Wnt proteins bind to and co-localized with Gpr177. Three-dimensional imaging analysis of immunostained C57MG cells expressing MT-Wnt1 reveals co-localization (C) of endogenous Gpr177 (A) with MT-Wnt1 (B) in both Golgi and vesicles. D and D′ display different angle views of C where the sectioned level is shown by purple rectangle. (E and F) IP-IB analysis identifies protein complexes containing Gpr177 and Wnt in cells transfected by HA-Wnt1 (E) or MT-Gpr177 (F). (G) A scheme of GST-Gpr177 fragments. Numbers indicate positions of amino acids. Endoplasmic reticulum signal sequence and transmembrane domains are highlighted in green and blue, respectively. (H) GST pull-down assay analyzes the association of Gpr177 with the Wnt1, Wnt3, or Wnt5a protein complex.

Disruption of Gpr177 impairs embryogenesis in mice. (A–H) In situ hybridization in whole mounts (A–D) and sections (E–H) reveals the expression of Gpr177 in E6.25 (A, E, and F) and E7 (B), and E7.25 (C, D, G, and H) embryos. The approximate positions of E–H are shown by the dotted line in A and C. Gross morphological analysis of Gpr177+/+ (I and K) and Gpr177-/- (J and L) embryos at E7.5 (I and J) and E8.5 (K and L). Sections of the Gpr177+/+ (M, N, P–R, and T) and Gpr177-/- (O, S, and U), E7.5 (M–S), and E8.5 (T and U) embryos were analyzed by histology (M–O, T, and U) and immunostaining of Gpr177 (P–S). Arrowheads and arrows indicate the anterior and posterior mesoderm, respectively. AVE, anterior visceral endoderm; Ch, chorion; Ect, ectoderm; Epc, ectoplacental cone; NE, neural ectoderm; PS, primitive streak; VE, visceral endoderm; XEct, extraembryonic ectoderm. [Scale bars, 300 μm (A–D, I, and J); 100 μm (E–H and P–S); 500 μm (K and L); and 200 μm (M–O, T, and U).]

Wnt regulates cellular distribution of Gpr177. (A–D) Three-dimensional images of immunostained cells reveal differential localizations of endogenous Gpr177 in NEP (A), Neurosphere cells (B), MSC (C), and C57MG (D). Cut view (E–I) and 3-D imaging (J–O) analyses show that Gpr177 co-localizes with a Golgi marker GM130 using superimposed imaging (H and L) or pseudocoloring of the co-localization signal in white (I and M–O). NEP cells were immunostained with Gpr177 (E and J) and GM130 (F and K), and counterstained with DAPI (G–O). Three serial sections along the front-view (Co Level 1, 2, and 3) reveal the co-localization signal on the surface of Golgi (M–O). (P–T) High levels of Gpr177 lead to its accumulations in Golgi. Three-dimensional imaging of the endogenous Gpr177 in the vesicles of C3H10T1/2 cells (P). Expression of a myc-tag full length Gpr177 (MT-Gpr177) shows alteration in subcellular distribution of Gpr177 (Q), co-localizing with GM130 (R–T). Co-immunostaining of a MT-Gpr177 mutant lacking the carboxyl terminal region (U) and Calnexin (V) reveals their co-localization (W).

Gpr177 is required for Wnt production and signaling in patterning of A-P axis. (A–N and P–T) Molecular marker analysis of control (+/+ and +/-) and Gpr177 mutant (-/-) littermates at E6.5 (A and B) and E7.0–7.5 (C–N and P–T) determines the role of Gpr177 in early embryogenesis using in situ hybridization of BMP4 (A and B), Cer1 (C and D), Otx2 (E and F), Hesx1 (G and H), Gsc (I and J), Brachyury (T) (K and L), Wnt3 (M and N), and Axin2 (P and Q), and GFP analysis of Axin2 (R–T). The control embryos are shown with the anterior facing to the left. (O) Gpr177 is essential for Wnt production and signaling. Immunoblot analysis of E6.5 and E7.5 embryos shows the level of Gpr177, Wnt3/3a, and β-catenin proteins affected by the Gpr177 mutation. Actin level is used as a loading control. The number represents the relative protein level of Wnt3/3a and β-catenin between Gpr177+/+, Gpr177+/-, and Gpr177-/-. (R–T) Axin2^GFP mouse strain, expressing GFP in the Axin2-expressing cells, was used to examine the activation of Wnt/β-catenin signaling in the Gpr177+/+ (R and S) and Gpr177-/- (T) embryos during gastrulation. AVE, anterior visceral endoderm; DE, definitive endoderm; PS, primitive streak. [Scale bars, 200 μm (A and B) and 300 μm (C–N and P–T).]

Gpr177 is regulated by the canonical Wnt pathway. (A) Graphs illustrate the luciferase reporter constructs for Gpr177 promoter with the wild-type or mutation (cross) of LEF/TCF binding consensus sequences. (B) Expression of dominant activated catCLEF1 or Δβ-cat protein stimulates the transcription activity of a 3-kb Gpr177 promoter (P1) in 293T cells. Relative luciferase activity (RLA) determined the transcriptional activation of the Gpr177 promoter-luciferase construct. The analysis of pGL3, a parent vector, shows background activity. (C) Fold of induction shows the effect of catCLEF1 on transcriptional activation of the deletion mutants. (D) 293T cells were transfected by increasing amounts of DNA plasmid (1, 3, and 9 μg) to express catCLEF1, analyzed by immunoblot (IB). (E) ChIP analysis reveals high affinity LEF/TCF binding sites (nos. 4–5 and 6–7) in the Gpr177 promoter. The order number of these sites is color coded with those shown in A. NC is a negative control, which analyzes the regulatory region of Gpr177 without LEF/TCF binding sequence. The controls are direct PCR analyses of LEF/TCF binding sites without ChIP. (F) Analysis of the promoter constructs containing point mutations further reveals the consensus sites required for β-catenin and LEF/TCF-dependent transcription. (G) IB analysis indicates the Gpr177 level elevated in the primary MEC cells by the MMTV-Wnt1 transgene. The expression level of Actin was analyzed as a loading control. (H–K) β-gal staining of the virgin 2-month mammary glands in whole mounts (H and I) and sections (J and K) reveals the Wnt-dependent activation of Gpr177 in the mammary glands. The reporter expression from the Gpr177-lacZ knock-in allele was detected in the MMTV-Wnt1 transgenics (I and K) but not the controls (H and J). Three-dimensional imaging of the immunostained control (L) and MMTV-Wnt1 transgenic (M) MEC reveals distinct localization patterns of endogenous Gpr177. The endogenous Gpr177 distribution in C3H10T1/2 cells (N) is also altered by high levels of HA-Wnt3A (O). The insets show co-immunostaining of Gpr177 with Golgi markers, GM130 (M) and GS28 (O). Immunostained cells were counterstained by DAPI (blue). G, Golgi; V, vesicle. [Scale bars, 500 μm (H and I) and 50 μm (J and K).]