Schmidt J et al. (1995), Drosophila short gastrulation induces an ectopi...

XB-ART-18964

Development 1995 Dec 01;12112:4319-28.

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Drosophila short gastrulation induces an ectopic axis in Xenopus: evidence for conserved mechanisms of dorsal-ventral patterning.

Schmidt J , Francois V , Bier E , Kimelman D .

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The Spemann organizer has long been recognized as a major source of patterning signals during the gastrula stage of amphibian embryogenesis. More recent evidence has suggested that the ventral side of the embryo also plays an important role in dorsal-ventral patterning during gastrulation through the action of signaling factors such as BMP-4. Bmp-4 is closely related to the Drosophila decapentaplegic (dpp) gene, and like Bmp-4, dpp is excluded from the neurogenic region. Recently we showed that Bmp-4 functions in an analogous role to that of dpp in Drosophila, suggesting that the mechanism of dorsal-ventral patterning in Xenopus and Drosophila embryos may be conserved. To further test this hypothesis, RNA of the Drosophila short gastrulation (sog) gene was injected into Xenopus embryos, since sog has been shown genetically to be an antagonist of dpp function. Overexpression of sog RNA in Xenopus dorsalizes the embryo by expanding neurogenic and dorsal paraxial tissue. When ectopically expressed on the ventral side of the embryo, sog induces a partial secondary axis. In addition, sog partially rescues embryos ventralized by ultraviolet irradiation. Since sog induces many similar changes in gene expression to that caused by truncated BMP receptors, we suggest that sog functions in part by opposing BMP-4 signaling. The recent identification of a possible Xenopus sog homolog, chordin, in conjunction with these results supports the hypothesis that dorsal-ventral patterning mechanisms are conserved between these two species.

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Species referenced: Xenopus
Genes referenced: bmp4 chrd dspp egr2 gsc myod1 not tbxt wnt8a

