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In spite of abundant evidence that Wnts play essential roles in embryonic induction and patterning, little is known about the expression or activities of Wnt receptors during embryogenesis. The isolation and expression of two maternal Xenopus frizzled genes, Xfrizzled-1 and Xfrizzled-7, is described. It is also demonstrated that both can activate the Wnt/beta-catenin signaling pathway as monitored by the induction of specific target genes. Activation of the beta-Catenin pathway has previously been shown to be necessary and sufficient for specifying the dorsal axis of Xenopus. beta-Catenin is thought to work through the cell-autonomous induction of the homeobox genes siamois and twin, that in turn bind to and activate the promoter of another homeobox gene, goosecoid. However, it was found that the beta-catenin pathway regulated the expression of both endogenous goosecoid, and a goosecoid promoter construct, in a cell non-autonomous manner. These data demonstrate that maternal Frizzleds can activate the Wnt/beta-catenin pathway in Xenopus embryos, and that induction of a known downstream gene can occur in a cell non-autonomous manner.
Fig. 1. Dendrogram and sequence alignment of Xfrizzled-1
and Xfrizzled-7. (A) Conceptual translations of the maternal
Xenopus frizzled coding regions were compared to published
Frizzled proteins using a ClustalW alignment (MacVector 6.5;
R, Rattus; M, murine; H, human; X, Xenopus; G, Gallus) and
found to cluster with Frizzled-1 and Frizzled-7, respectively. (B)
Xfrizzled-1 and Xfrizzled-7 proteins share 76% identity and 85%
similarity.
Fig. 2. Developmental
expression of Xfrizzled-1
and Xfrizzled-7. A reverse
transcriptionâpolymerase
chain reaction (RT-PCR)
analysis was used to assay
relative levels of Xfrizzled-1
and Xfrizzled-7 expression
at various stages of embryogenesis.
(A) Xfrizzled-1 is
abundant in unfertilized
eggs (UF) but is expressed
at very low levels during
gastrulation (10, 12). Zygotic
Xfrizzled-1 expression is
detected at neurula stages
and later in embryogenesis.
(B) Xfrizzled-7 is expressed
throughout embryogenesis.
Two Xfrizzled-7 polymerase
chain reaction (PCR) primer
pairs using a common
forward primer and distinct
reverse primers yielded
similar results. Histone H4 serves as a control for cDNA synthesis and gel loading. The -RT control demonstrates that contaminating
genomic DNA is not present in the samples analyzed.
Fig. 3. Xfrizzled-1 and Xfrizzled-7 are expressed in overlapping
and distinct regions of Xenopus embryos. All embryos are
oriented with anterior to the left and dorsal to the top. Embryos
in (A,G) were cleared in Murray’s clearing agent. (A) Xfrizzled-7
transcripts are broadly expressed in late gastrulae (stage 13),
and are intensely expressed in migrating cranial neural crest of
neurula-stage embryos (B, arrow). (C) A control tadpole treated
with a sense probe exhibits no staining. (D) Xfrizzled-7 is
expressed in the brain (arrowhead), neural crest (nc), ventral otic
vesicle (ov), and pronephros (P) of tailbud-stage embryos. (E)
Xfrizzled-7 transcripts are present in the brain (arrowheads), heart
(h), and pronephros (P) of tadpoles. Xfrizzled-1 transcripts are
first detected in neurula stages in the brain (data not shown),
and brain expression domains are maintained in later embryos
(F,G, arrowheads). (F,G) Xfrizzled-1 is expressed in the otic
vesicle (ov, unlabeled arrow in (G)), but in a pattern complementary
to Xfrizzled-7. Like Xfrizzled-7, Xfrizzled-1 is expressed
in the pronephros (P) and heart (h) of tailbud (F) and tadpolestage
(G) embryos, as well as the proctodeum (pd) of tadpoles
(F).
