Eph signaling is required for segmentation and differentiation of the somites

Abstract

Somitogenesis involves the segmentation of the paraxial mesoderm into units along the anteroposterior axis. Here we show a role for Eph and ephrin signaling in the patterning of presomitic mesoderm and formation of the somites. Ephrin-A-L1 and ephrin-B2 are expressed in an iterative manner in the developing somites and presomitic mesoderm, as is the Eph receptor EphA4. We have examined the role of these proteins by injection of RNA, encoding dominant negative forms of Eph receptors and ephrins. Interruption of Eph signaling leads to abnormal somite boundary formation and reduced or disturbed myoD expression in the myotome. Disruption of Eph family signaling delays the normal down-regulation of her1 and Delta D expression in the anterior presomitic mesoderm and disrupts myogenic differentiation. We suggest that Eph signaling has a key role in the translation of the patterning of presomitic mesoderm into somites.

Figure 1

(A) Amino acid sequence of zebrafish ephrin-A-L1. (B) Alignment of the protein sequences of zebrafish ephrin-B2 with mouse and human ephrin–B2. Identical residues shown by shading. (C) Amino acid sequence of zebrafish EphB-rtk8. Signal sequences are underlined; probable transmembrane domains are double underlined; putative GPI linkage signal is overlined; conserved cysteine residues are boxed.

Figure 2

Expression of ephrin-B2 (A–D), ephrin-A-L1 (E–H), and EphA4 (I,J). Embryos were hybridized with digoxigenin-labeled antisense RNA probes. Dorsal views (except E lateral view), anterior is to the left (except in A, E,F where anterior is to the top). ephrin-B2 expression. (A) 50% epiboly. Transcripts are detected around the germ ring. (B) Bud stage. Expression is seen in presumptive rhombomere 4 and in three stripes in the presomitic mesoderm (arrow). (C) Two somites. Transcripts are detected in rhombomere 4, in the somites, and in two stripes in the anterior presomitic mesoderm (arrows). (D) Six somites. Expression is seen in rhombomeres 4 and 7, in a posterior domain of the formed somites and in a posterior domain of the most anterior presumptive somites (lines mark the somite boundaries). ephrin-B2 expression is restricted to a lateral domain within the somites as these mature. Ephrin-A-L1 expression. (E) 70% epiboly. Transcripts are detected in the epibolizing margin and the hypoblast, expression is stronger on the ventral side of the embryo (arrowheads mark the edges of the expression domain). (F) 90% epiboly. Expression is seen in the epibolizing margin, more strongly around the tailbud, and in cells on either side of the presumptive notochord. (G) One somite. Transcripts are detected in the tailbud, adaxial cells (arrowhead), and in three weak stripes in the formed somite and anterior presomitic mesoderm (arrows). (H) Six somites. Expression is seen in the tailbud and adaxial cells (arrowhead). Ephrin-A-L1 is expressed throughout the somites and anterior presomitic mesoderm, more strongly in the posterior region of each segment (lines mark the somite boundaries). Transcripts are also detected in individual paired cells in the neural tube (white arrow). EphA4 expression. (I) One somite. Transcripts are detected in the notochord and adaxial cells, and in one stripe in the formed somite (arrow). (J) Three somites. Expression is seen in the notochord and adaxial cells (arrowhead), and in an anterior domain of the somites and most anterior presumptive somites (lines mark the somite boundaries). (D) dorsal; (nc) notochord; (pn) presumptive notochord; (r) rhombomere; (tb) tailbud; (V) ventral; Bars, 100 μm.

Figure 3

Analysis of the binding characteristics of Eph family proteins in vitro and in situ using alkaline phosphatase-tagged proteins. (A–D) The in vitro binding characteristics of ephrin-A-L1 (A,B) and ephrin-B2 (C,D). Ephrin–B2-AP and ephrin-A-L1–AP were put on Cos cells that had been transfected with EphA4 (A,C) or EphB–rtk8 (B,D). Staining of cells indicates that the ligand binds to the expressed receptor. Both ephrin-A-L1 and ephrin-B2 can bind to EphA4 (A,C), but only ephrin-B2 can bind to EphB–rtk8 (B,D). The presence of class A and B ephrins within the somites was investigated in situ using an AP-tagged protein representative for each of the receptor classes. Dorsal views, anterior is to the left (except in E where anterior is to the top). EphA–rtk6–AP binding to 95% epiboly (E) and six-somite (F) embryos is shown, and EphB–rtk8–AP binding to two somite embryos (G). The resulting binding patterns look very similar to the expression patterns of ephrin-A-L1 and ephrin-B2, respectively, at comparable stages (Fig. 2C,F,H). Bars, 100 μm.

Figure 4

Effect of disruption of Eph family signaling on somite formation and differentiation. Embryos were assessed at stages between 4 and 14 somites. Dorsal views, anterior is to the top (except in C and D where anterior is to the left). (A,B) Live embryos injected with ephrin-B2-WT. A camera lucida drawing adjacent to each photo shows the somite boundaries. Somites fail to form (A) or are abnormally shaped and not paired across the midline (B) in affected embryos. (C,D) Confocal microscope images of the somites of phalloidin-stained embryos. (C) A wild-type embryo. The intersomitic furrows (white arrow) and columnar-shaped epithelial cells on either side can be seen. (D) An experimental embryo injected with ephrin-B2–WT. Incorrectly positioned boundaries can be seen in the injected embryos (white arrows), these have columnar-shaped epithelial cells on either side. (E,F) Experimental embryos hybridized to a myoD RNA probe (purple). (E) An embryo injected with ephrin-B2–WT; (F) an embryo injected with ephrin-A-L1–Tru. Segmental myoD expression is either lost (E) or disrupted (F) on the injected side. The blue color in F is reacted β-galactosidase derived from coinjected RNA. (G) An embryo injected with ephrin-B2–WT reacted with EphB–rtk8–AP to show the presence and localization of ectopic protein. Ectopic ephrin-B2 is found in regions of the somites that are disrupted. Bars, 100 μm.

