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. 2010 Nov 18;5(11):e14047.
doi: 10.1371/journal.pone.0014047.

The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse

Helen Waller-Evans  1 , Simone Prömel, Tobias Langenhan, John Dixon, Dirk Zahn, William H Colledge, Joanne Doran, Mark B L Carlton, Ben Davies, Samuel A J R Aparicio, Johannes Grosse, Andreas P Russ
Affiliations

The orphan adhesion-GPCR GPR126 is required for embryonic development in the mouse

Helen Waller-Evans et al. PLoS One. .

Abstract

Adhesion-GPCRs provide essential cell-cell and cell-matrix interactions in development, and have been implicated in inherited human diseases like Usher Syndrome and bilateral frontoparietal polymicrogyria. They are the second largest subfamily of seven-transmembrane spanning proteins in vertebrates, but the function of most of these receptors is still not understood. The orphan Adhesion-GPCR GPR126 has recently been shown to play an essential role in the myelination of peripheral nerves in zebrafish. In parallel, whole-genome association studies have implicated variation at the GPR126 locus as a determinant of body height in the human population. The physiological function of GPR126 in mammals is still unknown. We describe a targeted mutation of GPR126 in the mouse, and show that GPR126 is required for embryonic viability and cardiovascular development.

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Conflict of interest statement

