell types, we monitor progress towards segregation driven by contact-mediated linkage of model cells, which is equivalent to the cell-cell adhesion of real cells. In comparison with real cell experiments, we show that this mechanical model can account for the observed extent of segregation obtained by differential adhesion in a 2D cell culture assay of cells with differentially expressed cadherin molecules. Calibration of virtual to real time allowed us to estimate a time course for these experiments that was within 50% agreement for the simulations compared to differential adhesion of cells. In contrast, simulations of differential adhesion do not account for the rate of segregation driven by interactions between EphB2 receptor and ephrinB1 expressing cells which occurs an order of magnitude faster. The latter result suggests that mechanisms additional or alternative to differential adhesion contribute to Eph-ephrin mediated cell segregation.

A gene regulatory network directed by zebrafish No tail accounts for its roles in mesoderm formation.

Using chromatin immunoprecipitation combined with genomic microarrays we have identified targets of No tail (Ntl), a zebrafish Brachyury ortholog that plays a central role in mesoderm formation. We show that Ntl regulates a downstream network of other transcription factors and identify an in vivo Ntl binding site that resembles the consensus T-box binding site (TBS) previously identified by in vitro studies. We show that the notochord-expressed gene floating head (flh) is a direct transcriptional target of Ntl and that a combination of TBSs in the flh upstream region are required for Ntl-directed expression. Using our genome-scale data we have assembled a preliminary gene regulatory network that begins to describe mesoderm formation and patterning in the early zebrafish embryo.