Christian JL and Moon RT (1993), Interactions between Xwnt-8 and Spemann organiz...

XB-ART-22961

Genes Dev 1993 Jan 01;71:13-28. doi: 10.1101/gad.7.1.13.

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Interactions between Xwnt-8 and Spemann organizer signaling pathways generate dorsoventral pattern in the embryonic mesoderm of Xenopus.

Christian JL , Moon RT .

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This study analyzes the hierarchy of signals that spatially restrict expression of Xenopus Xwnt-8 to mesodermal cells outside of the Spemann organizer field and examines the potential role that endogenous Xwnt-8 may play in dorsoventral patterning of the embryonic mesoderm. The effects of ectopic introduction of a Nieuwkoop center-like activity or of ectopic expression of goosecoid, on the distribution of endogenous Xwnt-8 transcripts were analyzed. The results of these studies are consistent with the hypothesis that maternally derived signals from the Nieuwkoop center function to positively regulate expression of the homeo box gene goosecoid in Spemann organizer cells, leading to a subsequent repression of Xwnt-8 expression in these cells. This exclusion of Xwnt-8 from cells of the organizer field may be important for normal dorsal development, in that ectopic expression of Xwnt-8 in organizer cells after the midblastula stage, by injection of plasmid DNA, ventralizes the fate of these cells. This is distinct from the previously observed dorsalizing effect of Xwnt-8 when expressed prior to the midblastula stage by injection of RNA. The effects of plasmid-derived Xwnt-8 on isolated blastula animal cap ectoderm were also analyzed. Expression of Xwnt-8 in animal pole ectoderm after the midblastula stage ventralizes the response of dorsal animal pole cells to activin and allows naive ectodermal cells to differentiate as ventral mesoderm in the absence of added growth factors. Collectively, these data are consistent with the hypothesis that Xwnt-8 plays a role in the mesodermal differentiation of ventral marginal zone cells during normal development. Furthermore, endogenous Xwnt-8 may ventralize the response of lateral mesodermal cells to dorsalizing signals from the organizer, thus contributing to the graded nature of the final body pattern.

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Species referenced: Xenopus
Genes referenced: cat.2 gsc myc wnt8a

