EphrinB/EphB signaling controls embryonic germ layer separation by contact-induced cell detachment

Abstract

Background: The primordial organization of the metazoan body is achieved during gastrulation by the establishment of the germ layers. Adhesion differences between ectoderm, mesoderm, and endoderm cells could in principle be sufficient to maintain germ layer integrity and prevent intermixing. However, in organisms as diverse as fly, fish, or amphibian, the ectoderm-mesoderm boundary not only keeps these germ layers separated, but the ectoderm also serves as substratum for mesoderm migration, and the boundary must be compatible with repeated cell attachment and detachment.

Principal findings: We show that localized detachment resulting from contact-induced signals at the boundary is at the core of ectoderm-mesoderm segregation. Cells alternate between adhesion and detachment, and detachment requires ephrinB/EphB signaling. Multiple ephrinB ligands and EphB receptors are expressed on each side of the boundary, and tissue separation depends on forward signaling across the boundary in both directions, involving partially redundant ligands and receptors and activation of Rac and RhoA.

Conclusion: This mechanism differs from a simple differential adhesion process of germ layer formation. Instead, it involves localized responses to signals exchanged at the tissue boundary and an attachment/detachment cycle which allows for cell migration across a cellular substratum.

Figure 1. Cell repulsion at ectoderm-mesoderm boundary.

(A) Tissue separation assay. Test explants are placed on BCRs, and the percentage remaining separate are scored after 45–60 min. Cell behavior at the boundary between tissues is examined by live confocal microscopy using test explants and BCRs with differently labeled membranes. (B–F) Live confocal microscopy. Test explants expressing mYFP (B–D) or mCherry (E, F) were combined with mGFP-expressing BCRs. Time between frames was 1 min for (B–D), 5 min for (E,F), frame number indicated. (B) Overview, mesoderm explant on BCR (Movie S1). Arrows show retractions. (C) Region indicated by arrow, asterisk in (B). Simultaneous retraction of mesoderm and BCR; a gap appears (14), spreads to upper right (26), then to opposite side (34). Protrusions extend into gap (26, short arrow). Insert 34a, double arrowheads: retraction fibers. Arrowheads: stable contacts within the tissues. (D) Ectoderm aggregate on BCR (Movie S2), showing stable contacts within the tissues (arrowheads) and at the interface between the two ectoderm explants (arrows). (E) Attachment/detachment of three mesoderm cells at BCR. Contact is first established by upper cell (long arrow, 9), then spreads through all cells (between long arrows, 10). Cells detach again from sides of frame (arrowheads, 16), central cell (*) (long arrow, 16) seems to resist and is last to detach (18). Arrow, insert in frame 9: cell protrusions. (F) Mesoderm on BCR. Initially in close contact (Frame 1), cells from both tissues retract (Frame 2) leaving retraction fibers behind (arrowheads, 2a). Four focal planes merged to capture entire fibers. (G) Quantification of attachment/detachment events. Numbers on top indicate total events/cells.

Figure 2. EphrinB/EphB signaling controls tissue separation.

(A) Soluble ephrin B fragments induce tissue separation between ectodermal cells. Separation assay was performed after a short pre-treatment of the ectodermal aggregates (ecto) or of the BCR substrate with soluble, pre-clustered ephrinA- or ephrinB-Fc fragments (eA*/eB*). Unless stated otherwise, pre-treatment was for 15 min. Untreated mesoderm aggregates (meso) on untreated BCR were used as positive controls for maximal separation behavior. Untreated ectoderm explants rapidly sunk into untreated BCR. Strong separation was induced when either ectodermal aggregates or BCR were treated with eB*. Separation was particularly strong when both the test aggregates and the BCR were treated by performing the assay in eB* solution. eA* had no effect. (B,C) EphrinB/EphB signaling is required for tissue separation. (B) Separation assay: EphrinB depletion inhibits separation. Mesoderm test explants injected with control MO (COMO) remained on the surface of the BCR (arrowheads); of those injected with ephrinB2 MO (eB2 MO), 2 out of 4 invaded the BCR in the example shown (arrows). (C) Quantification of loss-of-function (LOF) experiments for ephrinBs and EphBs. Injections in mesoderm (Meso) or ectodermal BCR are indicated below bars for each tissue combination; wt, uninjected, ephrinB1,2 MO (eB1,2MO), EphB4 MO (EphMO), cytoplasmically deleted EphB (ΔCEph). Numbers of test explants remaining separated/total number of explants on top of bars. ** indicates p<0.01 compared to controls. Simultaneous depletion/inhibition in the mesoderm and the BCR (eB2MO/ΔCEph) did not increase the phenotype compared to eB2MO or ΔCEph alone (p = 0.2 and 0.3).

Figure 3. Forward signaling across the boundary is required in both apposed tissues.

(A) Enhanced inhibition of separation by interference with EphB signaling in both tissues. ΔC-EphB was expressed in the mesoderm, in the BCR, or in both. Symbols as in Figure 2. (B–C) Rescue of separation by soluble ephrinB2-Fc fragment after ephrinB depletion. (B) Diagram of the experiment. Ephrins in one tissue and their cognate Eph receptors in the other tissue are represented as green and red plasma membranes, respectively. Ephrins were depleted in one of the tissues (eB1 and eB2 in the BCR and eB2 in the mesoderm, depletion symbolized by black membranes), and the signal was then restored at the surface of the other tissue by a 15 min treatment with ephrin-Fc fragment (eB2*, green dots) before assembly of the assay. (C) Quantitative data. Fc, control Fc fragments. Otherwise, symbols as in (B).

