Expression of Gpr177, a Wnt trafficking regulator, in mouse embryogenesis

Abstract

Wls/Evi/Srt encoding a multipass transmembrane protein has been identified as a regulator for proper sorting and secretion of Wnt in flies. We have previously demonstrated that Gpr177 is the mouse ortholog required for axis determination. Gpr177 is a transcriptional target of Wnt that is activated to assist its subcellular distribution in a feedback regulatory loop. We, therefore, proposed that reciprocal regulation of Wnt and Gpr177 is essential for the Wnt-dependent developmental and pathogenic processes. Here, we examine the expression pattern of Gpr177 in mouse development. Gpr177 is expressed in a variety of tissues and cell types during organogenesis. Furthermore, Gpr177 is a glycoprotein primarily accumulating in the Golgi apparatus in signal-producing cells. The glycosylation of Gpr177 is necessary for proper transportation in the secretory pathway. Our findings suggest that the Gpr177-mediated regulation of Wnt is crucial for organogenesis in health and disease.

Figure 1

Gpr177 transcript and protein are detected in multiple tissues and organs during mouse embryogenesis. In situ hybridization (A, B) and immunostaining (C, D) analyses show the expression pattern of Gpr177 in E14.5 and E13.5 embryos, respectively. Cb, cerebellum; ChP, choroid plexus; CC, cerebral cortex; CP, cartilage primordium; ccSC, central canal of spinal cord; DRG, dorsal root ganglia; Es, esophagus; Hc, hippocampus; Ht, hypothalamus; HB, hyoid bone; IE, inner ear; K, kidney; Lp, lip; Lu, lung; Mb, midbrain; MG, mid gut; MC, Meckel’s cartilage; OE, olfactory epithelium; Ov, ovary; PP, pancreatic primordium; RP, Rathke’s pouch; Tg, tongue; Th, thalamus; TP, tooth primordium; TyP; thymic primordium; Ur, urethra. Scale bar, 1 mm.

Figure 2

Gpr177 gene is active in neural development. The expression pattern of Gpr177 in various neural tissues and cell types of the developing embryos is analyzed by in situ hybridization (A–D, I–L; E14.5) and immunostaining (E–H; E13.5, M–P; E14.5). Cb, cerebellum; ChP, choroid plexus; CC, cerebral cortex; Hc, hippocampus; Ht, hypothalamus; Mb, midbrain; Me, medulla; SC, spinal cord; Th, thalamus. Scale bar, 200 μm.

Figure 3

Expression of Gpr177 during craniofacial morphogenesis. In situ hybridization (A, C, E, G, I, K, M, O, Q, S) and immunostaining (B, D, F, H, J, L, N, P, R, T) analyses reveal that a variety of craniofacial tissues and cell types express Gpr177 at E14.5. Co, cochlea; HF, hair follicle; Ln, lens; OE, olfactory epithelium; OM, ocular muscle; ON, optic nerve; OR, optic recess; P, palate; SC, semicircular canal; SG, salivary gland; SL, sublingual duct; SM, submandibular duct; Tg, tongue; TP, tooth primordium. Scale bars, 100 μm (A–D, G, H, S, T); 50 μm (Q, R); 200 μm (E, F, I–P).

Figure 4

Expression of Gpr177 in development of multiple organs. The mRNA and protein expression of Gpr177 is examined by in situ hybridization (A, B, E, G, I, K) and immunostaining (C, D, F, H, J, L) of E14.5 embryos, respectively. Insets in A, C denote higher magnification in B, D, respectively. Du, duodenum; Es, esophagus; K, kidney; Lu, lung; TyP, thymic primordium. Scale bars, 200 μm (A, C); 100 μm (B, E, F, I, K); 50 μm (D, G, H, J, L).

Figure 5

Glycosylation is required for Golgi distributions of Gpr177. (A) Neurosphere cells were treated with Tunicamycin (TM) for different concentrations and time periods as indicated. 3D imaging of the immunostained cells shows the localization of endogenous Gpr177 (green) and GM130 (red), counterstained with DAPI (blue). Note that the TM treatment disrupts the co-localization of Gpr177 and GM130 in Golgi. (B) Immunoblot analysis of Gpr177 reveals that TM abolishes the detection of Gpr177 with slow mobility (arrow) in neural stem cells. The number indicates the drug concentration (μg/ml) present in culture media. The expression level of Actin was analyzed as a loading control. (C) Immunoblot analysis shows that the slow migrating band (arrow) detected by the Gpr177 antibody disappears after the addition of Endo H_f. The number presents the amount of enzyme (μl: 1,000 units per μl) added to the in vitro assays. The expression level of Actin was analyzed as a loading control.