XB-ART-23685
Development
June 1, 1992;
115
(2):
463-73.
Analysis of Xwnt-4 in embryos of Xenopus laevis: a Wnt family member expressed in the brain and floor plate.
Abstract
This study characterizes the temporal and spatial expression during early Xenopus development of
Xwnt-4, a member of the Wnt gene family. The
Xwnt-4 protein contains all of the sequence motifs that are hallmarks of the Wnt gene family and is 84% identical to the mouse homolog,
Wnt-4. The highest level of
Xwnt-4 expression occurs during the early
neurula stage of development although its expression persists throughout embryogenesis and can be found in the adult
testis,
brain and
epithelium. Consistent with its localization to
head and dorsal regions of microdissected embryos, the expression of
Xwnt-4 is enhanced in anterodorsalized embryos resulting from treatment with LiCl, and the expression of
Xwnt-4 is suppressed in UV-ventralized embryos that lack
anterior neural
tissue. These results suggested that expression of
Xwnt-4 is dependent on the induction of neural
tissue. This idea was tested using induction experiments with dorsal or
ventral ectoderm from a
stage 10 embryo, recombined with
dorsal marginal zone mesoderm from the same
embryo. Recombinant
tissue and
ectoderm alone were cultured until
stage 14, when
Xwnt-4 expression was assayed using Northern analysis. In the recombinant assay,
Xwnt-4 expression does not occur in the uninduced
ectoderm but is expressed in both the dorsal and
ventral recombinants.
Xwnt-4 expression in
neural ectoderm was confirmed in isolated, induced
neural ectoderm, dissected away from the dorsal
mesoderm, in a
stage 12.5 embryo. Whole-mount in situ hybridization confirmed the dissection studies and demonstrated that
Xwnt-4 transcripts are expressed in the dorsal midline of the
midbrain,
hindbrain and the
floor plate of the
neural tube. Collectively, the data indicate that
Xwnt-4 is a unique member of the Wnt family whose expression is dependent on neural induction. The specific pattern of expression following neural induction suggests that
Xwnt-4 plays a role in the early patterning events responsible in the formation of the
nervous system in Xenopus.
PubMed ID:
1425335
Article link:
Development
Grant support:
[+]
Species referenced:
Xenopus laevis
Genes referenced:
krt18.1
prph
ube2k
wnt1
wnt4
Article Images:
[+] show captions
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Fig. 1. Comparison of the
predicted XwM-4, Xwflt-1 and
Wnt-4 protein Sequences. GapS
introduced to align the
sequences are shown as dashes
and identical residues are
marked with asterisks. A
putative hydrophobic leader
sequence
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Fig. 2. Expression of Xwnt-4 in adult tissues and during
early Xenopus development. (A) Northern analysis of 20
lig of total RNA isolated from each of the adult tissues
indicated and hybridized with ^P-labelled antisense Xwnt-4
RNA probe. The filters were rehybridized with an EFl-ar
cDNA clone to determine the integrity of RNA in each
lane. (B) Temporal expression of Xwnt-4 mRNA during
early development. Northern analysis of RNA from
different developmental stages (Nieuwkoop and Faber,
1967). 20 ug aliquots of total RNA were analyzed at each
stage. Equivalent loading of RNA was determined by
rehybridization of the filters in the presence of a Xenopus
5S cDNA probe. The 28S and 18S ribosomal RNA
markers are noted.
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Fig. 3. Xwnt-4 expression in UV-irradiated or lithiumtreated
embryos. Northern blot containing 20 ng of total
RNA from tailbud embryos (stage 25) that either received
no treatment (—UV, —Li), or were treated with UV
(+UV) or lithium (+Li) as described. The blot was
rehybridized with a ^P-labeled X3F3 antisense RNA probe
to determine the amount of anterior neural tissue in each
sample, followed by EFl-a cDNA probe to determine the
relative loading of RNA in each sample.
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Fig. 4. Xwnt-4 expression in neural recombinant tissues
cultured in vitro. (A) Dissected tissues were assembled
from a stage 10 embryo as diagrammed. (B) Recombinant
tissue and isolated ectodermal tissue were cultured until
control embryos reached stage 14, total RNA was isolated
and 20 /ig aliquots were examined using Northern analysis.
The integrity of the RNA in the sample was verified by
rehybridization of the filter with the 5S cDNA probe.
Control embryo, C.
