Cells Undergo Mesenchymal-to-Epithelial Transition at Somite Boundaries

(A–L) Dorsal views of the left-sided paraxial mesoderm of embryos labeled with Bodipy ceramide (which reveals cell morphology; [A], [E], and [I]) or with Bodipy 505-515 (which reveals nuclear position, [C], [G], and [K]) or immunostained for β-catenin ([B], [F], and [J]) or for γ-tubulin (which labels centrosomes) and stained with phalloidin (which labels actin) ([D], [H], and [L]). Anterior is oriented toward the top. (A–D) Cells at somite boundaries in wild-type embryos. The arrowheads point to the intersomitic boundary. (E–H) Cells in the presomitic mesoderm (PSM) of wild-type embryos. The arrows point to epithelial adaxial cells in which centrosomes are apically localized (H), as also seen in epithelial cells at somite boundaries (D). Centrosomes are randomly positioned in other PSM cells. (I–L) Cells in the somitic mesoderm of fss^-/- embryos. n, notochord; ac, adaxial cells. The scale bars represent 10 μm.

Expression of Eph Family Members in the Paraxial Mesoderm of Wild-Type and fss^-/- Embryos

Dorsal views of the paraxial mesoderm of 8-somite-stage wild-type and 10-somite-stage fss^-/- embryos and schematics with anterior oriented toward the top. The arrowheads indicate the position of the most recently formed intersomitic boundary.

(A and B) Living wild-type and fss^-/- embryos labeled with Bodipy ceramide. In the wild-type embryo, the positions of the last two somites formed (SII, SI), the forming somite (S0), and the two presumptive somites in the PSM (S-I, S-II) are indicated. The arrowhead points to the intersomitic boundary.

(C–J) Expression of (C and D) ephA4, (E and F) ephrin-B2b, (G and H) ephrin-B2a, and (I and J) ephrin-A1 in wild-type (top row) and fss mutant (bottom row) embryos in the region of the paraxial mesoderm shown in (A) and (B).

(C′–J′, K, and L) Schematics summarizing expression of Eph family members in the paraxial mesoderm of wild-type and fss^-/- embryos.

nt, neural tube; n, notochord. The scale bars represent 30 μm.

Eph/Ephrin Signaling Restores Morphologically Distinct Boundaries in fss^-/- Embryos

(A–E and A′–E′) (A–E) DIC and fluorescence overlays and (A′–E′) DIC images of the paraxial mesoderm of fss^-/- hosts into which wild-type (wt) or fss^-/- cells expressing various GFP-tagged reagents (green labeling in [A], [B], [D], and [E]) or containing rhodamine dextran (RD, red labeling in [C]) have been transplanted. Reagents are indicated at the bottom of the panels. (C′) ephA4 expression (blue) is absent from the transplanted cells. The arrowheads point to morphologically distinct boundaries formed at the interface between donor and host cells. nt, neural tube.

Boundaries Restored by Eph/Ephrin Signaling Are Maintained during Muscle Differentiation

(A–E) Lateral views of (A) wild-type and (B–E) fss^-/- somitic muscles immunostained for myosin (green). Anterior is oriented toward the left. (C), (D), and (E) show transplanted (C and E) wild-type or (D) fss^-/- cells (red) expressing (C and D) full-length or (E) truncated, dominant-negative EphA4. The arrowheads point at morphologically distinct furrows. The arrows point at fss^-/- muscle fibers that span adjacent segments.

Eph/Ephrin Signaling Rescues Epithelialization of Cells at Morphologically Distinct Boundaries

(A–L) (A, E, and I) Confocal images showing Bodipy ceramide-labeled somitic mesoderm of fss^-/- host embryos (green) containing rhodamine dextran-labeled donor cells (red). (B, F, and J) Confocal images of β-catenin immunolocalization (green) in transplanted rhodamine dextran-labeled cells (red) in fss^-/- host embryos. The arrows point to the basal surfaces of the transplanted cells at the interface at which morphologically distinct boundaries form (visible with DIC optics, not shown). In (B) and (J), β-catenin is reduced on the basal surfaces of these cells. (C, G, and K) Confocal images showing Bodipy 505-515-labeled somitic mesoderm of fss^-/- host embryos (green) containing rhodamine dextran-labeled donor cells (red). The white arrowhead points to nuclei localized at the basal pole of host fss^-/- cells, adjacent to the boundaries created between donor and host cells. (D, H, and L) Confocal images showing phalloidin-labeled somitic mesoderm of fss^-/- host embryos (red) containing CFP-labeled donor cells (blue) and immunostained for γ-tubulin (green). The white arrows point to centrosomes. These are localized at the apical pole of host fss^-/- cells adjacent to the boundary created between donor and host cells in (D) but are randomly positioned in (H) and (L). Donor cells are wild-type cells expressing (A–D) full-length or (E–H) truncated, dominant-negative EphA4 or fss^-/- cells expressing (I–L) full-length EphA4. n, notochord; ac, adaxial cells. The scale bars represent 10 μm.

Cells within the Core of the PSM Are Mesenchymal, and Epithelialization of Somite Boundary Cells Occurs Concomitantly with Boundary Formation

(A–C) Dorsal views of the PSM of wild-type embryos; anterior is oriented toward the top. Embryos have been stained with (A) Bodipy ceramide or (B) phalloidin (red) or (C) Bodipy 505-515. In (B), the embryo has been immunostained for γ-tubulin (green) to show centrosomes. The arrows point to presomitic cells that have epithelial morphology adjacent to the (A) neural tube, the (B) notochord, and the (C) lateral plate mesoderm. Other cells in the PSM have mesenchymal morphology. The arrowheads indicate the most recently formed or forming intersomitic boundary in each panel. Cells on either side of this boundary only begin to acquire epithelial morphology concomitant with boundary formation. Full maturation of epithelial morphology (see Figures 1A–1D in the main text) occurs after boundary furrow formation. lpm, lateral plate mesoderm; nt, neural tube; n, notochord. The scale bar represents 15 μm.

