Characterization of G protein-coupled receptor 56 protein expression in the mouse developing neocortex

Abstract

GPR56, one of the adhesion G-protein-coupled receptors (GPCRs), plays an important role in the development of the cerebral cortex. Mutations in GPR56 cause a severe human cortical malformation called bilateral frontoparietal polymicrogyria (BFPP), characterized by a global malformation of the cerebral cortex that most severely affects the frontal and parietal regions. To characterize the expression pattern of GPR56 in the developing cerebral cortex, we developed a mouse monoclonal antibody against mouse GPR56. We revealed that GPR56 is expressed in multiple cell types in the preplate, marginal zone, subventricular zone (SVZ), and ventricular zone (VZ). Most interestingly, the expression of GPR56 in preplate neurons showed an anterior-to-posterior gradient at embryonic day (E) 10.5-11.5. In contrast, the expression pattern of the GPR56 ligand, collagen III, revealed no visible gradient pattern. With the widespread expression of GPR56 in the developing cortex, it is difficult to draw a specific conclusion as to which of the GPR56-expressing cells are critical for human brain development. However, the correlation between GPR56 expression in neurons at E10.5-E11.5 and the anatomic distribution of the cortical malformation in both humans and mice suggests that its function in preplate neurons is indispensible.

Figure 1

Mouse monoclonal antibody specifically recognizes GPR56. A: Western blot analysis of whole cell lysates of E14.5 Gpr56^+/+ and Gpr56^−/− brains. Novex Sharp prestained protein marker in SDS-PAGE gels is indicated in kDa. A protein band between 60 and 80 kDa was recognized by antibody H11 in the Gpr56^+/+, but not the Gpr56^−/− whole cell brain lysates. B: GPR56 IHC staining on coronal sections of E12.5 Gpr56^+/+ and Gpr56^−/− neocortex using H11 antibody. GPR56 expression (left panel) was detected in Gpr56^+/+ but not in the Gpr56^−/− (right panel) brains. Scale bar = 50 μm in B (applies to both parts).

Figure 2

Postmitotic neurons express GPR56. A–F, J–L: Antigens were retrieved for either 8 minutes (E12.5), 12 minutes(E14.5), or 14 minutes (E18.5). Double immunostaining of Tuj1 (magenta) and GPR56 (H11, green) on coronal sections of E12.5 (A–C), E14.5 (D–F), and E18.5 (J–L) neocortex. G–I, M–O: Higher magnification of boxed regions in D–F and J–L. GPR56 is expressed in the Tuj1-positive cell population, most prominently in the preplate at E12.5. By E14.5, only scattered subpial neurons remain positive for GPR56 (arrows in I). P–R: Double immunostaining of Tuj1 and GPR56 on 1DIV progenitor cells fixed in 95% ethanol and 5% glacial acetic acid for 10 minutes at −20°C. Some Tuj1-positve neurons express GPR56 (arrowheads in R). Scale bar = 25 μm in C (applies to A–C) and I (applies to G–I); 50 μm in F (applies to D–F) and L (applies to J–L); 10 μm in O (applies to M–O) and R (applies to P–R).

Figure 3

Expression pattern of collagen III in developing mouse cortex. A–F: Antigens were retrieved for 8 minutes and double immunostaining of collagen III (magenta) and GPR56 (H11, green) was performed on E10.5 (A–C) and E11.5 (D–F) sagittal mouse cortex. Collagen III was expressed in the meninges and pial BM of both E10.5 and E11.5 brains without visible anterior-to-posterior gradient. A–P and D–V axes are shown in C. Abbreviations: A, anterior; P, posterior; D, dorsal; V, ventral. Scale bar = 200 μm in A (applies to A–F).

Figure 4

Gradient expression pattern of GPR56 in early neocortex. A: Schematic representation of embryonic brain showing plane of sectioning (sagittal plane). The shadowed area indicates the region where the brain sections were obtained and analyzed. B–J: IHC of GPR56 (H11, gray) was performed on sagittal sections of E10.5 (B), E11.5 (E), and E12.5 (H) brains followed by antigen retrieval for 8 minutes. Higher magnification views of the boxed regions in B, E, and H for anterior (C,F,I) and posterior (D,G,J) regions of the neocortex. K: The relative fluorescent intensities of GPR56 expression in the region between the two arrowheads in B, E, and H were evenly divided into 10 bins and the intensity was quantified in preplate (left panel) and ventricular zone (VZ; right panel) by using the Image QuantTL program. Data are presented as mean ± SD; n = 3 for all groups. Two-tailed Student’s t-tests were performed with P values denoted as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.005. Anterior (A) to posterior (P) and dorsal (D) to ventral (V) axis is shown in B. Scale bar = 200 μm in B,E,H; 25 μm in C (applies to C,D), F (applies to F,G), and I (applies to I,J).

Figure 5

GPR56 expression is gradually decreased from anterior to posterior in Tuj1-positive postmitotic neurons. A–C, G–I: Double IHC of Tuj1 (magenta) and GPR56 (H11, green) was performed on sagittal sections of E10.5 (A–C) and E11.5 (G–I) brains. GPR56 expression (B) is parallel with Tuj1-positive postmitotic neurons (A) in the anterior region at E10.5 (C). In the E11.5 brains, GPR56 expression is highly correlated with the Tuj1-positive cell population in the anterior, but not in the posterior region (G–I). D–F, J–O: Higher magnification views of the boxed regions in A–C for anterior (D–F) and in G–I for anterior (J–L) and posterior (M–O). Scale bar = 200 μm in A (applies to A–C) and G (applies to G–I); 20 μm in D (applies to D–F) and J (applies to J–O).

Figure 6

Cajal-Retzius cells express GPR56. A–R: Double IHC of sagittal E11.5 mouse neocortex shows that reelin-positive (A) and calretinin-positive (J) CR cells are densely distributed in the anterior region, where GPR56 (D,M) is highly co-expressed with them (arrows in H,Q), whereas CR cells are sporadically populated in the posterior region, where GPR56 is largely negative. Higher magnification views of boxed regions in A, D, G, J, M, and P for anterior (B,E,H,K,N,Q) and posterior (C,F,I,L,O,R). S–V: 1DIV progenitor cells were fixed in cold acetone for 10 minutes at −20°C and immunostained with reelin (S, magenta) and GPR56 (199, T, green). Reelin is detected in some cells expressing GPR56 (V, arrowheads). Scale bar = 200 μm in A (applies to A,D,G,J,M,P); 10 μm in B (applies to B,C,E,F,H,I,K,L,N,O,Q,R) and S (applies to S–V).

Figure 7

Intermediate progenitor cells express GPR56. A–F: Immunostaining of Tbr2 (magenta) and GPR56 (green) was performed on coronal sections of E12.5 (A–C) and E14.5 (D–F) neocortex. G–I: Higher magnification views of the boxed regions in D–F. J–L: 1DIV progenitor cells fixed in 95% ethanol and 5% glacial acetic acid were immunostained with Tbr2 (J, magenta) and GPR56 (K, green). GPR56 expression is observed in Tbr2-positive cells (arrowheads in L). Scale bar = 25 μm in C (applies to A–C) and I (applies to G–I); 50 μm in F (applies to D–F); 10 μm in L (applies to J–L).