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	Fig. 1. Drosophila sog induces a secondary axis in Xenopus embryos. The top three embryos were injected ventrally with sog RNA. The top two embryos have secondary tails and the third embryo’s secondary axis extends from the primary axis. The bottom embryo is an uninjected control embryo. All embryos are at stage 38.
	Fig. 2. Injection of sog RNA alters early gene expression. (A,B) Vegetal view of gsc expression in stage 10 embryos. Dorsal is up. (A) Uninjected embryo. (B) Embryo injected ventrally with 6 ng sog RNA. Gsc expression is only weakly enhanced on the ventral side. (C,D) Vegetal view of Xnot expression in stage 11 embryos. Dorsal is up. (C) Uninjected embryo. (D) Embryo injected ventrally with 6 ng sog RNA. Xnot expression is enhanced on the ventral side, however only in the non-notochordal domain of expression. (E,F) MyoD expression in mid-neurula-stage embryos. Anterior is up. (E) Uninjected embryo, dorsal view. (F) Embryo injected ventrally with 4 ng sog RNA, dorsolateral view. An additional domain of MyoD expression extends from the primary paraxial domain of expression (arrowhead). No midline clearing is present in this secondary axial expression. (G,H) Xwnt-8 expression in mid-neurula-stage embryos. Dorsal is up. (G) Uninjected embryo, dorsoposterior view. Xwnt-8 expression is found ventrally and laterally, and adjacent to the closed blastopore. (H) Embryos injected ventrally with 6 ng sog RNA, posterior view. Ventral Xwnt-8 expression has been eliminated and the lateral expression has been extended along the sides of the new secondary axis (2°). The primary axis is up (1°).
	Fig. 3. Secondary axes induced by sog RNA lack notochords. (A) Posterior view of Xbra expression in an uninjected mid-neurula-stage embryo. Xbra is expressed both posteriorly, surrounding the closed blastopore, and in the presumptive notochord. Dorsal is up. (B) Posterior view of a mid-neurula-stage embryo injected ventrally with 6 ng sog RNA. Posterior Xbra expression remains (although slightly altered) and the notochord expression along the primary axis (1°) is present. No axial expression of Xbra is seen in the secondary axis (2°). Dorsal is up. (C) Transverse section near the tail of a stage 38 embryo (Fig. 1, third embryo from the top). The vacuolated notochord (no) is visible in the primary axis, as are the somites (s) and neural tube (ne). The secondary axis (to the lower right) has no notochord, but instead contains neural tissue (ne) as well as somitic tissue (s) that is fused across the midline of the secondary axis, and with the somitic tissue on one side of the primary axis (arrowhead). (D) Transverse section of a stage 38 embryo in which the secondary axis is opposite and distinct from the primary axis (embryo similar to Fig. 1, top embryo). The secondary axis is on the ventral side of the embryo (bottom of picture), and does not contain a notochord. Labeling as in C.
	Fig. 4. sog partially rescues uv-irradiated embryos. (A) DAI scores obtained in uninjected (top), sog RNA-injected (middle), and ea-sog-injected (bottom) embryos. Each chart shows the range of values between completely ventralized (DAI=0) and normal (DAI=5) embryos, and the percentage of embryos with each DAI value. (B-D) Stage 34 embryos, anterior is to the left. (B) Uninjected embryo. (C). Uv-irradiated embryo injected with 4 ng wild-type sog RNA (DAI=2). (D) Uv-irradiated embryo injected with 4ng ea-sog (DAI=2). (E) Uninjected, uv-irradiated embryo (DAI=0). Closed blastopore is to the right.
	Fig. 5. Gene expression in uv-irradiated embryos rescued by sog RNA injection. (A-C) MyoD expression in early neurula-stage embryos. (A) Uninjected embryo. (B) Uv-irradiated embryo injected with sog RNA. MyoD is expressed in the induced axis. There is no midline clearing of expression. (C) Uninjected, uv-irradiated embryo. (D-F) Xbra expression in mid-neurulastage embryos. (D) Uninjected embryo. (E) Uv-irradiated embryo injected with sog RNA. Posterior Xbra expression is present, however no notochordal Xbra is present in the induced axis. (F) Uninjected, uv-irradiated embryo. Xbra is expressed around the blastopore, but there is no axial Xbra expression. (G,H) Hairy II expression in midneurula- stage embryos. (G) Uninjected embryo. (H) Uv-irradiated embryo injected with sog RNA. Hairy II expression is found in a stripe around the neural plate, and demonstrates a reduction in the size of the anterior neural region. No dorsal midline expression of Hairy II is observed. (I) Uninjected, uv-irradiated embryo. In the above panels, the uninjected and sog RNA-injected embryos are shown in a dorsal view with anterior up, whereas the uninjected, uv-irradiated embryos are shown in a posterior view. (J) Krox- 20 expression in a stage 27 control embryo (top embryo; black arrowhead marks the two stripes of expression in rhombomeres 3 and 5) and in a uvirradiated embryo injected with sog RNA (bottom embryo). In this embryo, both stripes of Krox-20 expression were observed (white arrowhead). (K) Cement gland was present in some uvirradiated embryos injected with sog RNA (bottom embryo, black arrowhead). In this embryo, only a single stripe of Krox-20 expression was observed.
	Fig. 6. uvirradiated, soginjected embryos do not contain a notochord. Transverse section through a stage 37/38 uv-irradiated embryo injected with sog RNA at the 8-cell stage. The somites (s) are fused across the dorsal midline beneath the neural tissue (ne).
	Fig. 7. Ubiquitous expression of sog dorsalizes the embryo. (A,B) Lateral view of MyoD expression at the mid-neurula stage in (A) an uninjected embryo and (B) an embryo injected with sog RNA in each blastomere at the 4-cell stage. Anterior is to the left. MyoD expression extends around the entire ventroposterior region of the injected embryo. (C,D) Transverse section of an embryo similar to the one in B. (C) Anterior section. MyoD is expressed in the paraxial mesoderm flanking the presumptive notochord (no). (D) Posterior section. MyoD is expressed circumferentially, except in the dorsal midline, the region of the presumptive notochord (no). (E) Dorsal view of late neurula-stage embryos. Top embryo is an uninjected control embryo. Bottom three embryos were injected in each blastomere at the 4-cell stage with ea-sog RNA (identical results were observed with wild-type sog RNA). The posterior region of the embryo was considerably elongated and narrowed. Anterior is to the left. (F-H) Hairy II expression at the late neurula stage in an uninjected embryo (F) and in an embryo injected with sog RNA in each blastomere at the 4-cell stage (G,H). Dorsal view in F, lateral view in G, and ventral view in H; anterior is to the left in all three panels. The stripe of Hairy II expression bordering the neural plate in the injected embryo continues around to the ventral side (arrowhead). (I) Transverse section of a sog-injected embryo stained for HairyII as in G and H, cut at the level of the stripe that circumscribes the embryo. HairyII expression was found to continue around the ventral side of the embryo in the ectodermal layer of cells. Expression of Hairy II in the floorplate was retained (arrowhead). (J) Top: Control stage 38 embryo; bottom: stage 38 embryo injected with sog RNA (third embryo from the top in E). A normal ventral tail fin has not formed.
	Fig. 8. BMP-4 prevents the axis-inducing capabilities of sog in uvirradiated embryos. Top: uninjected embryo; middle: uv-irradiated embryo injected with 5 ng sog RNA (DAI=2); bottom: uv-irradiated embryo co-injected with 5 ng sog RNA and 300 pg Bmp-4 RNA (DAI=0). Uninjected, uv-irradiated embryos were similar to the bottom injected embryo and had DAI values of 0 (data not shown). All embryos are at the equivalent of stage 37.