Massé K et al. (2010), The lysophosphatidic acid (LPA) and sphingosine...

XB-ART-41924

Int J Dev Biol 2010 Jan 01;548-9:1361-74. doi: 10.1387/ijdb.103068km.

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The lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) receptor gene families: cloning and comparative expression analysis in Xenopus laevis.

Massé K , Kyuno J , Bhamra S , Jones EA .

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Sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are endogenous bioactive lipids which mediate a variety of biological cell responses such as cell proliferation, migration, differentiation and apoptosis. Their actions are mediated by binding to the G-protein-coupled endothelial differentiation gene (Edg) receptor subfamily, referred to as S1P1-5 and LPA1-5, and regulate a variety of signalling pathways involved in numerous physiological processes and pathological conditions. Their importance during embryogenesis has been demonstrated by the generation of knock-out mice and specific roles have been assigned to these receptors. However, potential functional redundancy and the lethality of some mutants have complicated functional analysis in these models. Here we report the cloning of the S1P and LPA receptors in Xenopus laevis and tropicalis. Phylogenetic analyses demonstrate the high level of conservation of these receptors between amphibian and other vertebrate species. We have conducted a comparative expression analysis of these receptors during development and in the adult frog, by both RT-PCR and whole mount in situ hybridisation. In particular, we show that S1P1, 2 and 5 display distinct embryonic specific expression patterns, suggesting potentially different developmental roles for these receptors, and therefore for their ligands, during amphibian embryogenesis.

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Species referenced: Xenopus laevis
Genes referenced: en2 gast lpar1 lpar2 lpar3 lpar4 lpar5 nppa odc1 s1pr1 s1pr2 s1pr3 s1pr4 s1pr5

