A novel mechanism for the transcriptional regulation of Wnt signaling in development - PubMed (original) (raw)

A novel mechanism for the transcriptional regulation of Wnt signaling in development

Tomas Vacik et al. Genes Dev. 2011.

Abstract

Axial patterning of the embryonic brain requires a precise balance between canonical Wnt signaling, which dorsalizes the nervous system, and Sonic hedgehog (Shh), which ventralizes it. The ventral anterior homeobox (Vax) transcription factors are induced by Shh and ventralize the forebrain through a mechanism that is poorly understood. We therefore sought to delineate direct Vax target genes. Among these, we identify an extraordinarily conserved intronic region within the gene encoding Tcf7l2, a key mediator of canonical Wnt signaling. This region functions as a Vax2-activated internal promoter that drives the expression of dnTcf7l2, a truncated Tcf7l2 isoform that cannot bind β-catenin and that therefore acts as a potent dominant-negative Wnt antagonist. Vax2 concomitantly activates the expression of additional Wnt antagonists that cooperate with dnTcf7l2. Specific elimination of dnTcf7l2 in Xenopus results in headless embryos, a phenotype consistent with a fundamental role for this regulator in forebrain development.

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Figures

Figure 1.

Figure 1.

Vax2 binds to an extremely conserved region in Tcf7l2 intron 5. (A) The first three exons and exons 5–8 of the mouse Tcf7l2 gene (Weise et al. 2010) are shown. Exon 1 encodes the β-catenin-binding domain. The Vax2-bound intron 5 region 152 kb downstream from the Tcf7l2 TSS is extraordinarily conserved through evolution, as shown by the Vista Browser plots. Regions in which a 100-bp sequence window exhibits >70% conservation are shaded (Mayor et al. 2000). (B, top bar graph) ChIP–chip analysis of the intron 5 region demonstrates enrichment by the Vax2 antibody relative to input sample. (Lower bar graph) No enrichment is observed with rabbit IgG. (Middle ChIP panel) ChIP confirms binding of Vax2 to regions covered by primer pairs 2–5 and 9–10. Agarose gel panels containing ChIP-PCR products amplified with primer pairs 1–11 are turned 90° from running direction (indicated by the arrow) to allow for alignment. (Bottom box diagram) Vax2-bound ChIP-PCR-positive regions are shown in blue. The sequence conservation plots of A, the ChIP–chip graphs, the ChIP-PCR panels 1–11, and the bottom box diagram of B are all in exact alignment. (C) Potential homeodomain-binding sites in intron 5 segments bound by Vax2 are highlighted. Numbers correspond to primer pairs/box diagram in B.

Figure 2.

Figure 2.

Truncated Tcf7l2 mRNAs originate in Tcf7l2 intron 5. (A) 5′ RACE reveals the existence of four alternative Tcf7l2 first exons in Tcf7l2 intron 5. Exons 1–17 of the mouse Tcf7l2 gene are shown in white (Weise et al. 2010). Alternative exons 4, 13, 14, 15, and 16 are marked with asterisks. Alternative first exons of the truncated Tcf7l2 mRNAs are shown in blue. Black arrows depict primers used in the qRT–PCR experiments, and blue arrows depict alternative TSSs for exons 1b–e. (B) qRT–PCR analysis of the expression of the alternative Tcf7l2 mRNAs 1b, 1c, 1d, and 1e during embryogenesis, relative to Hprt. Error bars are mean ± SD (n = 3). (C) Northern analysis of Tcf7l2 mRNAs at E13.5. (Left two lanes) A probe against exons 1–3 detects only the full-length Tcf7l2 mRNAs (∼4.1 kb) in both the E13.5 head and body. (Right two lanes) A probe against exons 6–10 detects the full-length mRNAs in both the head and body, and also the truncated Tcf7l2 mRNAs (∼3.4 kb, arrow), but only in the E13.5 head. (D) Whole-mount in situ hybridization with a probe against exon 1b reveals a strong diencephalic expression (D) of the alternative Tcf7l2 mRNA 1b at E11.5. (E) In situ hybridization on a coronal section through an E13.5 head reveals the expression of the Tcf7l2 mRNA isoform 1b in ganglion cells of the developing retina (RGC), the optic stalk (OS), and diencephalon (D).