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Fig. 5. Xwnt-4 expression in neural ectoderm as compared
to the expression of XIF3, a marker of anterior neural
tissue and XK endo B, a marker of dorsal mesoderm
tissue. Tissues were dissected as described and cultured
until control embryos reached stage 23. 20 jig of total RNA
from each sample, including control embryos (lanel), were
hybridized to a Xwnt-4 antisense RNA probe followed by
successive rehybridizations to XIF3 antisense RNA probe,
XK endo B cDNA probe and the 5S ribosomal cDNA
probe.
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Fig. 6. Localization of Xwnt-4 transcripts during early
development using whole-mount in situ hybridization. (A)
Antisense Xwnt-4 probe hybridized to a late gastrula stage
embryo (stage 12; on the left), the blastopore is pointing
down (arrowhead), and hybridized to a slightly later stage
embryo (stage 13; on the right). Xwnt-4 transcripts are
localized to a thin band of cells above the archenteron
(arrow) in the embryo on the right. (B) Antisense Xwnt-4
probe hybridized to a neurula stage embryo (stage 14/15),
shown from the side. Xwnt-4 expression occurs along the
entire anteroposterior axis where it is localized to the inner
layer of the neural ectoderm (arrow). A distinct anterior
patch of staining is denoted by the arrowhead. (C)
Antisense Xwnt-4 probe hybridized to a neurula stage
embryo (stage 14/15), shown from the top. Two anterior
patches of Xwnt-4 expression are clearly seen (arrowhead)
on the medial region of the neural plate. At this stage,
there is an absence of Xwnt-4 expression along the midline
of the neural groove (arrow). (D) Antisense XwntA probe
hybridized to an early tailbud stage embryo (stage 23).
Xwnt-4 expression in the neural tube changes by this stage,
becoming concentrated along the length of the floor plate
and restricted to the posterior end of the embryo. Three
overlapping regions of expression are seen in the border of
the prosencephalon/mesencephalon (arrow 1), the anterior
rhombencephalon (arrow 2) and the posterior
rhombencephalon (arrow 3). (E) Antisense Xwnt-4 probe
hybridized to a late tailbud stage embryo (stage 27). The
same regions of expression in the brain are denoted as in
D. (F) Antisense Xwnt-4 probe hybridized to a tadpole
stage embryo (stage 38). (G) Sense Xwnt-4 probe
hybridized to early and late tailbud stage embryos. All
embryos were rendered transparent with Murray's clear
prior to photography. The intensity of the stain varies with
respect to the length of time the embryos are incubated in
the chromogenic reaction and should not be taken as a
measure of transcript abundance when compared between
different embryos.
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Fig. 7. Comparison of the
expression of Xwnt-4 and
Xwnt-1 in the neural tube of
a late tadpole stage embryo
(stage 28). (A) Antisense
Xwnt-4 and Xwnt-1 probes
hybridized to late tadpole
stage embryos and processed
for whole-mount in situ
hybridization. Embryos were
embedded in paraffin and
sectioned. Representative
sections are denoted by the
lettered arrows. (B) A
transverse section through an
Xwnt-4 hybridized embryo
taken at the level of the optic
vesicle (ov). Xwnt-4
expression occurs on the
dorsal midline of the
mesencephalon (me). (C) A
transverse section through an
Xwnt-4 hybridized embryo
taken at the level of the
auditory vesicle (av). At this
position in the
anteroposterior axis Xwnt-4
expression is seen in the
dorsolateral region of the
rhombencephalon (rh) and in
the ventral midline directly
above the notochord (nc).
The ventral portion of the
auditory vesicle is also
positive for Xwnt-4
expression. (D) A transverse
section through an Xwnt-4
hybridized embryo, taken at
the posterior edge of the
rhombencephalon. Xwnt-4
expression is localized to the
floor plate of the neural tube
(nt). (E) A transverse section
taken at the posterior edge of
the optic vesicle (ov),
through an embryo
hybridized with Xwnt-1.
Xwnt-1 expression occurs in
both the dorsal midline of
the mesencephalon (me) and
in the ventral-lateral margin
of the mesencephalon. (F) A
transverse section taken
through an Xwnt-1 hybridized
embryo in the
rhombencephalon (rh). All of
the Xwnt-1 expressing cells at
this point in the
rhombencephalon are
localized to the dorsal
midline. There is no Xwnt-1
expression in the ventral
midline overlying the
notochord (nc).
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