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Abstract
When Xenopus gastrulae are made to misexpress Xwnt-8, or are exposed to lithium ions, they develop with a loss of anterior structures. In the current study, we have characterized the neural defects produced by either Xwnt-8 or lithium and have examined potential cellular mechanisms underlying this anterior truncation. We find that the primary defect in embryos exposed to lithium at successively earlier stages during gastrulation is a progressive rostral to caudal deletion of the forebrain, while hindbrain and spinal regions of the CNS remain intact. Misexpression of Xwnt-8 during gastrulation produces an identical loss of forebrain. Our results demonstrate that lithium and Wnts can act upon either prospective neural ectodermal cells, or upon dorsal mesodermal cells, to cause a loss of anterior pattern. Specifically, ectodermal cells isolated from lithium- or Wnt-exposed embryos are unable to form anterior neural tissue in response to inductive signals from normal dorsal mesoderm. In addition, although dorsal mesodermal cells from lithium- or Wnt-exposed embryos are specified properly, and produce normal levels of the anterior neural inducing molecules noggin and chordin, they show a greatly reduced capacity to induce anterior neural tissue in conjugated ectoderm. Taken together, our results are consistent with a model in which Wnt- or lithium-mediated signals can induce either mesodermal or ectodermal cells to produce a dominant posteriorizing morphogen which respecifies anterior neural tissue as posterior.
FIG. Is Exposure of Xenopus gastrulae to hthium or ectoplc Xwnt-8 results in the loss of anterior structures. Embryos were treated with
lithium at gastrula stages 12 (B), 11 (C), 10 (D), and 9 (E) or injected with pCSKA-Xwnt-8 m the dorsal marginal zone at the 4-ceU stage
(F) and allowed to grow to the tailbud stage (stage 30). Control embryos (A) possess eyes (arrow) and cement gland (arrowhead) while these
structures are reduced or absent in the lithium-treated and Xwnt-8-misexpressing embryos. The scale of the extent of posteriorization
(P1-P4) corresponds to that described in Yamaguchl and Shinagawa (1989).
FIG. 2. Exposure to lithium during late-blastula through gastrula stages results in deletion of part or all of the forebrain. Untreated
embryos or embryos treated with lithium at stages 9-12 (as indicated to the right of each row) were cultured to the tailbud stage (stage
30) and hybridized with digoxygenin-labeled X-dll3, Xwnt-8b, En-2, and XKrox-20 probes, as indicated at the top of each column. All
embryos are oriented with anterior to the left of the figure and dorsal toward the top of the figure. (A) X-dll3 staining of branchial arches
(arrows), otic and olfactory placodes (arrowheads), telencephalon (open black arrow), and ventral diencepha.lon (open white arrow} is
indicated. (B) Arrow denotes staining of the dorsal diencephalon and arrowhead indicates staining of cells at the forebrain-midbrain
boundary following hybridization with an Xwnt-8b probe. (C) Arrow indicates En-2 signal in cells at the midbrain-hindbrain boundary.
(D) XKrox-20 staining of rhombomeres 3 and 5 of the hindbrain is indicated by arrowheads.
FIG. 3. Exposure to lithium at the late-blastula stage results in
the loss of both forebrain and midbram. Untreated embryos (A) or
embryos treated with lithium at stage 10 (B) or 9 (C) were cultured
to stage 32 and hybridized with digoxygenin-labeled Xotx2 riboprobes.