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	Figure 1. Xwnt-8 is expressed in all mesodermal cells except those of the Spemann organizer field. Whole-mount in situ hybridization studies in Xenopus embryos. (A) Early gastrulae (stage 10) hybridized with Xwnt-8 antisense (lower embryo) and sense (upper embryo) probes. Arrows denote the position of the dorsal lip of the blastopore. (B) Mid-gastrulae (stage 11, lower embryo) and late gastrulae (stage 12, upper embryo). Arrows denote lateral cells that express Xwnt-8 and are undergoing convergent extension movements toward the animal pole and dorsal midline. (C) Transverse section of a stage-20 neurula showing Xwnt-8 signal in lateral and ventral plate mesoderm. (Inset) Characteristic staining observed when embryos are treated for a longer period with proteinase K before hybridization. Note that the signal is more intense, but ectodermal surface cells are absent and lateral staining is lost. (D) Xwnt-8 expression in a UV-irradiated gastrula-stage embryo. (E-H) Early gastrulae (stage 10). Arrows represent the position of dorsal blastopore lips. Illustrated are Xwnt-8 expression in embryos injected ventrally with Wnt-1 (E) or goosecoid RNA (H) during cleavage stages and goosecoid expression in unperturbed (F) and Wnt-l-injected (G) embryos.
	Figure 2. Differential induction of Xwnt-8 and goosecoid expression by activin in ectoderm isolated from prospective dorsal or ventral halves of blastulae. Ectoderm isolated from the prospective dorsal half (D), ventral half (V), or whole (W} animal region of blastulae was cultured in the presence ( + ) or absence ( - ) of 10 ng/ml of activin A as indicated above each lane. RNA prepared from 10 explants in each group at control stage 11 was subjected to Northern blot analysis. The filter was successively hybridized to Xwnt-8 ar~t goosecoid riboprobes, as indicated at left, and then to an EF- 1 ~ cDNA probe to determine equivalence of loading.
	Figure 3. Expression of Xwnt-8 and CAT transcripts following injection of CSKA-X8 or CSKA-CAT DNA into Xenopus embryos. (A) RNA was isolated from injected or uninjected embryos at cleavage (stage 3), blastula (stage 9), gastrula (stage 11 ), neurula (stage 21), or tailbud (stage 32) stages, as indicated at the top of A [(N/F) Nieuwkoop and Faber 1967] and subjected to gel blot analysis. The filter was successively hybridized with the probes indicated at left. (B) Gastrulae (stage 10; lanes 1-6) or neurulae (stage 21; lanes 7-14) were dissected as illustrated. Northern blots containing RNA from the region indicated above each lane were successively hybridized with the probes indicated at left. The filter was exposed to film for 10 hr, or for 6 hr, to obtain the Xwnt-8 signals shown for lanes 7-14, and 1-6, respectively. (D)Dorsal; (V) ventral; (W) whole; (H) head. (C) Whole-mount in situ hybridization of Xwnt-8 probes to stage-11 gastrulae that had received dorsal injections of CSKAX8. Solid arrows denote signal from endogenous Xwnt-8 transcripts in ventrolateral maginal zone ceils; open arrows indicate staining of dorsal lip cells from plasmid-derived Xwnt-8. (A) Whole gastrulae; (B) sagittal section through a representative embryo.
	Figure 4. Deletion of anterior structures following dorsal injection of CSKA-X8 into Xenopus embryos. CSKA-CAT (A,C,E) or CSKA-X8 {B,D,F) DNA was injected as described in the text. (A,B) Whole-mount in situ hybridization of goosecoid probes to early gastrulae {stage 10 1/2). (C-F) Photographs of embryos fixed at the tailbud (stage 34, C,D) or tadpole (stage 45, E,F) stage.
	Figure 5. Injection of CSKA-X8 DNA into dorsal blastomeres of Xenopus embryos results in loss of forebrain and notochord. (A-F) Transverse sections taken at a comparable distance from the anterior end of CSKA/CAT (A-C}- or CSKA/X8 (D-F)-injected embryos. (G,H) Sagittal sections through CSKA/CAT (G)- or CSKA/X8 (H)-injected embryos. Abbreviations are as follows: (pros) Prosencephalon (forebrain); (soml somitic mesoderm (muscle); (not) notochord; (sc) spinal cord; (fp) floor plate cells of the ventral spinal cord; (rhom) rhombencephalon (hindbrain); (ot} otic vessicle (ear anlage); (vsc) ventral wall of the spinal cord. Bar in A represents 400 t~m in all panels except for C and F, where it represents 150 ~m. (/, J) Notochord immunostaining in stage 28 (I) or stage 21 (J) neurulae using monoclonal antiserum Tor70. Open arrows denote normal notochord staining in CSKA/CAT-injected (upper) embryos; solid arrows in I indicate anterior and (faint) posterior notochord staining of this CSKA/X8-injected embryo. Staining is not detected between the two arrows in the mid-body region. The lower embryo in J represents a CSKA/X8-injected embryo in which notochord staining is absent.
	Figure 6. Lineage analysis of Xw/'/t-8 myc expressing cells. CSKA-X8 myc was injected into the dorsal (A-C} or lateral (DI marginal zone of four-cell embryos, and expression of Xwr/t-8 myc protein was detected by wholemount immunocytochemical staining with monoclonal antiserum 9E10. {A) Whole gastrulae {stage 10). {B) Sagittal section through a representative stage-10 embryo; dorsal lip indicated by open arrow. (C-D) Transverse sections through neurulae {stage 21). Abbreviations are as in Fig. 5. Curved arrows in A denote dorsal marginal zone staining and, in other panels, denote representative Xwnt- 8myc-expressing cells.
	Figure 7. Expression of Xwnt-8 after the midblastula transition induces ventral mesoderm and inhibits induction of notochord by activin in ectodermal explants. Ectoderm was isolated from blastulae that had received no injection IA,D,G,J}, ventral injections {B,E,H,K), or dorsal injections (C,F,I,L) of CSKA-X8. Photographs of intact explants (A-C, G-I) or histological sections ID-F, J-L) of explants cultured in the absence {A-F) or presence [G-L} of 10 ng/ml of activin A until control stage 40 are shown. Abbreviations are as follows: {mese) Mesenchyme; {meso) mesothelium; (bl) cells resembling blood cells; (mus) muscle; (not) notochord. Bar in A, 1 mm [A-CJ, 310 Izm {D-F), 620 wm {G-/J, or 400 I~m [J-L}.