POU-V factors antagonize maternal VegT activity and beta-Catenin signaling in Xenopus embryos

Abstract

VegT and beta-Catenin are key players in the hierarchy of factors that are required for induction and patterning of mesendoderm in Xenopus embryogenesis. By descending the genetic cascades, cells lose their pluripotent status and are determined to differentiate into distinct tissues. Mammalian Oct-3/4, a POU factor of subclass V (POU-V), is required for the maintenance of pluripotency of embryonic stem cells. However, its molecular function within the early embryo is yet poorly understood. We here show that the two maternal Xenopus POU-V factors, Oct-60 and Oct-25, inhibit transcription of genes activated by VegT and beta-Catenin. Maternal POU-V factors and maternal VegT show an opposite distribution along the animal/vegetal axis. Oct-25, VegT and Tcf3 interact with each other and form repression complexes on promoters of VegT and beta-Catenin target genes. We suggest that POU-V factors antagonize primary inducers to allow germ layer specification in a temporally and spatially coordinated manner.

Figure 1

Maternal POU-V factors regulate transcription of Xnrs and Siamois. (A) Distribution of Oct-25, Oct-60 and VegT in eight-cell and blastula embryos. Animal and vegetal blastomeres were dissected from stage 4 embryos. Animal, equatorial and vegetal parts were excised from stage 8.5 embryos and subjected to real-time RT–PCR. Quantification of expression level in each part was normalized to the yield of RNA and to the respective expression level in whole embryos. (B) A total of 400 pg Oct-25, Oct-60 or mOct-3/4 mRNA was injected into all vegetal blastomeres at the eight-cell stage. Controls and injected embryos were grown to stage 10.5 and subjected to RT–PCR. (C) A mixture of 15 ng of Oct25MO and 40 ng of Oct60MO was injected into the equatorial region of four blastomeres at the four-cell stage. Controls and injected embryos were grown to stage 10.5 and subjected to RT–PCR.

Figure 2

Oct-25 and Oct-60 counteract the activities of VegT and β-Catenin. (A–C) Real-time RT–PCR showed that injection of 600 pg Oct-25 and Oct-60 RNA into the animal pole region of four-cell-stage embryos inhibited gene expression in animal caps activated by 500 pg VegT alone (A), by 500 pg β-Catenin alone (B) and, synergistically, by 200 pg VegT and 200 pg β-Catenin RNA together (C). (D–M) The phenotype of Oct-25 or Oct-60 overexpression was rescued by VegT. (D, E) Uninjected control embryos showed normal development at gastrula (D) and tail-bud stages (E). (F, G) Embryos injected vegetally with 400 pg Oct-25 RNA showed failure of gastrulation (F) and body axis formation (G). (H, I) The phenotypic effect of Oct-25 was rescued by co-injection of 700 pg VegT RNA, as shown by restored blastopore during gastrulation (H) and nearly normal body axis formation during tail-bud stage (I). (J, K) Embryos injected vegetally with 400 pg Oct-60 RNA showed failure of gastrulation (J) and body axis formation (K). (L, M) Co-injection with 700 pg VegT RNA restored gastrulation (L) and body axis formation (M). (N–P) POU-V factors repressed axis formation induced by β-Catenin. (N) Ventral–vegetal overexpression of 400 pg β-Catenin RNA induced axis duplication. The secondary axis was abolished by co-injection of 400 pg Oct-25 (O) or Oct-60 RNA (P).

Figure 3

GST pull-down and immunoprecipitation demonstrate interactions between Oct-25, VegT and Tcf3. (A) Radiolabeled VegT bound to GST/Oct-25 fusion protein and vice versa, but β-Catenin did not interact with GST/Oct-25. Radiolabeled Tcf3 interacted with GST/Oct-25 and vice versa. Radiolabeled β-Catenin was precipitated by GST/Oct-25, when in vitro translated unlabeled Tcf3 was simultaneously added. (B) Both VegT and Tcf3 bound to Oct-60, Oct-91 as well as mouse Oct-3/4. (C) Radiolabeled Tcf3 bound to GST/VegT fusion protein and vice versa. In contrast, VegT did not interact with GST/β-Catenin. (D) VegT did not interact with 1–190 aa, but interacted with 191–397 aa and 398–551 aa regions of Tcf3. Vice versa, Tcf3 interacted with the N-terminal region (1–120 aa) but did not interact with the C-terminal region (121–456 aa) of VegT. (E) Co-immunoprecipitation with anti-myc antibody (IP:α-myc) showed that both myc-tagged Tcf3 (Tcf3-MT) and VegT (VegT-MT) precipitated endogenous Oct-25, whereas anti-flag antibody (IP:α-flag) precipitated a complex of flag-tagged Tcf3 (flag-Tcf3) and VegT-MT and a ternary complex of flag-tagged Oct-25 (flag–Oct-25), Tcf3-MT and VegT-MT.

