Reciprocal regulation of Wnt and Gpr177/mouse Wntless is required for embryonic axis formation Jiang Fu1, Ming Jiang1, Anthony J. Mirando1, Hsiao-Man Ivy Yu, and Wei Hsu2 Department of Biomedical Genetics, Center for Oral Biology, James P Wilmot Cancer Center, University of Rochester Medical Center, 601 Elmwood Avenue, Box 611, Rochester, NY 14642 Edited by Kathryn V. Anderson, Sloan-Kettering Institute, New York, NY, and approved September 16, 2009 (received for review May 6, 2009) Members of the Wnt family are secreted glycoproteins that trigger Gpr177, the mouse orthologue of Drosophila Wls, required for cellular signals essential for proper development of organisms. Cel- embryogenesis. Disruption of Gpr177 disturbs axial patterning, a lular signaling induced by Wnt proteins is involved in diverse devel- phenotype resembling the loss of Wnt3. This disruption not only opmental processes and human diseases. Previous studies have affects Wnt production, but also interferes with Wnt signaling. As generated an enormous wealth of knowledge on the events in a Wnt transcriptional target, Gpr177 is elevated to promote Wnt signal-receiving cells. However, relatively little is known about the production in a positive feedback loop. Our results indicate that a making of Wnt in signal-producing cells. Here, we describe that reciprocal regulation of Wnt and Gpr177 is essential for the Gpr177, the mouse orthologue of Drosophila Wls, is expressed during establishment of the mammalian A-P axis. formation of embryonic axes. Embryos with deficient Gpr177 exhibit defects in establishment of the body axis, a phenotype highly remi- Results niscent to the loss of Wnt3. Although many different mammalian Wnt Gpr177 Is Essential for Mouse Embryogenesis. We investigated how proteins are required for a wide range of developmental processes, many Wls exist, and whether Wls regulates the Wnt pathway the Wnt3 ablation exhibits the earliest developmental abnormality. essential for mammalian development. By protein sequence This suggests that the Gpr177-mediated Wnt production cannot be analysis (NCBI HomoloGene) we found that Gpr177, which substituted. As a direct target of Wnt, Gpr177 is activated by ␤-catenin shows high percentages of identity with Wls of human (96.1%), and LEF/TCF-dependent transcription. This activation alters the cellu- Drosophila (44.9%), and C. elegans (40.9%), is likely the mouse lar distributions of Gpr177 which binds to Wnt proteins and assists orthologue. No additional gene product shared significant ho- their sorting and secretion in a feedback regulatory mechanism. Our mology with fly and human counterparts. We then performed findings demonstrate that the loss of Gpr177 affects Wnt production whole mount RNA in situ hybridization to examine Gpr177 in the signal-producing cells, leading to alterations of Wnt signaling expression in early embryogenesis. Gpr177 was expressed in the in the signal-receiving cells. A reciprocal regulation of Wnt and proximal epiblast at the junction between the embryonic and Gpr177 is essential for the patterning of the anterior-posterior axis extraembryonic tissue, and later was more restricted to the during mammalian development. primitive streak and mesoderm extending to the distal tip of the embryo (Fig. 1 A–D). Its strong presence was found in both A-P axis ͉ ␤-catenin ͉ developmental deformities ͉ primitive streak ͉ posterior visceral endoderm and epiblast at the prestreak, but Wnt production switched to the mesoderm at late-streak (Fig. 1 E–H). The expression pattern of Gpr177 is reminiscent of Wnt3, which is embers of the Wnt family are secreted glycoproteins which required for axial patterning (5, 23). To determine whether Gpr177 regulates Wnt production and trigger cellular signals essential for proper development of M this regulation is essential for mouse development, we created a organisms (1, 2). Aberrant regulation of an evolutionary conserved mutant strain Gpr177lacZ, carrying an insertion of ␤-geo into the Wnt signal transduction pathway has been implicated in a variety of ninth intron of Gpr177 (Fig. S1 A and B). The transgene insertion cancers and congenital diseases (3, 4). There is no question that disrupted the seven transmembrane domain which results in Wnt signaling is intimately involved in human health and disease. generation of a fusion transcript (Fig. S1C). PCR analyses Genetic analysis in mice has revealed the essential role of different further confirmed that the Gpr177 locus was altered by trans- Wnt proteins in a wide range of developmental processes (http:// gene-mediated mutagenesis (Fig. S1D). The mutant lacking the www.stanford.edu/ϳrnusse/wntwindow.html). Wnt3 deficiency ap- carboxyl-terminal region of Gpr177 