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.2012 Jun;139(12):2118-29.
doi: 10.1242/dev.076067. Epub 2012 May 9.

Function of Wnt/β-catenin in counteracting Tcf3 repression through the Tcf3-β-catenin interaction

Affiliations

Function of Wnt/β-catenin in counteracting Tcf3 repression through the Tcf3-β-catenin interaction

Chun-I Wu et al. Development.2012 Jun.

Abstract

The canonical Wnt/β-catenin signaling pathway classically functions through the activation of target genes by Tcf/Lef-β-catenin complexes. In contrast to β-catenin-dependent functions described for Tcf1, Tcf4 and Lef1, the known embryonic functions for Tcf3 in mice, frogs and fish are consistent with β-catenin-independent repressor activity. In this study, we genetically define Tcf3-β-catenin functions in mice by generating a Tcf3ΔN knock-in mutation that specifically ablates Tcf3-β-catenin. Mouse embryos homozygous for the knock-in mutation (Tcf3(ΔN/ΔN)) progress through gastrulation without apparent defects, thus genetically proving that Tcf3 function during gastrulation is independent of β-catenin interaction. Tcf3(ΔN/ΔN) mice were not viable, and several post-gastrulation defects revealed the first in vivo functions of Tcf3-β-catenin interaction affecting limb development, vascular integrity, neural tube closure and eyelid closure. Interestingly, the etiology of defects indicated an indirect role for Tcf3-β-catenin in the activation of target genes. Tcf3 directly represses transcription of Lef1, which is stimulated by Wnt/β-catenin activity. These genetic data indicate that Tcf3-β-catenin is not necessary to activate target genes directly. Instead, our findings support the existence of a regulatory circuit whereby Wnt/β-catenin counteracts Tcf3 repression of Lef1, which subsequently activates target gene expression via Lef1-β-catenin complexes. We propose that the Tcf/Lef circuit model provides a mechanism downstream of β-catenin stability for controlling the strength of Wnt signaling activity during embryonic development.