Figure 5

Visualization of the rescue experiments using myoD expression. Embryos were hybridized with a digoxigenin-labeled antisense RNA myoD probe. Embryos are between 10 and 14 somites. Dorsal views, anterior is to the top. Blue staining marks the localization of coinjected β-galactosidase RNA. Embryos shown were injected with (A) ephrin-B2–Tru, (B) EphA4 and ephrin-B2–Tru, (C) ephrin-A-L1–Tru, and (D) EphB–rtk8 and ephrin-A-L1–Tru. Disrupted segmental myoD expression is seen on the injection of truncated forms of ephrin-B2 (A) and ephrin-A-L1 (C). Coinjection of EphA4 with ephrin-B2–Tru rescues the myoD expression (B); whereas coinjection of EphB–rtk8 has no effect on the ephrin-A-L1–Tru phenotype (D). Bars, 50 μm.

Figure 6

Effect of the disruption of Eph family signaling on the segmental pattern in the presomitic mesoderm and on patterning of the somites. Dorsal views, anterior is to the top. Blue staining (E,F,H,K,O) marks localization of coinjected β-galactosidase RNA. (A) Wild-type her1 expression in a two-somite embryo. (B,C) her1 expression in two-somite embryos injected with ephrin-B2–WT. The anterior stripes of her1 expression are either disrupted (arrows in B), or expression is maintained anteriorly such that extra stripes are seen (arrows in C), on the injected side. (D) Wild-type expression of her1 in a 12-somite embryo. (E,F) her1 expression in 12-somite embryos injected with ephrin-B2–WT and EphB–rtk8DN, respectively. Once again the anterior stripes of her1 expression are either disturbed (injected side in E), or expression is not switched off correctly, resulting in the presence of an extra stripe anteriorly (arrows in F). (G,J) The dynamic nature of Delta D expression in 12-somite control embryos, either one (G) or two (J) stripes are seen in the anterior presomitic mesoderm. (H,I,K) Delta D expression in embryos injected with ephrin-B2–WT, ephrin-B2–Ic, and EphB–rtk8DN, respectively. Delta D expression is incorrectly regulated in injected embryos, resulting in disrupted stripes of expression (arrow in H), or persistence of expression such that extra stripes are seen anteriorly on the injected side (arrows in I and K). (L)Wild-type expression of par1 in a 12-somite embryo. (M) par1 expression in a 12-somite embryo injected with ephrin-B2–WT. Par1 is not restricted to its usual segmental pattern on the injected side where somites have failed to form. (N) Wild-type FGF-8 expression in a 12-somite embryo. (O) FGF-8 expression in a 12-somite embryo injected with ephrin-B2–Ic. FGF-8 is expressed in an anterior domain of abnormally shaped somites in the injected embryo (arrows in O). (A–C) Bar, 50 μm; (D–O) bar, 100 μm.

Figure 7

Relationship of ephrin-B2, EphA4, and her-1 expression in the anterior presomitic mesoderm and posterior somites. Embryos were hybridized with digoxigenin- and fluorescein-labeled antisense RNA probes. Dorsal views, anterior is to the top, of six-somite (A,B,C,D,E,G) and five-somite (F) stage embryos. (A) EphA4 (blue) is expressed in an anterior domain of the formed somites and most anterior presumptive somites, whereas ephrin-B2 (red) is expressed in a posterior domain of these segments (lines mark the two most posterior somite boundaries). In the anterior presomitic mesoderm a row of nonexpressing cells is found at the position where the next somite boundary will form (arrow). (B,D,E) ephrin-B2 (blue) and her-1 (red) expression. Her1 expression is lost in an anterior to posterior direction in the most anterior presumptive somite (line marks the last formed somite boundary), which corresponds with the posterior expansion of the ephrin-B2 expression domain in the same region, such that cells expressing both genes are seen (arrow in B). Expression of ephrin-B2 is seen between the two most anterior stripes of her1 expression (arrowhead in B). (C,F,G) EphA4 (blue) and her-1 (red) expression. EphA4 and her1 expression are not seen juxtaposed in the anterior presomitic mesoderm (line marks the last formed somite boundary). Low level expression of EphA4 is also seen throughout the paraxial mesoderm (stars in F and G). (a) Anterior; (nc) notochord; (p) posterior; bars, 50 μm.

Figure 8

Diagrammatic representation of the relationship between her1, EphA4, and ephrin-B2 in a single file of cells along the anteroposterior axis of the paraxial mesoderm during somitogenesis. The three columns represent the cells over a period of time during which two somites are added. her1 expression is shown in hatched circles, EphA4 expression in light shading, and ephrin-B2 expression in dark shading. Where her1 and EphA4 expression overlap lightly shaded hatched circles are used, and where her1 and ephrin-B2 expression overlap darkly shaded hatched circles are used.