Figure 3.

Figure 3.

The truncated Tcf7l2 mRNAs encode dominant-negative Tcf7l2. (A) Tcf7l2 and dnTcf7l2 exon structure, with important protein domains highlighted. Asterisks mark alternatively spliced exons. (B) A complex alternative usage of exons 13–16 gives rise to two major groups of Tcf7l2 isoforms (long proteins of ∼79 kDa and short proteins of ∼58 kDa), all of which are recognized by both the C9B9 and the C48H11 antibodies. (C) The alternative dnTcf7l2 mRNAs 1b and 1c encode a protein of ∼35 kDa, while the rare 1e isoform encodes a protein of ∼40 kDa. All of these truncated proteins are recognized only by the C48H11 antibody. (D) C9B9 antibody recognizes the 79-kDa and 58-kDa proteins in both the E13.5 head and body (first two lanes), but no protein translated in vitro from the truncated Tcf7l2 mRNA (third lane). The C48H11 antibody detects the 79-kDa and 58-kDa proteins in both the E13.5 head and body (fourth and fifth lanes) and also the 35-kDa protein, but only in the E13.5 head (fourth lane). (Sixth lane) This same 35-kDa protein is detected by C48H11 in protein translated in vitro from the pCS2-exon 1b + exon 6–14 + exon 16–17 expression vector. (E) Diagram of the TOPflash Wnt-responsive reporter construct, consisting of five Tcf/Lef-binding sites fused to a minimal promoter and the luciferase gene. (Top) Expression of this reporter is activated by β-catenin binding to full-length Tcf/Lef proteins. (Bottom) The truncated 35-kDa Tcf7l2 protein acts as a dominant-negative antagonist (dnTcf7l2) that cannot bind β-catenin; it instead interacts with corepressors. (F) Activation of the TOPflash luciferase reporter by transfected β-catenin is abolished by dnTcf7l2. β-Catenin expression vector was transfected into HEK293 cells in the absence (second bar) or presence (third bar) of the same amount (50 ng) of the dnTcf7l2 expression construct used for the in vitro transcription/translation reactions in D. Fold induction is the ratio between the normalized luciferase activity of the TOPflash and FOPflash constructs. Error bars are mean ± SD (n = 3).

Figure 4.

Figure 4.

Vax2-dependent expression of dnTcf7l2 and transcriptional corepressors. (A) In situ hybridization reveals that the Vax2 (left) and dnTcf7l2 1b (middle) mRNAs are coexpressed in the wild-type ventral retina at E13.5. (Right) dnTcf7l2 expression is lost in this region in the Vax2−/− mutant retina. (D) Dorsal; (V) ventral. (B) Truncated Tcf7l2 mRNAs 1b and 1c are down-regulated in Vax2−/− eyes, as determined by qRT–PCR. Expression of the full-length Tcf7l2 mRNA is unchanged, as determined using primers spanning exons 1–3 and exons 5–6. Fold change is the ratio between the relative mRNA expression normalized to Gapdh in the Vax2−/− mutant versus wild-type eyes. (C) Tle1, Hdac2, Gsk3β, Apc, and Ctbp1 mRNAs are expressed in the wild-type (WT) retina at E13.5, with strongest expression in the RGC layer, as shown by in situ hybridization. Their expression is lost in the Vax2−/− ventral retina. (D) Dorsal; (V) ventral. (D, left, top) Wnt signaling is not active in the E13.5 wild-type retina, except in the dorsal RPE, as shown by X-Gal staining of retinal sections from a BATgal reporter mouse. (Left, bottom) The BATgal reporter is derepressed in the ventral neural retina of a Vax2−/−/BATgal mouse. A schematic of the BATgal reporter transgene containing seven Tcf/Lef consensus binding sites fused to the siamois promoter and the lacZ gene (Maretto et al. 2003) in repressed (right, top) and activated (right, bottom) states. (E) Vax2 binds to the potential regulatory regions of the Wnt antagonists (A–E), as shown by ChIP analysis. Gel panels containing ChIP-PCR products are turned 90° from running direction, as indicated by the arrow. Candidate regulatory regions A–E are located at the indicated positions and contain the indicated clusters of potential Vax2 (homeodomain)-binding sites. A′–E′ indicate negative control regions surrounding each gene.

