Seigfried FA et al. (2018), Expression of the adhesion G protein-coupled re...

XB-ART-54608

Gene Expr Patterns 2018 Jun 01;28:54-61. doi: 10.1016/j.gep.2018.02.002.

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Expression of the adhesion G protein-coupled receptor A2 (adgra2) during Xenopus laevis development.

Seigfried FA , Dietmann P , Kühl M , Kühl SJ .

Abstract
The adhesion G protein-coupled receptor A2 (Adgra2) is a seven transmembrane receptor that has been described to be a regulator for angiogenesis in mice. Furthermore, the zebrafish ouchless mutant is unable to develop dorsal root ganglia through a disrupted trafficking of Adgra2. Besides RNA sequencing data, nothing is reported about Adgra2 in the south African crawled frog Xenopus laevis. In this study, we investigated for the first time the spatio-temporal expression of adgra2 during early Xenopus embryogenesis in detail. In silico approaches showed that the genomic adgra2 region as well as the Adgra2 protein sequence is highly conserved among different species including Xenopus. RT-PCR experiments confirmed that embryonic adgra2 expression is primarily detected at the beginning of neurulation and is then present throughout the whole Xenopus embryogenesis until stage 42. Whole mount in situ hybridization approaches visualized adgra2 expression in many tissues during Xenopus embryogenesis such as the cardiovascular system including the heart, the migrating neural crest cells and the developing eye including the periocular mesenchyme. Our results indicate a role of Adgra2 for embryogenesis and are a good starting point for further functional studies during early vertebrate development.

PubMed ID: 29462671
Article link: Gene Expr Patterns

Species referenced: Xenopus laevis
Genes referenced: adgra1 adgra2 adgra3 brf2 erlin2 gprc6a plpbp prl rab11a rab11fip1 shf slc12a3 XB982628 znf703

