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The Sphingosine-1-phosphate Receptors S1P1, S1P2, and S1P3 Function Coordinately during Embryonic Angiogenesis*

      Sphingosine-1-phosphate (S1P) elicits diverse cellular responses through a family of G-protein-coupled receptors. We have shown previously that genetic disruption of the S1P1 receptor, the most widely expressed of the family, results in embryonic lethality because of its key role within endothelial cells in regulating the coverage of blood vessels by vascular smooth muscle cells. To understand the physiologic functions of the two other widely expressed S1P receptors, we generated S1P2 and S1P3 null mice. Neither the S1P2 null mice nor the S1P3 null mice exhibited significant embryonic lethality or obvious phenotypic abnormalities. To unmask possible overlapping or collaborative functions between the S1P1, S1P2, and S1P3 receptors, we examined embryos with multiple S1P receptor mutations. We found that S1P1 S1P2 double null and S1P1 S1P2 S1P3 triple null embryos displayed a substantially more severe vascular phenotype than did embryos with only S1P1 deleted. We also found partial embryonic lethality and vascular abnormalities in S1P2 S1P3 double null embryos. Our results indicate that the S1P1, S1P2 and S1P3 receptors have redundant or cooperative functions for the development of a stable and mature vascular system during embryonic development.
      Sphingosine-1 phosphate (S1P)
      The abbreviations used are: S1P, sphingosine-1 phosphate; BSA, bovine serum albumin; E, embryonic day; ES cells, embryonic stem cells; PBS, phosphate-buffered saline; PBST, PBS plus Triton X-100; PECAM-1, platelet/endothelial cell adhesion molecule-1.
      1The abbreviations used are: S1P, sphingosine-1 phosphate; BSA, bovine serum albumin; E, embryonic day; ES cells, embryonic stem cells; PBS, phosphate-buffered saline; PBST, PBS plus Triton X-100; PECAM-1, platelet/endothelial cell adhesion molecule-1.
      is a sphingolipid metabolite that is present at high levels in the blood (
      • Hla T.
      ,
      • Watterson K.
      • Sankala H.
      • Milstien S.
      • Spiegel S.
      ,
      • Yatomi Y.
      • Ozaki Y.
      • Ohmori T.
      • Igarashi Y.
      ). Through the interaction with a family of five G-protein-coupled receptors (S1P1–5), originally known as EDG receptors, sphingosine-1-phosphate triggers diverse cellular responses, including cytoskeletal changes, proliferation, and migration (
      • Hla T.
      ,
      • Chun J.
      • Goetzl E.J.
      • Hla T.
      • Igarashi Y.
      • Lynch K.R.
      • Moolenaar W.
      • Pyne S.
      • Tigyi G.
      ,
      • Fukushima N.
      • Ishii I.
      • Contos J.J.
      • Weiner J.A.
      • Chun J.
      ,
      • Goetzl E.J.
      ,
      • Kluk M.J.
      • Hla T.
      ,
      • Spiegel S.
      • Milstien S.
      ). The S1P1, S1P2, and S1P3 receptors are widely expressed, including on embryonic endothelial cells (Table I) (
      • MacLennan A.J.
      • Browe C.S.
      • Gaskin A.A.
      • Lado D.C.
      • Shaw G.
      ,
      • McGiffert C.
      • Contos J.J.
      • Friedman B.
      • Chun J.
      ,
      • Okazaki H.
      • Ishizaka N.
      • Sakurai T.
      • Kurokawa K.
      • Goto K.
      • Kumada M.
      • Takuwa Y.
      ,
      • Yamaguchi F.
      • Tokuda M.
      • Hatase O.
      • Brenner S.
      ,
      • Zhang G.
      • Contos J.J.
      • Weiner J.A.
      • Fukushima N.
      • Chun J.
      ,
      • Liu Y.
      • Wada R.
      • Yamashita T.
      • Mi Y.
      • Deng C.X.
      • Hobson J.P.
      • Rosenfeldt H.M.
      • Nava V.E.
      • Chae S.S.
      • Lee M.J.
      • Liu C.H.
      • Hla T.
      • Spiegel S.
      • Proia R.L.
      ). S1P4 and S1P5 receptor expression is more restricted and found on the cells of the immune and nervous systems (
      • Graler M.H.
      • Bernhardt G.
      • Lipp M.
      ,
      • Im D.S.
      • Heise C.E.
      • Ancellin N.
      • O'Dowd B.F.
      • Shei G.J.
      • Heavens R.P.
      • Rigby M.R.
      • Hla T.
      • Mandala S.
      • McAllister G.
      • George S.R.
      • Lynch K.R.
