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Vertebrate Bicaudal-C (Bicc1) has important biological roles in the formation and homeostasis of multiple organs, but direct experiments to address the role of maternal Bicc1 in early vertebrate embryogenesis have not been reported. Here, we use antisense phosphorothioate-modified oligonucleotides and the host-transfer technique to eliminate specifically maternal stores of both bicc1 mRNA and Bicc1 protein from Xenopus laevis eggs. Fertilization of these Bicc1-depleted eggs produced embryos with an excess of dorsal-anterior structures and overexpressed organizer-specific genes, indicating that maternal Bicc1 is crucial for normal embryonic patterning of the vertebrate embryo. Bicc1 is an RNA-binding protein with robust translational repression function. Here, we show that the maternal mRNA encoding the cell-fate regulatory protein Wnt11b is a direct target of Bicc1-mediated repression. It is well established that the Wnt signaling pathway is crucial to vertebrate embryogenesis. Thus, the work presented here links the molecular function of Bicc1 in mRNA target-specific translation repression to its biological role in the maternally controlled stages of vertebrate embryogenesis.
Fig. 1. Identifying antisense oligonucleotides that degrade Xenopus
bicc1 mRNA. (A) Identification of antisense oligonucleotides (oligos) that
degrade the bicc1 mRNA. Oligos (9460 and 9463) were injected into oocytes,
then total RNA was isolated from injected cells and analyzed by RNA blot
hybridization. The bicc1 mRNA was detected with a radiolabeled probe
generated from the 3′UTRof bicc1 cDNA. The same filter was hybridized with a
radiolabeled probe to detect ornithine decarboxylase 1 (odc1) mRNA as a
negative control. (B) In a separate experiment, oligos (9460 and 9463) were
injected into oocytes, then total RNA was isolated from injected cells, and
analyzed by qRT-PCR. (C) Expression of the Bicc1 protein during early
Xenopus development. Proteins from an equivalent number or oocytes, eggs
or embryos were analyzed by immunoblotting probing with an antibody
generated against the N-terminal half of the Xenopus Bicc1. The same blot
was analyzed with an anti-actin antibody as a loading control. ST, stage.
(D) Antisense oligos block accumulation of the Bicc1 protein during oocyte
maturation. Oocytes were injected with either the 9460 or the 9463 oligo and
incubated for two hours. The injected oocytes were matured overnight with
progesterone then the proteins analyzed by immunoblotting and probing with
the Bicc1 antibody. Error bars represent s.e.m.
Fig. 2. Embryos depleted of maternal Bicc1 exhibit expanded dorsalanterior
structures and increased organizer-specific gene expression.
Phosphorothioate derivatives of the 9460 and 9463 oligos were injected into
oocytes and the oocytes matured overnight. Matured oocytes were treated with
vital dyes, transferred to an ovulating host female, and the laid eggs from
manipulated oocytes were fertilized. (A-C) Phenotypes of control embryos and
sibling bicc1-depleted embryos. (A) Control (left, green) and maternal bicc1-
depleted (blue; as9463) embryos at stage 13, showing delayed gastrulation
and deep blastopores. (B) Stage 28 embryos depleted of maternal bicc1
mRNA (as9463) develop expanded dorsal-anterior structures (DAI 7).
(C) Stage 30 embryos depleted of maternal bicc1 mRNA using either the 9460
or the 9463 oligo. Embryos treated with either oligo develop with expanded
dorsal-anterior structures (DAI 7). (D) The defects from depleting embryos of
bicc1 can be rescued by injecting bicc1 mRNA. Embryos depleted of
maternal bicc1 mRNA were injected at the vegetal pole with HA-bicc1 mRNA
(20 pg). (E) Summary of the phenotypes of control and antisense oligo-injected
host-transfer embryos. The (+) samples received an injection of HA-bicc1
mRNAwhereas the (−) samples did not. (F) The expression of organizer genes
increased in embryos depleted of maternal bicc1. Total RNAwas isolated from
bicc1-depleted stage 11 embryos and controls. qRT-PCR was used to analyze
the expression of different organizer-specific genes and the ventx1.2 and
wnt8a genes, which are markers of ventral-posterior development. The
expression changes were significantly different (P<0.05, except for wnt8 in
9463-treated embryos for which P<0.1). Error bars represent s.e.m.
