Mtgr1^−/− mice display an accumulation of cycling cells in the crypts of the small intestines and colons. (A) Proliferation in the small intestine. Proliferating cells were detected in sections of the jejunum from 8-week-old wild-type (WT) and Mtgr1-null mice by immunohistochemistry with anti-Ki-67. Positive staining is brown, with a blue hematoxylin counterstain. Graph shows the quantification of Ki-67-positive cells. Ki-67-positive cells from 100 consecutive histologically complete crypts were counted, and the average number of positive cells per crypt was calculated. Error bars, standard deviations. (B) Inactivation of Mtgr1 leads to the accumulation of cycling cells in the crypts of the colon. Proliferating cells were detected in sections of the colon from 8-week-old WT and Mtgr1-null mice by immunohistochemistry with anti-BrdU 2 h after administration of BrdU. Graph shows the quantification of the average number of positive cells per crypt. Error bars, standard deviations.

Mapping of TCF4 repression domains and TCF4 motifs that mediate MTG binding. (A) Schematic diagram of Gal4-TCF4 fusion proteins used. (B) Identification of TCF4 repression domains. NIH 3T3 cells were transfected with the Gal4-TK-luciferase reporter plasmid (0.2 ng) and Gal4-TCF4 or the indicated mutant (0.15 ng). A plasmid expressing secreted alkaline phosphatase was used to normalize for transfection efficiency. Con, control; Vec, vector. (C) Identification of Mtg16 binding sites on TCF4. Mtg16 and the GAL4-TCF4 deletion mutants were coexpressed in Cos7 cells and immunoprecipitated (IP) from whole-cell lysates using anti-GAL4 agarose beads, followed by Western blot (WB) analysis to detect the associated proteins.

(A) TCF4 associates with all of the members of the MTG family. Cos7 cells were cotransfected with pCMV5-HA-TCF4 and either vector alone (control [Con]), pCMV5-GAL-MTG8, pCMV5-GAL-Eto2 (MTG16), or pCMV5-GAL-Mtgr1 and were analyzed as described in the legend to Fig. 3B. An anti-HA immunoblot was used to detect the presence of HA-TCF4 (top panel) in lanes where an MTG family member was coexpressed (middle panel), but not in the vector-only lane. (B) Mtgr1 associates with TCF1, Lef1, and TCF4. GAL-Mtgr1 was coexpressed with the indicated TCF family members. The level of expression of the TCF was determined by immunoblot analysis of whole-cell lysates (WCL) (bottom panel). The expression and purification of Mtgr1 were confirmed by immunoblotting using anti-GAL4 after immunoprecipitation (IP) (middle panel). The Mtgr1-associated TCF family members were detected by immunoblotting (top panel). IgH, immunoglobulin H.

Loss of Mtgr1 activates the TCF4-regulated gene c-Myc. (A) RNA blot analysis of total RNA isolated from the small intestines of 8-week-old wild-type (WT) and Mtgr1-null littermates using probes for the TCF targets c-Myc, Cyclin D1, Id2, and c-Jun as indicated. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to control for RNA loading (lower panels). (B) c-Myc is overexpressed in the crypts of Mtgr1-null mice. Laser capture microdissection was used to isolate crypt epithelial cells, and quantitative RT-PCR was used to assess the levels of c-Myc mRNA in WT and Mtgr1-null small intestines. C_T, threshold cycle; the change in the C_T was calculated relative to β-actin. Shown are C_T values from two experiments each. The difference in C_T is equivalent to a 2.9-fold induction.

Mtgr1 associates with TCF4. (A) Mtgr1 interacts with TCF4 but not with Math1 or Hes1. Cos7 cells were cotransfected with plasmids encoding GAL-Mtgr1 and either FL-Math1, FL-Hes1, or HA-TCF4. Coimmunoprecipitation (IP) analysis using an anti-GAL4 monoclonal antibody linked to agarose beads was followed by immunoblot analysis using a mixture of anti-HA and anti-FLAG, which detected the associated TCF4 (top panel). Immunoblot analysis using anti-GAL4 and anti-HA plus anti-FLAG confirmed the expression of Mtgr1 (middle panel) and of Math1, Hes1, and TCF4 (bottom panel). (B) Endogenous Mtgr1 copurifies with TCF4. Whole-cell lysates (WCL) from K-562 cells were immunoprecipitated with anti-TCF4, and the associated MTGR1 was detected by immunoblot analysis. Lysates in the control lane (Con) were immunoprecipitated with normal mouse immunoglobulin G (IgG). Asterisks indicate background bands that were consistently observed in more than five experiments.