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	Fig. 5. Embryonic spatial expression profile of S1P genes. Whole mount in situ hybridisation with DIG-labelled RNA probe was performed on embryos from stages 6-41 for S1P genes, except for S1P4. (A) Whole mount in situ hybridisation analysis of S1P1 expression. Dorsal and lateral views of a cleared embryo at stage 23 (i, ii), of uncleared embryos at stage 27 (iii), at stage 32 (v, vi), at stage 41 (vii, ix). Sagital section (ix of the head at stage 41. Tranverse sections (ix, ix at stage 41. The dotted lines through the embryo (ix) correspond to planes of the transverse sections. Dorsal view of a stage 27 (iv) and stage 41 (viii) embryo stained with En2 antisense probe. The arrow indicates the midbrain/hindbrain boundary marked by En2 expression. For the lateral views, dorsal is up and anterior is left. fb: forebrain; hb: hindbrain; mb; midbrain; ns: neural system; nt: neural tube; op: otic placode; sc: spinal cord.
	Fig. 5. Embryonic spatial expression profile of S1P genes. Whole mount in situ hybridisation with DIG-labelled RNA probe was performed on embryos from stages 6-41 for S1P genes, except for S1P4. (B) Whole mount in situ hybridisation analysis of S1P2 expression. Lateral view of cleared em- bryos at stage 24 (i), stage 27 (ii). Dorso-lateral view of an uncleared embryo (iii) and lateral view of a cleared embryo (iv) at stage 32. Lateral view of a uncleared embryo at stage 40 (v). Detail of the stained somites of a cleared stage 40 embryo 40 (v. For the lateral views, dorsal is up and anterior is left. ba: branchial arches; h: hypophyseal anlagen; s: somites.
	Fig. 5. Embryonic spatial expression profile of S1P genes. Whole mount in situ hybridisation with DIG-labelled RNA probe was performed on embryos from stages 6-41 for S1P genes, except for S1P4. (C) Whole mount in situ hybridisation analysis of S1P5 expression. Anterior view of an embryo at stage 17 (i), lateral view at stage 24 (ii). Details of the head (iii) and lateral view (iv) of a stage 27 embryo. Details of the head (v) and lateral view (vi) of a stage 32 embryo. Lateral views at stage 37/38 of an embryo hybridized with the antisense probe (vii) or with the sense probe (viii). Lateral view of the stained stage 32 embryo (ix) used for the transverse sections (ix ix, ix. The dotted line through the embryo corresponds to plane of the sections. The arrow indicates the expression in the posterior notochord in the tail tip. For lateral views, dorsal is up and anterior is left. anp: anterior neural plate; df; dorsal fin; hb: hindbrain; l:lens; mb: midbrain; olp: olfactory placode; op: otic placode; s: somites.
	Fig. 6. Embryonic spatial expression profile of the LPA5 gene. Whole mount in situ hybridisation with DIG-labelled antisense (i) and sense (ii) LPA5 probes was performed. Expression was only detected at stage 6 in the animal pole (ap) of the embryos. Animal pole is up.
	Fig. 4. Temporal expression profile of LPA and S1P families genes. RT-PCR analysis showing expression pattern of the receptor transcripts in Xenopus laevis unfertilised eggs and embryos. Expres- sion of the LPA1.1, LPA1.2, LPA4 and S1P3 genes is constant during develop- ment. Expression of S1P1, 2 and 5 is first detected at stage 10.5 and increases until stage 45, the latest stage tested whereas the expression of S1P5 remains at approximately the same level during development. LPA2 is a maternal gene; its zygotic expression is also switched on at stage 10.5 and expressed until stage 45. Expression of LPA3 can be weakly detected during neurulation and increases until stage 45 whereas LPA5 is expressed at its highest level during maternal stages. S1P4 transcripts cannot be detected in this experiment. ODC was used as a loading control. The linearity was per- formed with doubling dilutions of cDNA from stage 45 embryos except stage 5 stage tested in this study. Maternal expression of LPA2 de- creases quickly to an almost undetectable level at stage 8 and its zygotic expression is detected from gastrulation throughout de- velopment. LPA5 expression is the highest before MBT but its zygotic transcripts can be amplified until stage 45. Zygotic expres- sion of LPA3 is switched on weakly during neurulation with an increase of expression at stage 37. However, its expression can be weakly detected during gastrulation with an increase of cycle numbers. Spatial expression of LPA and S1P gene families during development of Xenopus laevis The spatial expression of these genes in the embryo was assessed by two different complementary techniques. In situ hybridisation allows the identification of domains of high expres- sion of genes; RT-PCR on the other hand identifies regions of both high and low expression domains in dissected embryos. In situ hybridisation was performed on embryos from stage 6 to stage 40/41 with specific antisense probes (see Supplementary Table 3). Sense probes were used as controls. Zygotic expression of S1P1, S1P2 and S1P5 genes can be detected by in situ hybridisation and these receptors display distinct and different expression profiles during development (Fig. 5 A-C). S1P4 expression was not analysed by in situ hybridisation as its expression was only very weakly detected by RT-PCR. S1P1 expression is detected from stage 23 in the nervous system, in the brain and the neural tube as seen on cleared embryos (Fig. 5Ai,ii). This expression in the nervous system is more intense at stage 27 (Fig. 5-Aiii). Expression in the otic placode can also been observed from this stage. Neural expression can be more pre- cisely observed in the forebrain, midbrain, hindbrain and spinal cord from stage 32 (Fig. 5-Av, vi). Strong expression can be detected in all the rhombomeres of the hindbrain from stage 37 for LPA5, stage 15 for S1P5 and stage 25 for LPA2. The major phases of embryogenesis are indicated. Seg: Segmentation; Gast: Gastrulation.
	Fig. 8. Spatial expression profile of LPA and S1P families genes in the adult frog. RT-PCR was performed with total RNA extracted from Xeno- pus laevis adult tissues. Each gene of the two families shows a distinct ex- pression pattern. LPA1.1 and LPA1.2 present a similar ubiquitous expres- sion profile. LPA2, 4 and 5 were found expressed in almost all tissues stud- ied whereas the expression of LPA3 is very specific being expressed in only 3 tissues. The S1P family members are also expressed in almost all tissues studied, with the exception of S1P4 which has high levels of expression in spleen and bladder. EF1α was used as a loading control. The linearity was performed with doubling dilutions of cDNA from the following tissues: blad- der for LPA3; brain for S1P1; kidney for LPA1.1, LPA1.2 and LPA2; lung for S1P5; muscle for S1P2; ovary for S1P3, LPA4 and LPA5; spleen for S1P4.
	s1pr1 (sphingosine-1-phosphate receptor 1 ) gene expression in X. laevis, NF stage 41, dorsal view of the head, anterior left. Key: fb= forebrain; mb= midbrain; hb= hindbrain; sc= spinal cord. Note the segmental nature of the hiindbrain, with each rhombomere (R1-R8) visible.
	s1pr1 (sphingosine-1-phosphate receptor 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 23, (i) lateral view, anterior left, dorsal up, and (ii) sagittal section, lateral view, anterior left, dorsal up. ( nt=neural tube; ns=nervous system/brain).
	s1pr1 (sphingosine-1-phosphate receptor 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, dorsal view, anterior left. (op= otic vesicle, arrow at midbrain-hindbrain boundary).
	s1pr1 (sphingosine-1-phosphate receptor 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.
	s1pr1 (sphingosine-1-phosphate receptor 1 ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 41, lateral view, anterior left, dorsal up. (ix') sagittal section (ix’) of the head, (ix’’, ix’’’) transverse sections corresponding to dotted lines.
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, dorsal view, anterior left. Key: anp, anterior (pre-chordal) neural plate.
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up. key: olp: olfactory placode; op: otic placode
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, dorsoanterior view. Key :olp, olfactory placode; op, otic placode.
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 27, lateral view, anterior left, dorsal up.
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, dorsal view of head region, anterior left. Key: hb: hindbrain; l:lens placode; mb: midbrain; olp: olfactory placode; op: otic placode.
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up. Arrow indicates the expression in the posterior notochord in the tail tip.
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 37, lateral view, anterior left, dorsal up.
	s1pr5 (sphingosine-1-phosphate receptor 5) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up, and in cross-section at marked planes.
	s1pr2 (sphingosine-1-phosphate receptor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up. Key:ba: pharyngeal/branchial arches; h: hypophyseal anlagen; s: somites.
	s1pr2 (sphingosine-1-phosphate receptor 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 40, lateral view, anterior left, dorsal up (left), close up of tail (right)