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Figures

Fig. 1.
Fig. 1.
Tcf3–β-catenin interaction is required after gastrulation. (A) Three-dimensional structure of the Tcf3–β-catenin interaction (Graham et al., 2000). Amino acid sequence of Tcf3 proteins fromXenopus and mouse are aligned above the structure. Red denotes Tcf3 residues of the core β-catenin interaction domain mutated in Tcf3ΔN. (B) Immunofluorescent staining detects nuclear co-localization in Cos-7 cells of endogenous β-catenin (green) and transiently transfected Tcf3 (red, top panels only). Expression of the Tcf3ΔN mutant protein (+Tcf3ΔN, right panels) failed to cause nuclear β-catenin localization. (C) Co-immunoprecipitation (IP) using a Tcf3-specific antibody and total cell lysate (TCL) from Cos-7 cells transiently transfected with the Tcf3 expression plasmid for wild-type (WT) (Tcf3), a mutant lacking the first 71 residues (Tcf3ΔN71), or theTcf3ΔN knock-in mutation. (D) Whole-mount in situ hybridization with a labeled brachyury cRNA probe onTcf3+/+ (WT),Tcf3ΔN/ΔN (knock-in, KI) andTcf3–/– (knockout, KO) embryos. Expression patterns and morphology of KI embryos resembled those of WT embryos, and KI embryos did not display the defects observed in KO embryos. (E) Recovery of embryos of the indicated genotypes obtained from timed pregnancies ofTcf3+/ΔN ×Tcf3+/ΔN matings.
Fig. 2.
Fig. 2.
Diverse requirements for Tcf3–β-catenin interaction during mouse embryogenesis. (A) Representative images showing externally visible abnormal phenotypes of E15.5 to E18.5 KI embryos. (B) The penetrance of abnormal phenotypes observed in KI mutants. NA, not applicable or not determined. (C-D′) IntactTcf3+/+ (C,D) and KI (C′,D′) E18.5 embryos. Arrow (C′,D′) points to externally visible hemorrhage. Low (C,C′) and high (D,D′) magnification views of the same region are shown. (E-F′) Hematoxylin and Eosin (H&E) staining of tissue sections of the neck from embryos in C-D′. The boxed region in E,E′ is shown at higher magnification in F,F′. Arrows (F,F′) indicate the internal carotid artery. Chevrons (E′) point to abnormal accumulation of blood in the KI embryo at the site of the externally visible hemorrhage. (G,H) H&E-stained tissue sections from submandibular salivary gland (G) and liver (H) of separate KI E18.5 embryos showing abnormal red blood cell accumulation. Sections of WT embryos are shown insupplementary material Fig. S1D,E.
Fig. 3.
Fig. 3.
Tcf3–β-catenin interaction is required for post-axial digit formation. (A-B′) Gross morphology of autopod limb segment in WT (A,B) and KI (A′,B′) mouse limbs at E16.5 (A,A′) and P1 (B,B′). Limbs were stained with Alizarin Red (B,B′) and numbers correspond to anterior (#1) to posterior (#5) digits. (C,C′) In situ hybridization of limb buds from E10.5 (C) and E11.5 (C′) WT embryos using a cRNA probe forTcf3. (D-E′) Whole-mount in situ hybridization using a cRNA probe forTbx2 mRNA on E11.5 (D,D′) and E12.5 (E,E′) embryos. The posterior edge ofTbx2 expression (arrows) in WT limb buds (D,E) was absent in KI limb buds (D′,E′). (F,F′) Tcf3–β-catenin activity was detected with the BAT-Gal transgene in WT (F) and KI (F′) limb buds at E10.0 [31-somite stage (ss)]. Arrows point to the high BAT-Gal activity in the posterior mesenchyme region of WT limb buds (F), which is absent from KI limb buds (F′).
Fig. 4.
Fig. 4.
Maintenance of Shh expression in the zone of polarizing activity requires Tcf3–β-catenin interaction. (A-B′) Whole-mount in situ hybridization using a cRNA probe specific forShh mRNA in E9.5 (A,A′) and E10.0 (B,B′) mouse embryos. Higher magnification views of forelimbs are shown in C-D′. (C-N′) Whole-mount in situ hybridization of WT (C-N) and KI (C′-N′) limb buds using labeled cRNA probes forShh (C-D′), patched 1 (E-F′),Fgf8 (G-H′), gremlin 1 (I-J′),Msx1 (K-L′) andMsx2 (M-N′). Two stages of development observed in E10 embryos are shown that correspond to the 30- to 31-somite (C,C′,E,E′,G,G′,I,I′,K,K′,M,M′) and 33- to 34-somite (D,D′,F,F′,H,H′,J,J′,L,L′,N,N′) stages.
Fig. 5.
Fig. 5.