Fig. 4. Xfrizzled-1 and Xfrizzled-7 activate the Wnt/b-catenin
pathway. (A,B) Overexpression of Xfrizzled-1 or Xfrizzled-7
in animal cap explants can induce expression of the Wnt/bcatenin-
responsive genes Xnr-3 and siamois. Whole-embryo total
ribonucleic acid (RNA) serves as a positive control in each
experiment (left lane in each panel). Animal caps from uninjected
embryos express neither of the b-catenin-responsive genes
(center lanes), but overexpression of either Xfrizzled-1 (A, right
lane) or Xfrizzled-7 (B, right lane) results in expression of both
Xnr-3 and siamois. Histone H4 and elongation factor (EF)1a
serve as loading controls. (C). Xfrizzled-7 induces neural crest
markers in neuralized animal caps. Embryos were injected with
RNA encoding Noggin and a control (green fluorescent protein)
RNA or Xfrizzled-7 RNA. Late blastula (stage 9) animal
caps were cultured until sibling embryos reached stage
18â20. Total RNA was prepared and was subjected to reverse
transcriptionâpolymerase chain reaction (RT-PCR) analysis.
While embryos express all markers used, uninjected control
explants express only the ubiquitous EF1a. Noggin-expressing
explants express the anterior neural marker otx-2, while
coexpression of Xfrizzled-7 results in the induction of a more
posterior marker, engrailed-2. These caps also express the neural
crest markers slug and twist.
Fig. 5. Xfrizzled-1 and Xfrizzled-7 recruit Xwnt-8 and Dishevelled but not Xwnt-5a to the plasma membrane. The indicated
ribonucleic acid (RNA) was overexpressed in animal caps and the ectopic Wnt and Dishevelled proteins were analyzed by confocal
microscopy. (A,D,F) Ectopic Wnt proteins are detected at the plasma membrane and in the secretory apparatus in animal caps. (B,C)
Coexpression of Xfrizzled-1 or Xfrizzled-7 results in a recruitment of Xwnt-8 to the plasma membrane. (E) Xwnt-5A is not recruited by
Xfrizzled-1. (G) In an independent experiment, Xfrizzled-7 does not recruit Xwnt-5A from the secretory apparatus to the plasma
membrane. (H) Overexpressed Dishevelled-GFP localizes to punctate structures throughout the cytoplasm but is recruited to the plasma
membrane by Xfrizzled-1 (I) and Xfrizzled-7 (J).
Fig. 6. Cell non-autonomous induction of endogenous goosecoid
by b-catenin. Ribonucleic acid was injected into ventral
vegetal cells or regions at the 4â32-cell stages, encoding
b-catenin mutated to accumulate in nuclei and tagged with a
c-myc epitope. Embryos at the gastrula stage were then processed
for whole-mount in situ hybridization to localize goosecoid
transcripts (blueâpurple) and the expression of the
c-myc-tagged b-catenin was visualized by immunostaining
(brown). (AâC) The in situ hybridizations reveal a normal endogenous
domain of goosecoid expression (E) as well as an ectopic
site (arrow). This ectopic goosecoid expression extends several
cell diameters away from the nearest cells expressing b-catenin
(b), indicating that b-catenin can induce goosecoid in a cell
non-autonomous manner. (D) A fixed embryo was cut in half prior
to analysis, revealing that expression of b-catenin deep within
the embryo (b) induces goosecoid (arrow) in the overlaying cells
in a cell non-autonomous manner.
Fig. 7. Cell non-autonomous induction of a goosecoid reporter construct by b-catenin signaling. (A) The goosecoid-1500gsc/LUC
Luciferase construct (Watabe et al. 1995) was injected into the marginal zone of a single ventral (V) or dorsal (D) cell at the 4-cell
stage. b-Catenin was then injected into the same cell (Vb) or the neighboring lateral cell in some of the ventral-injected embryos (V/b)
and Luciferase activity was determined at the gastrula stage. (B) The goosecoid-1500gsc/LUC Luciferase construct was injected into
a ventral vegetal (V) or dorsal (D) cell at the 16â32-cell stage, and where indicated (V//b) embryos injected ventrally with the reporter
construct were then injected with b-catenin two cells away laterally. This left one uninjected cell between the two injected cells.
(C) The siamois S01234 Luciferase construct (Brannon et al. 1997) was injected into ventral (V) or dorsal (D) cells as in (A). Where
indicated ventral injection of the siamois reporter construct was followed by injection of b-catenin into the adjacent ventral cell (V/b).
This level of b-catenin is sufficient to induce this promoter when coexpressed in the same cell (data not shown). Relative light units
are normalized to that of the dorsal side for each experiment, and two to three sets of embryos were used for each condition in each
experiment. Data are also representative of multiple independent experiments.