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	Fig. 1. Synteny and phylogenetic analyses. a. Synteny analysis of the adhesion G protein-coupled receptor a2 (Adgra2) gene and its neighboring genes in Homo sapiens (Chr. 8), Rattus norvegicus (Chr. 16), Mus musculus (Chr. 8), Gallus gallus (Chr. 22), Xenopus laevis (Chr. 3), Xenopus tropicalis (Chr. 3) and Danio rerio (Chr. 8). Schematic overview of the adgra2 gene region within different species. Arrows indicate the orientation of the genes, conserved genes are depicted in similar colors, non-conserved genes are drawn in grey. Gene length as well as the distances between the single genes are not in scale. adrb3: adrenoceptor beta 3; brf2: RNA polymerase II transcription factor 50â€¯kDa subunit; Chr.: chromosome; eif4ebp1: eukaryotic translation initiation factor 4E binding protein; erlin2: ER lipid raft associated 2; got1l1: glutamic-oxaloacetic transaminase 1-like 1; pdlim2: PDZ and LIM domain 2; prlhr2a: prolactin releasing hormone receptor 2a; prlhel: prolactin releasing hormone receptor like; npm2a: nucleophosmin/nucleoplasmin 2a; prosc: proline synthetase co-transcribed homolog; rab11fip1: Rab11 family interacting protein 1; sorbs3: sorbin and SH3 domain containing 3; zfp703: zinc finger protein 703; znf703: zinc finger protein 703. b. Phylogenetic tree of all three Adgra amino acid sequences within different species. Individual branch lengths are given in the panel. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 2. Adgra2 protein. a. Schematic overview of X. laevis Adgra2 protein precursor (NP_001083670.1). Individual domains are highlighted in different colors and amino acid (aa) numbers are given for orientation within the protein. The region of binding of the WMISH probe is highlighted in purple. b. Primary protein sequence of X. laevis Adgra2. Different domains are highlighted as in a. c. Amino acid comparison of Adgra2 sequences between different species. Given are the total numbers of aa as well as the percentage of identity of the overall protein sequences and different domain sequences compared to Homo sapiens ADGRA2. Colors are used similar to a. GPCR: G protein-coupled receptor; GPS: GPCR proteolysis site; HRD: hormone receptor domain; IG: immunoglobulin; LRR: leucine rich repeat; PDZ: PDZ binding domain; SP: signal peptide; 7TMR: seven transmembrane receptor domain. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
	Fig. 3. Temporal expression pattern of adgra2 during Xenopus laevis development analyzed by reverse transcriptase (RT)-PCR. A weak adgra2 signal is firstly detected at stage 15 which becomes stronger at stage 22. Starting from stage 24, adgra2 is continuously expressed during the whole embryonic development until stage 42. Glyceraldehyde-3-phosphate dehydrogenase (gapdh) was used as loading control, and reactions lacking reverse transcriptase (RT) as negative control. As template DNA, cDNA of indicated embryonic stages were used.
	Fig. 4. Spatial expression pattern of adgra2 mRNA during Xenopus laevis embryogenesis. Spatial expression pattern of adgra2 is visualized by WMISH. Embryonic stages are indicated in each panel. a-c. Lateral views with animal to the top. d-f. Anterior views with dorsal to the top. g-j. Lateral views with anterior to the left. f, g. The first tissue specific adgra2 expression starts at stage 23 in the periocular mesenchyme (POM), the migrating neural crest cells (NCC) and the intersomitic arteries (IA). h. At stage 25, adgra2 is expressed in the periocular mesenchyme (POM), the migrating cranial neural crest cells (NCC) and the intersomitic arteries (IA). i. At stage 30, adgra2 was detected in the branchial (BA), hyoidal (HA) and mandibular (MA) arches, the periocular mesenchyme (POM), the intersomitic arteries (IA) and the posterior cardinal vein (PCV). j. At stage 35 adrga2 transcripts were detected in the pharyngeal arches (BA, HA and MA), in the intersomitic arteries (IA), the vascular system (VS), the posterior cardinal vein (PCV) and the proctodeum (PD). a, animal; A, anterior; BA, branchial arches; BP, blastoporus; D, dorsal; HA, hyoidal arch; IA, intersomitic arteries; MA, mandibular arch; NCC, neural crest cells; P, posterior; PCV, posterior cardinal vein; PD, proctodeum; POM, periocular mesenchyme; v, vegetal; V, ventral. Scale bars: 500â€¯Î¼m (aâ€“h): 1â€¯mm (i, j).