XETOR acts at the level of TCFs. (A) Epistasis analysis places XETOR downstream of β-catenin. β-catenin RNA (25 pg) induces secondary-axis formation (51%; 52/102; four independent experiments) when injected into one of the two ventral blastomeres at the 4-cell stage. Secondary-axis induction by β-catenin RNA injection (25 pg) was significantly impaired (25%; 29/118 embryos duplicated; four independent experiments) when XETOR RNA (3 ng) was coinjected. Results of a representative experiment are shown. (B) XETOR impairs β-catenin (β-cat)-mediated axis duplication in a dose-dependent manner. Embryos were injected ventrally at the 2- to 4-cell stage with 25 pg of β-catenin and increasing amounts of XETOR, and the percentage of axis duplication was determined. Expt., experiment. (C) XETOR impairs XLef1-induced axis duplication. Embryos at the 2- to 4-cell stage were injected in both ventral blastomeres with 0.75 ng XLef-1 mRNA either alone or together with 3 ng XETOR. XLef-1 resulted in axis duplication, which was not observed for uninjected sibling controls. Coinjection with XETOR mRNA significantly decreased the number of embryos with a duplicated axis. (D) XETOR impairs XLef-1 axis duplication in a dose-dependent manner. Embryos were injected ventrally with 0.75 ng XLef-1 and increasing amounts of XETOR mRNA as indicated, and the percentage of axis duplication was determined.

Mtgr1 is a corepressor for TCF4. (A) Expression of β-catenin disrupts the interaction between Mtgr1 or Mtg16 and TCF4. GAL-Mtgr1 or GAL-Mtg16 and HA-TCF4 were transfected into Cos7 cells with or without a β-catenin expression plasmid. Immunoprecipitation (IP) with anti-GAL was followed by anti-HA (αHA) immunoblotting to detect TCF4 (top panel). Anti-GAL immunoblot analysis was used to detect the expression of GAL-Mtgr1 in the immune complexes, while anti-HA immunoblotting confirmed that equal amounts of TCF4 were present in whole-cell lysates (WCL). The bottom panel shows an immunoblot with anti-β-catenin (αβ-cat), which detects both endogenous and overexpressed protein. (B) Mtgr1 impairs β-catenin-induced activation of a TCF reporter plasmid in a dose-dependent fashion. NIH 3T3 cells were transfected with the TOP-FLASH or FOP-FLASH (containing mutant TCF sites) reporter and either TCF4 alone, TCF4 plus β-catenin, or TCF4 plus β-catenin and either 100 or 250 ng of an Mtgr1 expression plasmid, as indicated. A plasmid expressing cytomegalovirus-secreted alkaline phosphatase was used to normalize for transfection efficiency. Numbers below the graphs indicate the amount of DNA in micrograms.

XETOR affects axis formation in Xenopus embryos. (A) Microinjection of XETOR ventralizes Xenopus embryos. Injection of XETOR RNA into the marginal zones of both dorsal blastomeres at the 4-cell stage (3 ng each) resulted in 92% (123/133; six independent experiments) of the embryos having a ventralized phenotype compared to uninjected sibling controls (left panel). As a positive control, injection of GSK-3β RNA (500 pg each) into the marginal zones of both dorsal blastomeres at the 4-cell stage also resulted in embryos having a ventralized phenotype (82%; 14/17). Embryos were injected with mRNA encoding β-galactosidase (β-Gal) as an injection control. An embryo stained for β-Gal is shown above an uninjected embryo for comparison. (B) Mammalian Mtg16 and MTG8 act similarly to XETOR. Microinjection of 3 ng of mRNA encoding Mtg16 (75%; 12/16 embryos) or MTG8 (61%; 37/60 embryos; four independent experiments) into the marginal zones of both dorsal blastomeres at the 4-cell stage ventralized the embryos as in the experiment for which results are shown in panel A, in contrast to uninjected sibling controls (left panel). (C) Xenopus embryos at the 2- to 4-cell stage were injected with increasing amounts of XETOR mRNA, as indicated, in both dorsal blastomeres, and DAI scores were determined. GSK3β was included as a positive control. The numbers below the graph indicate the amount of mRNA injected (in nanograms). (D) XETOR impairs β-catenin-dependent transactivation in vivo. Embryos at the 2-cell stage were either mock injected, injected with β-catenin alone, or coinjected with mRNAs encoding β-catenin and XETOR, and the levels of the Wnt target genes Siamois and Xnr3 were determined by RT-PCR analysis of the animal caps. Con (−RT), PCR lacking reverse transcriptase, used as a PCR control; Con (+RT), positive control for the PCR. (E) In vivo analysis of TCF-dependent transcription. The TOP-FLASH reporter plasmid was coinjected with mRNA encoding the indicated transcription factors. Luciferase activity was determined, in triplicate, from lysates made from pooled embryos (typically 5 embryos). β-cat., β-catenin. Results of one representative experiment out of four performed are shown.