Figure 5.

Figure 5.

Vax2 activates endogenous dnTCF7L2 and represses Wnt signaling. (A) Expression of full-length TCF7L2 mRNA (exons 5–6) is unaffected by overexpressing mouse Vax2 in HEK293 cells, as determined by qRT–PCR. In contrast, the truncated TCF7L2 mRNAs 1b and 1e are up-regulated by full-length Vax2, but not by Vax2 lacking the activation SAFEPY motif (Vax2ΔAD). Fold change is the ratio between expression normalized to GAPDH in cells transfected with pCS2-Vax2 versus cells transfected with empty pCS2 vector. Error bars are mean ± SD (n = 3). (B) Vax2 up-regulates the enhancer activity of the F1R1 and F2R2 Vax2-bound regions in a luciferase reporter assay. The Vista Browser plot shows sequence conservation of the dnTcf7l2 regulatory regions between humans and chickens. The alternative Tcf7l2 first exons 1b and 1c lie immediately downstream from the Vax2-bound regions (blue squares). Fold induction is the ratio between the normalized luciferase activity of the insert containing reporter construct activated with Vax2 and that activated with empty expression vector. Error bars are mean ± SD (n = 3). (C) Both dnTcf7l2 and Vax2 are strong repressors of β-catenin activation of Wnt targets. While full-length Vax2 is able to repress the β-catenin-activated Wnt reporter with a potency comparable with dnTcf7l2, Vax2 lacking the SAFEPY activation motif (Vax2ΔAD) is not. Fold induction is the ratio between the normalized luciferase activity of the TOPflash and FOPflash reporter constructs. Error bars are mean ± SD (n = 3). (D) Vax2 is unable to repress an activated Notch-responsive CBF1-pGL2 reporter containing eight CBF1-binding sites. The reporter was activated by overexpressing the Notch intracellular domain (NICD). Fold induction is the ratio between the normalized luciferase activity of the wild-type and mutated reporter. Error bars are mean ± SD (n = 3).

Figure 6.

Figure 6.

DnTcf7l2 is essential for Xenopus forebrain development. (A) A 5′ RACE experiment in X. laevis reveals the existence of an alternative XTcf7l2 first exon in intron 5, which contains an ATG start codon in-frame with Ser 180 in XTcf7l2 exon 6. The positions of the translation-blocking (MO-ATG) and splicing-blocking (MO-SPL) morpholinos are indicated. Arrows represent the primers used for qRT–PCR. (B) In situ hybridization analysis of dnTcf7l2 expression at stages 13, 15, and 28 of X. laevis development. (A) Anterior; (P) posterior; (D) dorsal; (V) ventral; (bp) blastopore. (C) Knockdown of XdnTcf7l2 with MO-ATG or MO-SPL leads to embryos with severely truncated anterior head regions at stage 40. Phenotype ranges from moderate (gray bars), showing small head and small eyes, to strong (black bars), where no head and no eyes are present.

Figure 7.

Figure 7.

A transcriptional mechanism through which Sonic hedgehog may antagonize Wnt/β-catenin signaling in the developing eye. In the developing ventral diencephalon and eye, Vax proteins are induced by Shh (left), and these transcription factors in turn activate expression of the truncated, dominant-negative isoform of Tcf7l2 (middle). (Right) DnTcf7l2 then shuts down genes that are targets of canonical Wnt signaling. The extreme conservation of the dnTcf7l2 intron 5 enhancer/promoter suggests that additional transcription factors and signaling pathways may impinge on and regulate its activity elsewhere in the developing forebrain.

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