Partially complementarily expressed ephrins and Ephs control notochord–paraxial mesoderm separation.

(A) Summary of ephrin and Eph expression and of the functional pairs (double arrows) in the dorsal mesoderm at stage 14 (see Figure S1B). (B–M) Manipulations were targeted to a restricted region of the dorsal mesoderm. Embryos were fixed at stage 14, and the dorsal structure was analyzed on sections. Injected cells were detected by the tracer Myc-GFP (red). Only the strongest signal is visible on these images but is sufficient to indicate the position of the injected area. Membranes were labeled with an anticadherin antibody (green). In (D) and (E), FoxA4-positive nuclei appear in green–cyan. FoxA4 is used as the notochord marker. (B) Control. Normal boundaries (highlighted by dashed lines) were characterized by a smooth alignment of the notochord and paraxial mesoderm cells. (C) Strong boundary disruption in an ephrinB2 MO-injected embryo. The position of the left boundary can still be located (arrowheads), but the alignment is jagged (arrowhead). On the right side, the two tissues are continuous, without a detectable boundary. Arrows point to the approximate limit of the notochord. (D and E) The boundary is effectively disrupted by ephrinB3 MO targeted to the paraxial mesoderm (arrows in D), but not to the notochord (E). (F–H) EphA4 MO injection in the notochord, the tissue fused with the paraxial mesoderm (F, arrows). A boundary can be rescued by the coinjection of mRNA coding for the AB Eph chimera (G, dashed lines) but not by the BA chimera (H, boundaries lack on both sides, arrows). Thus, the extracellular domain of EphA4 is necessary and sufficient for its function in the notochord. (I) Inhibition of separation by EphB4 MO targeted to the paraxial mesoderm. Left boundary is missing (arrows). (J) Rescue by the BA chimera. The injection was targeted to the right boundary. (K) The AB chimera fails to rescue. The injection was targeted to the left side (arrows). The right boundary is intact (dashed line). (L) Quantification of boundary disruption by ephrin/Eph depletion. Individual boundaries were scored as follows: 1, intact boundary; 0.5, partially disrupted boundary (rare cases); 0, fully disrupted boundary. The first column (t) compiles data of all embryos, (no) and (pm) the number of boundaries where injection was mainly targeted to the notochord or to the paraxial mesoderm. Numbers on top are numbers of embryos (2–6 independent experiments). EphrinB2, which can interact with both EphA in the notochord and EphB4 in the paraxial mesoderm, was required in both tissues. EphrinB3 and EphB4 depletion strongly disturbed the boundary when targeted to the paraxial mesoderm, but had no effect in the notochord. The opposite was observed for EphA4, consistent with the expression patterns and the selective interactions. (M) Quantification of rescues by wild type and chimera forms of EphA4 and EphB4. In all cases, complete rescue was obtained with the corresponding wild-type proteins and with the chimera containing the correct extracellular domain. The nature of the intracellular domain was indifferent. Numbers on top indicate total number of boundaries in each category.