Journal: PLoS biology

Article Title: Variable combinations of specific ephrin ligand/Eph receptor pairs control embryonic tissue separation.

doi: 10.1371/journal.pbio.1001955

Figure Lengend Snippet: Figure 3. Ectoderm–mesoderm separation relies on asymmetric expression of specific ephrin/Eph pairs across the boundary, irrespective of the direction of the asymmetry. (A, A9, A0) Reciprocal replacement of mesodermal ephrinB3 and ectodermal EphA (mRNA injection). (A, A9) Diagram describing the experiment. (A) Endogenous ephrinB3 and EphA4 were depleted in the mesoderm and ectoderm, respectively (ghost labels). (A9) 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). (A0) 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, B9) 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. doi:10.1371/journal.pbio.1001955.g003

Article Snippet: Recombinant Proteins Recombinant mouse ephrinB2-Fc, ephrinB1-Fc, human ephrinB3, and mouse EphB4-Fc and EphA4-Fc (R&D Systems) comprising the extracellular domain of ephrin/Eph fused to Cterminal 66 histidin-tagged Fc region of human IgG were preclustered by 1 h incubation with anti-human Fc antibody (Jackson ImmunoResearch Laboratories) at a 1:2 ratio in MBSH [4] before application.

Techniques: Expressing, Injection, Incubation, Over Expression, Control

Journal: PLoS biology

Article Title: Variable combinations of specific ephrin ligand/Eph receptor pairs control embryonic tissue separation.

doi: 10.1371/journal.pbio.1001955

Figure Lengend Snippet: Figure 6. Schematic representation of the three boundaries forming during Xenopus gastrulation and simplified diagrams of the preferential ephrin–Eph pairs. (A) Dorsal ectoderm–mesoderm boundary. (B) Ventral ectoderm–mesoderm boundary. (C) Notochord–paraxial mesoderm boundary. The top drawings represent sagittal sections of the corresponding stages and highlight the two tissues forming the corresponding boundary. At stage 14, the boundaries between the notochord (no) and the paraxial mesoderm (pm) form perpendicular to the plane of the section. Cross-section is shown in insert. ar, archenteron roof; ne, neuroderm. In the lower diagrams, the position of the boxes representing each molecule symbolizes its general distribution: A box placed on one side of the boundary corresponds to strong asymmetric distribution. Weakly asymmetric or homogenous distributed molecules are drawn overlapping the boundary. The double arrows link the functional pairs. The patterns at the dorsal and the ventral boundaries are similar, with two prominent differences: EphrinB1 shifts from equal in the two dorsal tissues to more mesodermal in the ventral side. EphB4, on the contrary, shifts from mainly ectodermal to equally distributed. At the end of gastrulation, the dorsal mesoderm experiences significant changes: Compared to the earlier dorsal mesoderm, the notochord (no) keeps EphA4 and ephrinB2, but the paraxial mesoderm (pm) loses EphA4 and acquires ephrinB3 and EphB4, which were until then typically ectodermal. Note that the major asymmetries concern EphA4 and B4; the other components are only slightly enriched in one or the other sides of the boundary. doi:10.1371/journal.pbio.1001955.g006

Article Snippet: Recombinant Proteins Recombinant mouse ephrinB2-Fc, ephrinB1-Fc, human ephrinB3, and mouse EphB4-Fc and EphA4-Fc (R&D Systems) comprising the extracellular domain of ephrin/Eph fused to Cterminal 66 histidin-tagged Fc region of human IgG were preclustered by 1 h incubation with anti-human Fc antibody (Jackson ImmunoResearch Laboratories) at a 1:2 ratio in MBSH [4] before application.

Techniques: Functional Assay