Discrete requirement for Tcf3–β-catenin interaction in the eyelid. (A-B′) H&E staining of the eye and eyelids in WT (A,B) and KI (A′,B′) mouse embryos at E15.5 (A,A′) and E16.5 (B,B′). (C,C′) Tissue sections of E14.5 eyelids from BAT-Gal transgenic mice were stained with X-gal. The mucocutaneous junction (MCJ) stains positively for BAT-Gal activity (dotted line) in WT (C) and is reduced in size in KI (C′) eyelids. Solid lines at the basement membrane denote palpebral epidermis (PE) and palpebral conjunctiva (PC) regions (C). (D,D′) Immunofluorescence staining for β-galactosidase (red) and Ki67 (green) in E14.5 eyelids from BAT-Gal+ WT (D) and BAT-Gal+ KI (D′) embryos. The BAT-Gal activity (bar) is in Ki67-negative cells and its size is reduced in KI eyelids. (E-F″′) Immunofluorescence staining for Lef1 (E,F; red), Tcf3 (E′,F′; green), and β-galactosidase (E″,F″; blue) in E14.5 eyelids from BAT-Gal transgenic WT (E-E″′) and KI (F-F″′) embryos. Arrows point to Lef1-positive nuclei with low levels of Tcf3. Arrowheads point to Tcf3 and Lef1 double-positive nuclei, which were observed in WT (E-E″′) but not KI (F-F″′). The dotted line demarcates the BAT-Gal-positive region of the MCJ.
Fig. 6.
Fig. 6.
Wnt3a stimulates gene expression through regulation of Lef1 levels in ESCs. (A) Quantitative real-time PCR (qPCR) analysis of cDNA made after treating WT ESCs with 50 ng/ml Wnt3a for 12 hours. Values represent fold-changes to the mRNA levels ofTcf3 andLef1 relative to starting levels. (B,C) Quantitative ChIP-qPCR analysis of Tcf3 binding to target genesAxin2 andCdx1 in ESC chromatin from WT (B) or KI (C) ESCs treated with control or Wnt3a-conditioned media for 24 hours. Values represent the mean of five biological replicates ± s.d. Neg indicates negative control sites not bound by Tcf3. (D) SuperTOPFlash luciferase reporter assay in ESCs.Tcf3 KO ESCs were transiently transfected with expression plasmids for stable ΔNβ-catenin, Lef1 and/or Tcf3. SuperTOPFlash activity is shown compared with the Renilla luciferase control for each transfection. Values represent the mean of biological triplicates ± s.d. (E) The humanLEF1 promoter constructs used for transfections. Numbers refer to base pairs from theLEF1 translation start site. Numbers in parentheses refer to the equivalent sites in the mouseLef1 gene. (F) Quantitative ChIP-qPCR analysis of Tcf3 binding to different regions of the mouseLef1 promoter. Chromatin from KO ESCs was used as a negative control. Values represent the mean of three biological replicates ± s.d. (G) Quantitative ChIP-qPCR analysis of β-catenin binding to different regions of the mouseLef1 promoter. WT ESCs were treated with 50 ng/ml Wnt3a for 24 hours. Values represent the mean of three biological replicates ± s.d. (H,I)Lef1 promoter luciferase reporter plasmids (illustrated in E) were transiently transfected into KO ESCs with a Tcf3 expression plasmid or empty vector control. Cells were treated with Wnt3a-conditioned media or control conditioned media for 24 hours prior to processing for luciferase activity. Values represent the mean of biological triplicates ± s.d. (J,K)Lef1 promoter luciferase reporter constructs were cotransfected with three concentrations of Tcf3 expression constructs and treated for 24 hours with three concentrations of Wnt3a-conditioned media. Values represent the mean of biological triplicates ± s.d. (L) Model illustrating the role of balancing Tcf3 and Lef1 levels in mediating Wnt/β-catenin signaling. Genes are boxed; proteins are in ovals. Wnt/β-catenin-responsive target genes (WRG) are repressed (red line) by Tcf3 and activated (green line) by Lef1–β-catenin. Wnt inhibits Tcf3 repression of WRG andLef1 gene expression in a β-catenin-dependent manner.
Fig. 7.
Fig. 7.
Full-length Tcf3 represses Lef1 and BAT-Gal in the MCJ. (A-B″′) Immunofluorescent staining of E14.5 eyelids from Tcf3-overexpressing double-transgenic (A-A″′) and control single-transgenic (B-B″′) mouse embryos. Transgenic Tcf3 (A; green) was expressed in a mosaic pattern in double-transgenic embryos. BAT-Gal activity (A′; blue) and Lef1 expression (A″; red) was low in Tcf3-overexpressing cells (white arrowheads). In neighboring cells that did not overexpress Tcf3 (yellow arrows), BAT-Gal activity and Lef1 expression were similar to levels in the control embryo (B′,B″). (C,C′) The eye region of a control single-transgenic embryo (C) and a Tcf3-overexpressing bi-transgenic littermate (C′) at E16.5.
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