Competing Interests: JDi, DZ, JDo, MBLC, and JG are employees of Takeda Cambridge Ltd. SAJRA is a consultant for Takeda Cambridge Ltd. MBLC, SAJRA, and APR are founders of Paradigm Therapeutics Ltd., which was acquired by Takeda Cambridge Ltd. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. The domain architecture and genomic conservation of GPR126.
a. The domain architecture of GPR126 in comparison to other Adhesion-GPCRs. Sketches are drawn to scale of primary protein structure, the conserved domains of the N-terminus are colour-coded. The shaded box in mouse GPR126 depicts the region deleted in the targeted allele GPR126LacZ. The arrow in zebrafish GPR126 shows the point of truncation in gpr126(st49) . The closest homolog of GPR126 is GPR112, which shares the Pentaxin domain but lacks the CUB domain. In zebrafish, GPR126 and GPR112 are structurally more similar than in mammals, where GPR112 contains a very long coiled domain in its N-terminus. CUB: C1r/C1s, Uegf, Bmp1 domain; EGF: Epidermal Growth Factor domain; HRM: Hormone-binding motif. b. Schematic depiction of the conserved synteny surrounding the GPR126 locus in diverse vertebrate species. Note that the synteny of the zebrafish locus is highly conserved to reptiles, birds, and mammals, while other fish species show higher divergence. NMBR: neuromedin B receptor; GJE-1: gap junction protein epsilon 1 (pseudogene gje-1 in humans); VTA-1 Vps20-associated 1 homolog (S. cerevisiae); HIVEP-2: HIV enhancer binding protein 2; AIG-1: androgen-induced gene 1; E2F6: E2F transcription factor 6; MSRA: methionine sulfoxide reductase A; ASAP2; ArfGAP with SH3 domain, ankyrin repeat and PH domain 2; ITGB1BP1: integrin beta 1 binding protein 1: EDN-1; endothelin 1; NACHT: NACHT-NTPase containing protein; KIAA1737: unknown novel protein; ZNF410: zinc-finger protein 410.
Figure 2
Figure 2. Disruption of the mouse GPR126 gene by homologous recombination in embryonic stem cells.
a. Schematic depiction of the wild-type GPR126 locus, the targeting vector, and the targeted locus after homologous recombination. Numbered boxes represent GPR126 coding exons 15–19, LacZ reporter and neomycin-resistance gene expression cassettes are shown as shaded boxes. KpnI, and BglII restriction enzymes sites as used in b. Arrows depict PCR primers used in c. b. Southern blot showing correct targeting of the GPR126 locus in embryonic stem cells, using the hybridization probe shown in a. Correct integration creates a new KpnI fragment of ∼6 kb and increases the size of the BglII fragment to ∼ 12 kb. c. PCR analysis demonstrating the absence of GPR126 transcript (top row) and the deletion of the GPR126 genomic locus (bottom row). HPRT transcript was used as a control for cDNA concentration in RT-PCR. The genomic PCR used three primers to amplify the wild-type and mutant fragments in a single reaction. +/+; wild-type samples; +/−: heterozygote samples; −/−: homozygous mutant samples.
Figure 3
Figure 3. The expression pattern of the GPR126LacZ reporter allele.
Expression pattern of GPR126. Embryos carrying the GPR126LacZ allele were stained for β-galactosidase activity as whole-mount preparations. Heterozygous (a–c) and homozygous (d–f) embryos at 10.5, 11.5, and 12.5 dpf, respectively. (g–j): higher magnification view of expression in somitic region in a heterozygous embryo. Note the faint and transient expression. (k, l): comparison of heterozygous and homozygous embryo at 11.5 dpf. Note the change in expression pattern and intensity in the homozygous specimen. Scalebar (a–f) 2 mm, (g–l) 500 µm.
Figure 4
Figure 4. Expression of GPR126LacZ in the somite.
GPR126 is expressed in the somite. Heterozygous (a, b, d, e, g, h) and GPR126 null (c, f, i) embryos at 10.5 dpf were stained as whole-mount specimens for β-galactosidase activity to visualise GPR126 expression. Transverse cryostat sections of 20 µm thickness were then counter-stained with an α-SMA antibody. Note the interspersed expression of GPR126 and α-SMA with few double-positive cells. Overlay images (g–j) have been post-processed electronically to enhance visualisation. Scalebar = 50 µm.
Figure 5
Figure 5. GPR126 is expressed in the placenta.
Gpr126 is expressed in trophoblast giant cells, but the anatomy of the placenta is normal in GPR126LacZ homozygotes. 2 mm vibratome sections of placentas from wild-type, heterozygous and GPR126 null embryos at e10 and e11 were stained with X-Gal to visualise GPR126LacZ expression, cryosectioned at 20 µm and counterstained with Nuclear Fast Red. TGC - trophoblast giant cells, Sp - spongiotrophoblast, Lab - labyrinth. Scalebar = 100 µm.
Figure 6
Figure 6. The heart phenotype of GPR126LacZ mutants.
(a–d) GPR126LacZ mutants show dilation of the heart ventricle. Histological sections through the heart of heterozygous (a, b) and homozygous (c, d) specimens at 11.5 dpf, H&E stain. (b) and (d) are higher magnification views of the boxed area in (a) and (c). Note the thinning of the ventricular wall in (d) compared to (b) (arrows) and the presence of myocardial trabeculation. Scalebar = 100 µm. (e–h) GPR126LacZ is weakly expressed in embryonic heart. Homozygous embryos at 11.5 dpf were stained for β-galactosidase activity as whole-mount specimens to visualise GPR126 expression (e), cryosectioned at 20 µm and counter-stained with an α-SMA (f) or α- PECAM (h) antibodies. In (g) the α-SMA in green is overlayed with the lacZ signal of (e) false-coloured in red, showing little overlap between the two channels. The panels (e) and (g) were post-processed electronically to enhance the visualisation of the very weak lacZ signal.
Figure 7
Figure 7. The developing vasculature in GPR126LacZ embryos.
Patterning of blood vessels is normal in GPR126LacZ embryos. Wild-type (a, c, e, g, i) and homozygous GPR126LacZ (b, d, f, h, j) embryos at 10.5 dpf were stained as whole-mount specimens with an α-PECAM (a–h) or α-SMA (i, j) antibodies. (a, b) Overall patterning of the vasculature is not disrupted in mutant embryos. (c, d) The complexity of branching of cranial vessels is normal, as is the development of intersomitic vasculature (e, f). (g, h) The dorsal aortae form normally and smooth muscle cells are recruited to stabilize the vessels (i, j). Scalebar1 mm (a, b) 200 µm (e, f) and 50 µm (g–j).
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