Figure 4. Ephrin/Eph loss-of-function inhibits repulsion at the boundary.

(A–E) Effect of ephrin/Eph MO on repulsion. Time lapse spinning disc confocal microscopy using mesoderm explants expressing mCherry on mGFP-expressing BCRs. EphrinB2 MO or EphB4 MO in mesoderm (B,C), or ephrinB1 MO in BCR (D) inhibited repulsion compared to control MO (A). The interface between inhibited mesoderm explants and BCR resembled ectoderm-ectoderm contacts (E). (F) Incubation of mesoderm test explants with ephrinB1-Fc fragments rescues repulsion from ephrinB1-depleted BCR in a dose-dependent manner. (G) Quantification of attachment/detachment events per cell per hour. Numbers on top indicate total events/cells.

Figure 5. Involvement of RhoA and Rac downstream of ephrin/Eph signaling.

(A–C) Rac and RhoA activities are required for tissue separation. (A, B) Expression of dominant negative forms N17Rac and N19RhoA either in the mesoderm test aggregates or in the BCR blocked separation. (C) Separation was strongly inhibited when assays with wt mesoderm and wt BCR were performed in the presence of the Rac inhibitor NSC23766 (18 µM) or the Rok inhibitor Y26732 (50 µM). (D–E) Rac or RhoA activation can rescue loss of ephrin signaling. Signaling was inhibited by injecting EphB4 MO (D) in the mesoderm or expressing ΔCEphB in the BCR (E). In both cases, separation was rescued by co-expression of constitutively active V12Rac or V14RhoA. Constitutively active cdc42 (V14cdc42) had no effect (D).

Figure 6. Effect of Rho/Rac modulation on cell behavior at the boundary.

Live confocal microscopy of mesoderm explants expressing mCherry combined with mGFP-expressing BCRs. (A–F) Selected frames from Movies S7 and S8. (A–D) Inhibition of repulsion by ΔCEph in the BCR (A) is recued by co-expression of constitutively active forms of RhoA (V14Rho) (B) and Rac (V12Rac) (C). (D) Control wild type mesoderm and BCR. Arrowheads in (C) and (D) point at mesoderm cell membranes that are initially in contact with BCR cells and detach in subsequent frames. The arrow in Frame 12 of (C) points to a protrusion emanating from the BCR cell after detachment. In the example shown in (B) cells remain detached for a prolonged period (compare the two parallel but separated membranes in the enlarged field B1a and B7a with the merged signals from the closely apposed membranes for ΔCEph alone, A3a, and A12a). Asterisks indicate the relative positions of two mesoderm and ectoderm cells sliding along the boundary, and the antiparallel arrows show the relative translation of these cells. This phenotype is observed in both V14RhoA and V12Rac rescues. (E–F) Inhibition of repulsion by dominant negative forms of RhoA and Rac. N19RhoA and N17Rac caused ectoderm and mesoderm cells to remain stably attached. Note disruption of the boundary through intercalation of cells from both tissues (F, arrows). (G, H) Quantification of attachment/detachment events per cell per hour. Numbers on top indicate total events/cells.

Figure 7. Subcellular localization of activated GTPases at the boundary.

Explants expressing GFP-Rhothekin-GBD (A), GFP-Wasp-GBD (B), or control GFP (C) were combined with mCherry expressing BCRs and analyzed by live confocal microscopy. Both GDBs accumulated preferentially at sites of contact with the BCR in wt mesoderm (arrows), but not in wt ectoderm (arrowheads). Control GFP in wt mesoderm did not accumulate at the boundary. EphB4 depletion (EphMO) in the mesoderm or ephrinB1 depletion (eB1 MO) in the BCR both strongly decreased accumulation at most contact sites (arrowheads), similar to expression of dominant negative forms of RhoA (N19Rho) for GFP-Rhothekin-GBD or Rac (N17Rac) for GFP-Wasp-GBD. (D) Quantification: percentage of cells showing accumulation at contact sites with the BCR.

Figure 8. Dynamic activation of RhoGTPases at the ectoderm-mesoderm boundary.

Explants expressing GFP-Rhothekin-GBD (A) and GFP-Wasp-GBD (B) were combined with mCherry expressing BCRs. Selected frames from Movies S9 and S10. Time between frames was 2 min for (A) and 1 min for (B). (A',B') Pseudocolors for the GFP signal intensity. (A, A') The mesoderm cell labeled with an asterisk establishes contact with a BCR cell in Frames 14 and 15 (arrow), then retracts in Frame 16. GFP-Rhothekin-GBD concentrates near the site of contact in Frames 14 and 15, and the signal decreases after detachment (Frame 16). A second cell in the lower part of the field accumulates progressively higher levels of GFP-Rhothekin-GBD but fails to retract. (B,B') Two cells are initially in close contact with the BCR. While the upper cell maintains the contact throughout this sequence (Frame 13, thin arrow), the lower cell (thick arrow) detaches progressively starting from the lower edge (arrowheads). The intense GFP-Wasp-GBD signal at the sites of contact (Frame 8) dissipates in both cells (Frames 10–15), while the signal in the cytoplasm and near the membrane separating both cells (small arrows) remains relatively constant (small arrow Frames 8 and 15).

Figure 9. Model for tissue separation.

Cells at boundary alternate between attachment and detachment: contact at boundary triggers signaling through membrane-bound ephrinB and EphB receptors, which induces repulsion. Once cells are apart, the signal decays, cells emit protrusions, and re-establish contacts. This prevents mesoderm cells from invading the BCR but allows them to use the BCR as substrate for migration.