Park S et al. (2016), A gradient of maternal Bicaudal-C controls vert...

XB-ART-51779

Development 2016 Mar 01;1435:864-71. doi: 10.1242/dev.131359.

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A gradient of maternal Bicaudal-C controls vertebrate embryogenesis via translational repression of mRNAs encoding cell fate regulators.

Park S , Blaser S , Marchal MA , Houston DW , Sheets MD .

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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.

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Species referenced: Xenopus laevis
Genes referenced: bicc1 ccnb1 cripto.3 dand5 ddx5 gsc nodal3.1 nog sia1 ventx1.2 wnt11b wnt8a
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	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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