EphA4 Is Unlikely to Induce Boundary Formation by Restoring “Anterior Segmental Identity” to Donor Cells
(A–D) Expression of (A and B) papc and (C and D) deltaD in the paraxial mesoderm of 11-somite-stage (A and C) wild-type and (B and D) fss^-/- embryos; anterior is oriented toward the top. Expression in the anterior region of the prospective somites is lost for papc and is reduced for deltaD in fss mutants. The rectangles indicate the areas of the paraxial mesoderm of fss^-/- embryos where transplanted cells are shown in (E)–(H).
(E, F, E′, and F′) (E and F) DIC and (E′ and F′) DIC and fluorescent overlays of the paraxial mesoderm of fss^-/- embryos in which wildtype cells containing rhodamine dextran (red) were transplanted. The arrows indicate restoration of expression of (E′) papc and high levels of (F′) deltaD expression in transplanted wild-type cells. These data show that the wild-type Fss protein is sufficient to cell-autonomously induce papc and high levels of deltaD expression in wild-type cells within fss^-/- hosts. Despite expression of these markers of “anterior segmental identity” in donor cells, ephA4 expression is absent (see Figure 3C in the main text) and boundaries are not induced in the somitic mesoderm (see Figure 3B in the main text).
(G, H,G′, and H′) (Gand H) DIC and (G′ and H′) DIC and fluorescent overlays of the paraxial mesoderm of fss^-/- embryos in which EphA4- expressing fss^-/- cells containing rhodamine dextran (red) were transplanted. (G′ and H′) Expression of papc or enhanced deltaD expression is not observed in EphA4-expressing fss^-/- cells. Therefore, restoration of EphA4 activity in fss^-/- cells has no obvious effect on papc or deltaD expression. Despite the absence of these markers of “anterior segmental identity” in donor cells, boundaries are induced in the paraxial mesoderm (see Figure 3D in the main text). These data suggest that EphA4 is acting downstream of the events that specify anterior character in the PSM.
(I and J) Dorsal views of the paraxial mesoderm of a (I) wild-type and a (J) dominant-negative XDeltaSTU-injected embryo. The antimorphic Delta construct is able to disrupt somite boundary formation (asterisk; [S4]). (K and K′) (K) DIC and (K′) DIC and fluorescent overlay of the paraxial mesoderm of an fss^-/- embryo where wild-type cells expressing EphA4 and XDeltaSTU (green) have been transplanted. The arrows point to the morphologically distinct boundary at the interface between donor and host cells (seen in nine of ten cases). In fss^-/- cells, expression of Notch pathway genes is disrupted [S5] and the XDeltaSTU construct is likely to perturb Notch pathway signaling in the transplanted wild-type cells. In this experiment, Eph/Ephrin signaling still induces boundary formation despite the likely disruption to Notch-Delta signaling (implying that Eph/Ephrin signaling is unlikely to induce boundaries by restoring Notch/Delta signaling interfaces).
From these results, we conclude that the apposition of ephA4-expressing cells and ephrin-expressing cells is sufficient to restore morphologically evident boundaries without requiring the full restoration of anterior segmental identity (or Notch/Delta signaling) in EphA4-expressing donor cells. We therefore predict that EphA4/Ephrin signaling is most likely functioning at the final step of boundary formation.

Epithelialization of Host Cells May Be Independent of Ephrin Reverse Signaling and Independent of Cell-Nonautonomous Activity of Papc
(A–D) Confocal images showing Bodipy 505-515-labeled somitic mesoderm of fss^-/- host embryos (green) containing rhodamine dextranlabeled donor cells (red). Donor cells are wild-type cells expressing (A) EphA4, (B) fss^-/- cells expressing EphA4 and Papc, (C) wild-type cells expressing EphA4 and dominant-negative Papc, or (D) wild-type cells expressing EphA4 and containing Papc morpholino. In (A), fss^-/- host cells are expressing truncated, dominant-negative Ephrin-B2a-eGFP. The arrows point to basally localized nuclei of fss^-/- host cells at boundaries created between donor and host cells. The scale bar represents 5 μm.
(E) Summary of experiments investigating the role of Ephrin-B2a and Papc upon epithelialization.
(F and G) Controls for the use of the papc morpholino (see the Supplemental Experimental Procedures).
(H and I) Dorsal views of somitic mesoderm of a wild-type embryo and an embryo injected with papc morpholino. Somite boundaries are not properly formed, and myoD expression is downregulated in the somites of Papc morphant embryos.

EphA4 Is Removed from the Basal Cell Membrane during Somite Boundary Formation
(A–F and A′–F′) (A–F) Confocal fluorescence and DIC overlays and (A′–F′) DIC images of EphA4eGFP-expressing cells (green) in the paraxial mesoderm of a (A–C) wild-type and a (D–F) fss^-/- embryo during somite furrow formation. The localization of EphA4eGFP is monitored through time (indicated in minutes at the bottom left). The arrowheads point at the interface between EphA4-expressing cells and nonexpressing cells, where the furrow of de-adhesion is forming and EphA4eGFP protein is being cleared. These observations suggest that Eph/Ephrin signaling may be terminated after boundary formation by the removal of receptor or receptor/ligand signaling complexes in both wild-type intersomitic boundaries and boundaries restored in fss^-/- embryos. The scale bar represents 10 μm.