Ectoderm–mesoderm separation relies on asymmetric expression of specific ephrin/Eph pairs across the boundary, irrespective of the direction of the asymmetry.

<p>(A, A′, A″) Reciprocal replacement of mesodermal ephrinB3 and ectodermal EphA (mRNA injection). (A, A′) Diagram describing the experiment. (A) Endogenous ephrinB3 and EphA4 were depleted in the mesoderm and ectoderm, respectively (ghost labels). (A′) EphrinB3 (or B2) was then overexpressed in the ectoderm and EphA4 (or B4) in the mesoderm, thus effectively swapping the ligand and receptor (red double arrow). (A″) Quantification. Swapping ephrinB3 and EphA4 efficiently restored separation. EphrinB3 could be replaced by ephrinB2, consistent with the latter also being a ligand for EphA4. However, EphA4 could not be substituted by EphB4, in agreement with EphA4 being the only receptor of ephrinB3. The weak nonsignificant rescue was likely due to a slight boost in the ephrinB2–EphB4 signal. (B, B′) Similar experiment, but with the ephrin ligand substituted by direct incubation of the receiving explant with the corresponding soluble Fc fragment. EphrinB3 and EphA4 were depleted, EphA4 was overexpressed in the ectoderm, and ectoderm explants were incubated with the indicated Fc fragments. Overexpression of EphA4 led to a partial rescue of separation (control Fc), likely by activating ephrinB2–EphA4 signaling across the boundary (red dashed double arrow). Incubation with ephrinB3-Fc fully rescued separation. EphrinB2-Fc but not the ephrinB1-Fc fragment could also rescue, in agreement with the selectivity of EphA4.