disabled its function as the pears to cause the earliest abnormality during embryogenesis, truncation interrupts its subcellular distribution and protein suggesting the importance of Wnt signaling in axis determination interaction (see below). Mice heterozygous for Gpr177lacZ ap- (5). Three body axes develop sequentially to generate embryo peared normal and were fertile. We were not able to recover orientations (6–8). At the egg cylinder stage, the dorsal-ventral axis Gpr177 homozygous (Gpr177-/-) newborns or embryos after is the first to form. The anterior-posterior (A-P) axis is established E10.5, suggesting that they died during early embryogenesis. to form the primitive streak at the posterior end before gastrulation. From E6.5 to E8.5, Gpr177ϩ/ϩ and ϩ/- embryos consisted of Lastly, the left-right asymmetry is formed, followed by embryo three germ layers and underwent gastrulation to form organized turning. Wnt signaling is critical for initiation of the embryonic axes in structures, including extraembryonic ectoderm, chorion, allan- early development (9–11). Disruptions of key molecules necessary for tois, head folds, and primitive streak (Fig. 1 I, K, M, N, and T). Wnt signaling regulation lead to defects in axial patterning (12–15). Studies in the past have generated an enormous wealth of knowledge on the events in signal-receiving cells. Before initiating Author contributions: J.F., M.J., A.J.M., H.-M.I.Y., and W.H. designed research; J.F., M.J., their effects on the signal-receiving cells, Wnt proteins undergo A.J.M., H.-M.I.Y., and W.H. performed research; J.F., M.J., A.J.M., H.-M.I.Y., and W.H. proper modification, sorting and secretion processes in the signal- analyzed data; and A.J.M. and W.H. wrote the paper. producing cells (16–19). Recent identification of Wntless (Wls/Evi/ The authors declare no conflict of interest. Srt) (20–22), a regulator for Wnt production in Drosophila, has This article is a PNAS Direct Submission. directed attention to the maturation, sorting and secretion pro- 1J.F., M.J., and A.J.M. contributed equally to this work. cesses in signal-producing cells. Given the extensive Wnt family in 2To whom correspondence should be addressed. E-mail: wei࿝[email protected]. higher organisms, it is not clear how many Wls genes are present and This article contains supporting information online at www.pnas.org/cgi/content/full/ whether Wls is essential for Wnt production. This study describes 0904894106/DCSupplemental. 18598–18603 ͉ PNAS ͉ November 3, 2009 ͉ vol. 106 ͉ no. 44 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0904894106 Downloaded by guest on September 27, 2021 A BCD ABC D E FGH E F GH I J K L I J K L MNOT M NO P Q S U PQRST Fig. 1. 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 Gpr177ϩ/ϩ (I and K) and Gpr177-/- (J and L) Fig. 2. Gpr177 is required for Wnt production and signaling in patterning of A-P embryos at E7.5 (I and J) and E8.5 (K and L). Sections of the Gpr177ϩ/ϩ (M, N, axis. (A–N and P–T) Molecular marker analysis of control (ϩ/ϩ and ϩ/-) and BIOLOGY Gpr177 mutant (-/-) littermates at E6.5 (A and B) and E7.0–7.5 (C–N and P–T) P–R, and T) and Gpr177-/- (O, S, and U), E7.5 (M–S), and E8.5 (T and U) embryos DEVELOPMENTAL were analyzed by histology (M–O, T, and U) and immunostaining of Gpr177 determines the role of Gpr177 in early embryogenesis using in situ hybridization (P–S). Arrowheads and arrows indicate the anterior and posterior mesoderm, of BMP4 (A and B), Cer1 (C and D), Otx2 (E and F), Hesx1 (G and H), Gsc (I and J), respectively. AVE, anterior visceral endoderm; Ch, chorion; Ect, ectoderm; Epc, Brachyury (T)(K and L), Wnt3 (M and N), and Axin2 (P and Q), and GFP analysis of ectoplacental cone; NE, neural ectoderm; PS, primitive streak; VE, visceral Axin2 (R–T). The control embryos are shown with the anterior facing to the left. endoderm; XEct, extraembryonic ectoderm. [Scale bars, 300 ␮m(A–D, I, and J); (O) Gpr177 is essential for Wnt production and signaling. Immunoblot analysis of 100 ␮m(E–H and P–S); 500 ␮m(K and L); and 200 ␮m(M–O, T, and U).] E6.5 and E7.5 embryos shows the level of Gpr177, Wnt3/3a, and ␤-catenin pro- teins affected by the Gpr177 mutation. Actin level is used as a loading control. The number represents the relative protein level of Wnt3/3a and ␤-catenin between However, Gpr177-/- embryos did not exhibit distinct structures Gpr177ϩ/ϩ, Gpr177ϩ/-, and Gpr177-/-. (R–T) Axin2GFP mouse strain, expressing but remained to grow as egg cylinders (Fig. 1 J and O). The GFP in the Axin2-expressing cells, was used to examine the activation of Wnt/␤- ϩ ϩ mutants consisted of two layers of tissue, ectoderm and visceral catenin signaling in the Gpr177 / (R and S) and Gpr177-/- (T) embryos during gastrulation.