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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 ventralmesoderm 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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8422982
???displayArticle.link???Genes Dev ???displayArticle.grants???[+]
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 ventralspinal 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 ventralmesoderm 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}.