	Fig. 5. Sections of adgra2 stained Xenopus early tadpoles. Spatial expression pattern of adgra2 is visualized by WMISH followed by cross sectioning of Xenopus embryos. a, h. Lateral views with anterior to the left at indicated stages. Dashed lines indicate the level of sections. b-g. Transversal (b-d, g) and horizontal (e, f) vibratome sections at stage 23. b. adgra2 is present in the periocular mesenchyme (POM). c, d, f. The migrating cranial neural crest cells (NCC) show adgra2 expression. e, g. adgra2 transcripts in the intersomitic arteries (IA). i-s. Horizontal (iâ€“m) and transversal (nâ€“s) vibratome sections at embryonic stage 25. i, j, p, r. adgra2 is visible in the intersomitic arteries (IA). k, m. At stage 25, adgra2 is detected in the branchial (BA) and hyoidal arches (HA). l. adgra2 transcripts in the posterior cranial vein (PCV). n. adgra2 is present in the periocular mesenchyme (POM). o. adgra2 is visualized in the mandibular arches (MA). q. Adgra2 transcripts are present in the second heart field (SHF). s. adgra2 is expressed in the developing myocardium (MC). A, anterior; BA, branchial arch; CG, cement gland; D, dorsal; HA, hyoidal arch; HG, hindgut; IA, intersomitic arteries; MA, mandibular arch; P, posterior; PCV, posterior cardinal vein, POM, periocular mesenchyme; S, somite; SHF, second heart field; SC, spinal cord; V, ventral. Scale bars: 1â€¯mm (a, g).
	Fig. 6. Sections of adgra2 stained Xenopus late tadpoles. Spatial expression pattern of adgra2 is visualized by WMISH followed by cross sectioning of Xenopus embryos. a/j/q. Lateral views with anterior to the left at indicated stages. Dashed lines indicate the level of sections. b-g. Transversal (b, d, e, h, i) and horizontal (c, f, g) vibratome sections at stage 30. b. adgra2 is present in the periocular mesenchyme (POM). c. adgra2 transcripts are localized in the intersomitic arteries (IA). d. adgra2 is visualized in the mandibular arches (MA). e. adgra2 is expressed in the myocardium (MC) of the developing heart. f. adgra2 expression in the hyoidal (HA) and branchial arches (BA). g. Caudal section with adgra2 transcripts in the intersomitic arteries (IA). h. adgra2 is expressed in the myocardium (MC). i. adgra2 is detected in the posterior cardinal vein (PCV) and the intersomitic arteries (IA) surrounding the somites (S). k-p. Transversal vibratome sections at stage 33/34. k. adgra2 expression is detected in the periocular mesenchyme (POM). l. adgra2 expression in the intersomitic arteries (IA) and the endothelium of the posterior cardinal vein (PCV). m. adgra2 is present in the mandibular arch (MA). n-p. Transcripts of adgra2 are visualized in the heart including the second heart field (SHF), the myocardium (MC), the pericardial roof (PR), the endocard (EC) and the pericard (PC). r-u. Transversal sections at stage 35/36. r. adgra2 is visualized in the mandibular arch (MA). s-t. adgra2 expression is visible in the second heart field (SHF), myocardium (MC), the pericardial roof (PR), and the pericard (PC). r-u. adgra2 transcripts are found in the vascular system (VS) nearby the pronephric tubule (PT). A, anterior; BA, branchial arch; CG, cement gland; D, dorsal; DL, developing lens; DR, developing retina; EC, endocard; HA, hyoidal arch; HG, hindgut; IA, intersomitic arteries; MA, mandibular arch; MC, myocardium; OC, optic cup; P, posterior; PC, pericardium; PCV, posterior cardinal vein; POM, periocular mesenchyme; PR, pericardial roof; PT, pronephric tubule; S, somite; SC, spinal cord; SHF, Second heart field; V, ventral; VS, vascular system; YE, yolk endoderm. Scale bars: 1â€¯mm (a, j, q).
	Fig. S1. Staining using an adgra2 antisense and sense mRNA probe. Comparison of the staining using an adgra2 antisense (a-d) and sense (aâ€™-dâ€™) probe by WMISH. Embryonic stages are indicated in each panel. Shown are lateral views with anterior to the left. Scale bars: 500â€¯Î¼m (a, b, aâ€™, bâ€™): 1â€¯mm (c, d, câ€™, dâ€™) A, anterior; D, dorsal, P, posterior, V, ventral.
	Fig. S2. Vibratome sections of adgra2 stained early Xenopus embryos. Spatial expression pattern of adgra2 was investigated by WMISH followed by cross sectioning of Xenopus embryos. a. animal view. c, f. Lateral views with anterior to the left at indicated stages. Dashed lines indicate the level of sections. Transversal (b, g, h) and horizontal (d, e, i) vibratome sections. No adgra2 transcript could be detected in this early stages. A, anterior; BC, blastocoel; BL, blastopore lip; BP, blastopore; D, dorsal; NC, notochord; NG, neural groove; NT, neural tube; OV, optic vesicle; P, posterior; V, ventral.
	adgra2 (adhesion G protein-coupled receptor A2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, anterior view, left, dorsal up.
	adgra2 (adhesion G protein-coupled receptor A2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, anterior view, left, dorsal up.
	adgra2 (adhesion G protein-coupled receptor A2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, lateral view, anterior left, dorsal up.
	adgra2 (adhesion G protein-coupled receptor A2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anterior left, dorsal up.
	adgra2 (adhesion G protein-coupled receptor A2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, transverse section through the pharyngeal region, dorsal up.
	adgra2 (adhesion G protein-coupled receptor A2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, transverse section through the posterior pharyngeal region, dorsal up.


	adgra2 (adhesion G protein-coupled receptor A2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 33 & 34, lateral view, anterior left, dorsal up