Figure 4

EMSAs with the Xnr1 promoter. (A) Xnr1 promoter sequence. The first nucleotide in front of the start codon ATG is designated as −1. The Oct binding site, TCF/LEF binding site and the two half T-box sites, TBX1 and TBX2, are boxed and labeled. The promoter fragments used for EMSAs are indicated below each panel. (B) Oct-25 bound specifically to the promoter fragment that contains the Oct binding site. Nonspecific competitive probe (p(dIC)) did not interfere with the binding, while the unlabeled target probe (u.t.) competed with the labeled target probe for binding with Oct-25. (C) EMSA with embryonic protein extracts (stage 12) showed that a probe containing the Oct binding site formed a complex with Oct-25. Arrow indicates specific protein/DNA complex, whereas arrowheads indicate unspecific complexes. The specific complex became more intense using extracts from embryos after injecting increasing doses of Oct-25. In contrast, the protein/DNA complex was not formed in extracts from embryos injected with increasing doses of Oct25MO and Oct60MO, or when a probe containing a mutated Oct binding site was used. (D) Tcf3 bound to the promoter fragment containing the TCF/LEF binding site. The labeled probe containing the TCF/LEF binding site was incubated with increasing amounts of Tcf3. (E) Oct-25 and VegT formed a complex on the Oct binding site. Oct-25 but not VegT bound to the promoter fragment containing the Oct binding site. When Oct-25 was incubated with increasing amounts of VegT, the mobility of the Oct-25/DNA complex was reduced. (F) Oct-25 and VegT formed a complex on the T-box site as revealed by the reduced mobility of the VegT/DNA complex. (G, H) Oct-25 and Tcf3 formed a complex on the promoter fragment containing either the Oct binding site (F) or the TCF/LEF binding site (G).

Figure 5

EMSAs with the Siamois promoter and ChIP. (A) Siamois promoter sequence. The first nucleotide in front of the transcription start site is designated as −1. The Oct binding site and the four TCF/LEF sites (S1, S2, S3, S4) are boxed and labeled. The promoter fragments used for EMSAs are indicated below each panel. (B) Oct-25 bound to the promoter fragment that contains the Oct binding site. (C) Tcf3 bound to the promoter fragment that contains the S1 TCF/LEF binding site. (D) Oct-25 and Tcf3 formed a complex on the S1 TCF/LEF binding site. (E, F) VegT and Tcf3 formed a complex on both S1 (E) and S3 (F) TCF/LEF sites. (G) ChIP assays revealed that the Siamois promoter region spanning the Oct and TCF/LEF binding sites, as well as the Xnr1 promoter region including the Oct, TCF/LEF and T-box sites, were amplified from chromatin precipitated by an Oct-25 antibody (α-Oct-25) but not in the absence of antibody (no Ab). When Oct-25 translation was blocked by Oct25MO, PCR products for Siamois and Xnr1 promoter regions were reduced as compared with ctrlMO injections.

Figure 6

Regulation of Xnr1 promoter/reporter activity by VegT, β-Catenin and Oct-25. (A) Wild-type and mutant Xnr1 promoter/reporter constructs used for luciferase assays. (B) The wild-type −907 promoter was stimulated by overexpression of VegT, Xwnt8 or β-Catenin. Stimulation was enhanced by co-injection of VegT and β-Catenin. In all cases, stimulation was repressed by overexpression of Oct-25. (C) In whole embryos, knockdown of Oct-25/Oct-60 led to an increased stimulation of Xnr1 promoter. (D, E) Promoter/reporter mutants lacking the T-box binding site were not stimulated by overexpression of VegT (D) and those lacking the TCF/LEF binding site were not stimulated by overexpression of β-Catenin (E). (F–I) The wild-type −279 promoter/reporter (F) or the mutants lacking the Oct binding site (G), TCF/LEF binding site (H) or both (I) were stimulated by overexpression of VegT. Stimulation was repressed by overexpression of Oct-25 or dnTcf3, and even more drastically, by co-injection of Oct-25 and dnTcf3. (J–M) The wild-type −279 promoter reporter (J) or the mutants lacking the Oct binding site (K), the T-box binding site (L) or both (M) were stimulated by overexpression of β-Catenin or, much more strongly, by overexpression of both β-Catenin and Tcf3. The stimulation was drastically repressed by overexpression of Oct-25. (N) Oct-25 stimulated slightly the promoter mutant containing only the Oct binding site, which by co-injection of VegT was in turn repressed.

Figure 7

Regulation of Siamois promoter/reporter activity by VegT, β-Catenin and Oct-25. (A) Wild-type and mutant Siamois promoter reporter constructs used for luciferase assays. (B) The promoter mutant lacking the TCF/LEF sites was not stimulated by overexpression of β-Catenin. (C, E) Both wild type −802 (C) and −250 (E) promoter/reporters were significantly stimulated by overexpression of either Xwnt8 or β-Catenin, and, much more strongly, by co-injection of β-Catenin and Tcf3. Stimulation was repressed by co-injection of Oct-25. (D) Knockdown of Oct-25/Oct-60 in whole embryos resulted in enhanced stimulation of Siamois promoter. (F) The promoter/reporter mutant lacking the Oct binding site was stimulated by injection of β-Catenin or by injection of both β-Catenin and Tcf3. However, overexpression of Oct-25 repressed this stimulation. (G) Overexpression of VegT activates the −250 promoter/reporter. When VegT and β-Catenin were co-injected, a higher luciferase activity was obtained. This activity was drastically inhibited by overexpression of Oct-25. (H) Overexpression of Oct-25 stimulated the Siamois promoter/reporter mutant that contains only the Oct binding site. Co-injection of VegT led to a repression. (I) Model for mesendoderm specification and patterning. POU-V factors antagonize VegT and β-Catenin, which induce and pattern the mesendodermal germ layer. In the animal region, Oct-25 promotes formation of neuroectoderm (see text for details). Org, organizer.