Fig. 3. The wnt11b maternal mRNA is a target of Bicc1 repression.
(A) Animal cell assay for Bicc1 translational repression. Animal cells of 8-cell
Xenopus embryos were injected with luciferase reporter mRNAs. Some of the
embryos were given a second injection of mRNA encoding full-length Xenopus
Bicc1. When embryos reached stage 7-8, luciferase assays were performed.
Repression, as measured by the ratio of luciferase exhibited by a reporter
mRNA with and without Bicc1 expression, was calculated and plotted.
(B) Diagram of 3′UTR fragments incorporated into luciferase reporter mRNAs
and analyzed for repression. (C) The wnt11b maternal mRNA is repressed by
Bicc1. Error bars represent the s.e.m. from three separate experiments.
(D) Immunoblot analysis of HA-Bicc1 expression in repression assays.
(E) qRT-PCR analysis of luciferase reporter mRNAs in repression assays.
Error bars represent s.e.m.
Fig. 4. Bicc1 functions by binding to the 3′UTR of the wnt11b mRNA.
(A) Animal cell assay for in vivo Bicc1 binding. Animal cells of 8-cell Xenopus
embryos were injected with mRNA encoding HA-tagged Bicc1. Some injected
samples included luciferase reporter mRNAs. When embryos reached stage
7-8, Bicc1 was immunoprecipitated with an HA antibody and the associated
RNA isolated for analysis. RNA samples were reverse transcribed and the
cDNA used as template for q-PCR. (B) The endogenous Xenopus maternal
wnt11b mRNA was bound by Bicc1. The wnt11b mRNA was enriched in Bicc1
IP samples, similar to the known mRNA target cripto1. (C) The binding of Bicc1
to wnt11b mRNA occurs via sequences in the 3′UTR. Embryos were injected
with mRNA encoding HA-tagged Bicc1 along with different reporter mRNAs.
Bicc1 was immunoprecipitated and the associated RNAs analyzed as
described above (Fig. 3A). The reporter mRNAs containing the 3′UTR of the
wnt11b mRNA associated with Bicc1, as did the positive control reporter
mRNA containing the 3′UTR of the cripto1 mRNA. Error bars represent s.e.m.
Fig. 5. Bicc1 translational repression activity is present as an animal vegetal gradient in the embryo. (A) Bicc1 protein is present in an animal vegetal gradient. Stage 7 embryos were manually dissected into three parts; animal (AN), marginal zone (MZ) and vegetal (VG). The proteins from these regions were analyzed by immunoblotting and probing with a Bicc1 antibody.
The same filter was also probed with an antibody recognizing cytoskeletal
actin. (B) The Bicc1 repression activity is present in an animal-vegetal gradient.
Xenopus embryos (16-cell) were injected with luciferase reporter mRNAs
containing different 3′UTRs (Fig. 2B): wnt11b, cripto1 and cyclin B1. Each
reporter was injected into animal cells, marginal zone cells or vegetal cells of
separate groups of embryos. When injected embryos reached stage 8-9 they
were used to prepare extracts for the assaying of luciferase activity. For each
mRNA, the activity when injected into animal cells was set as 1 and used as a
reference point for the activities in other cells. (C) Changes in luciferase for
each reporter mRNA were due to changes in translation and not changes in
mRNA degradation. RNA isolated from embryos from each injection was
analyzed for presence of luciferase reporter mRNAs using qRT-PCR.
(D) Animal or vegetal cells of 8-cell Xenopus embryos were injected with
luciferase reporter mRNA containing the 3′UTR from the cripto1 mRNA. Some
of the embryos were given a second injection of mRNA encoding full-length
Xenopus Bicc1. When embryos reached stage 8-9, luciferase assays were
performed. Repression as measured by the ratio of luciferase exhibited by a
reporter mRNA with and without Bicc1 expression was calculated and plotted.
(E) Immunoblot analysis was used to analyze the expression of HA-Bicc1 in
animal and vegetal cells. Error bars represent s.e.m.
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