Staining of the telencephalon (open black arrow), the ventral
dieneephalon (open white arrow), and the midbrain (between
filled arrows) is indicated
FIG. 4. Misexpresslon of Xwnt-8 in the dorsal mesoderm results in the loss of both forebrain mad mldbram. Untreated embryos (A-C) or embryos
made to misexpress Xwnt-8 in the dorsal mesoderm during gastrulation (D-I) were cultured to tailbud stage and hybridized with digoxygeninlabeled
X-dll3, Xwnt-8b, and Xotx-2 probes as indicated above each column. All embryos are oriented with anterior to the left, and dorsal toward
the top of the figure. (A) X-dlI3 staining of branchial arches (small black arrows), ouc (large black arrow) and olfactory (black arrowhead) placodes,
telencephalon (open black arrow), and ventraldiencephalon (open white arrow) is indicated. Staining of the ventraldiencephalon is partially out
of the plane of focus in this figure and thus appears reduced relative to that shown in Fig. 2A. (B} Arrow denotes staining of the dorsal diencephalon
and arrowhead indicates staining of cells at the forebram-midbram boundary following hybndizatlon with an Xwnt-Sb probe. (C) Xotx-2 staining
of the telencephalon (open black arrow), and the rnidbrain (between filled arrows) is indicated.
FIG. 5. Exposure to lithium during gastrulation or misexpresslon of Xwnt-8 retards involution of the dorsoantenor mesoderm. Mldsagittal
confocal images of an untreated embryo (A) and of embryos treated with lithium at stage 12 (B), 11 (C), 10 (D), or 9 (E) or made to
misexpress Xwnt-8 in the dorsal mesoderm (F) and then cultured to stage 13. Arrowhead in each panel marks the position of the yolk
plug while the archenteron is denoted by an arrow and the blastocoel by an asterisk. Dorsal is toward the top of the micrograph.
FIG. 6. Expression of goosecoid in the dorsoanterior mesoderm is
not perturbed by exposure to lithium. Control embryos (A, C, E,
G, I) and embryos treated with lithium at stage 9 (B, D, F, H, J) were
fixed at stages 9-13 and hybridized with a digoxygenin-labeled
antisense goosecoid (gsc) probe. (A, B) Stage 9, vegetal view. (C, D)
Stage 10, vegetal view. (E, F) Stage 10.5, vegetal view. (G, H) Stage
11.5, lateral view. (I, J) Stage 13, lateral view. Arrows indicate the
dorsal blastopore lip and arrowheads mark the yolk plug.
FIG. 7. Exposure to LiC1 or misexpression of Xwnt-8 does not alter
expression of chordin or goosecoid. (A). Control embryos, embryos
made to misexpress Xwnt-8 in dorsal mesodermal cells (Xwnt-8
DMZ) and embryos treated with lithium at stage 9 were cultured
to stage 13 and RNA extracted from 2 embryos in each group.
Expression of chordin, gsc, and EF-la was assayed by RT-PCR. All
data shown in A are from a single experiment. (B). The dorsal marginal
zone (DMZ) was explanted from stage 10 embryos and was
either left untreated (control explant) or was treated with lithium
for 20 rain (LiC1 explant}. Explants were cultured to sibling stage
13 following which RNA was extracted from 4 explants in each
group and the expression of chordin, gsc, and EF-I~ assayed by RTPCR.
All data shown in B are from a single experiment.
FIG. 8. Expression of Xotx2 and NCAM m recombinants of ectoderm
and mesoderm from lithium-treated or Xwnt-8 misexpressmg
embryos. Embryos were made to misexpress Xwnt-8 by rejection
of a plasmid expression construct into dorsal marginal zone (DMZ)
or animal pole cells at the 4-cell stage, or were treated with hthium
at stage 9. In some cases, explants of mesoderm (N) or ectoderm
(O), rather than whole embryos, were treated with lithium. Ectoderm
and mesoderm isolated from control, hthlum-treated, or
Xwnt-8-mlsexpressing embryos at stage 10 were conjugated as illustrated
above Table 3 and cultured until sibling embryos reached
stage 23 (A-I) or 27 (J-P). RNA was extracted from whole embryos
(embryo) or from mesoderln/ectoderm conjugates (recombinants)
and expression of Xotx2, NCAM, and EF-la was assayed by RTPCR.
Eqmvalent aliquots of RNA from each sample (whole control
embryo RNA shown) were analyzed in the presence and absence
(RT-) of reverse transcriptase {RT) to demonstrate that the signal
was not due to contamination of RNA with genomic DNA. Data
shown in A-I are from one single experiment and those shown in
J-P are from a second experiment.