      ). The S1P1 receptor couples selectively to the Gi signaling pathway, whereas the S1P2 and S1P3 receptors both couple to the Gi, Gq, and G12/13 pathways (
      • Watterson K.
      • Sankala H.
      • Milstien S.
      • Spiegel S.
      ,
      • Hla T.
      ,
      • Pyne S.
      • Pyne N.
      ,
      • Siehler S.
      • Manning D.R.
      ,
      • Windh R.T.
      • Lee M.J.
      • Hla T.
      • An S.
      • Barr A.J.
      • Manning D.R.
      ). In addition to these five S1P receptors, GPR3, GPR6, GPR12, and GPR63 have been characterized as G-protein-coupled receptors that interact with sphingosine-1phosphate (
      • Uhlenbrock K.
      • Gassenhuber H.
      • Kostenis E.
      ,
      • Ignatov A.
      • Lintzel J.
      • Kreienkamp H.J.
      • Schaller H.C.
      ,
      • Niedernberg A.
      • Tunaru S.
      • Blaukat A.
      • Ardati A.
      • Kostenis E.
      ).
      Table ICharacteristics of S1P receptors
      ReceptorCoupled G-proteinsExpression pattern
      S1P1/EDG-1Gi/0Widely expressed
      S1P2/EDG-5Gi/0Gq/12/13Widely expressed
      S1P3/EDG-3Gi/0Gq/12/13Widely expressed
      S1P4/EDG-6Gi/0Lymphoid tissues
      S1P5/EDG-8Gi/0Gq/12Brain, spleen
      The major physiological effects of S1P receptor signaling defined thus far have been localized to the immune and vascular systems. A global deletion of the S1P1 receptor in mice results in lethality beginning at E12.5 due to severe hemorrhage as the result of deficient coverage of vessels by vascular smooth muscle cells, a process that occurs during the last stages of angiogenesis and is necessary for stabilizing the vascular system (
      • Liu Y.
      • Wada R.
      • Yamashita T.
      • Mi Y.
      • Deng C.X.
      • Hobson J.P.
      • Rosenfeldt H.M.
      • Nava V.E.
      • Chae S.S.
      • Lee M.J.
      • Liu C.H.
      • Hla T.
      • Spiegel S.
      • Proia R.L.
      ). Through analysis of endothelial cell-specific S1P1 receptor knock-out mice, we have shown that the S1P1 receptor functions within endothelial cells to regulate vascular smooth muscle cell coverage (
      • Allende M.L.
      • Yamashita T.
      • Proia R.L.
      ). The function of the S1P1 receptor in the developing vasculature is also essential for proper limb development (
      • Chae S.S.
      • Paik J.H.
      • Allende M.L.
      • Proia R.L.
      • Hla T.
      ). Deletion of the S1P1 receptor in T-cells has revealed a critical role in lymphocyte trafficking (
      • Allende M.L.
      • Dreier J.L.
      • Mandala S.
      • Proia R.L.
      ,
      • Matloubian M.
      • Lo C.G.
      • Cinamon G.
      • Lesneski M.J.
      • Xu Y.
      • Brinkmann V.
      • Allende M.L.
      • Proia R.L.
      • Cyster J.G.
      ).
      To learn the physiologic functions of the S1P2 and S1P3 receptors, we have disrupted these genes in mice, and, in agreement with earlier reports (
      • Ishii I.
      • Friedman B.
      • Ye X.
      • Kawamura S.
      • McGiffert C.
      • Contos J.J.
      • Kingsbury M.A.
      • Zhang G.
      • Brown J.H.
      • Chun J.
      ,
      • Ishii I.
      • Ye X.
      • Friedman B.
      • Kawamura S.
      • Contos J.J.
      • Kingsbury M.A.
      • Yang A.H.
      • Zhang G.
      • Brown J.H.
      • Chun J.
      ), neither the S1P2 nor S1P3 receptor null mice showed an obvious phenotype. To uncover functions that might be masked by redundancy, we produced mice carrying different combinations of the deleted S1P1, S1P2, and S1P3 receptor genes. Indeed, this analysis indicates that these three receptors function coordinately to promote vessel stability during embryonic angiogenesis.

      EXPERIMENTAL PROCEDURES

      Generation of S1P2 and S1P3 Null Mice—Genomic fragments containing the entire open reading frames of the S1P2 and S1P3 genes were cloned from a 129/Sv mouse genomic library. To construct the S1P2 targeting vector, a 4.8-kb cassette containing a neomycin-resistant gene (
      • Nehls M.
      • Kyewski B.
      • Messerle M.
      • Waldschutz R.
      • Schuddekopf K.
      • Smith A.J.
      • Boehm T.
      ) was inserted into an EcoRV site within the open reading frame in an orientation opposite of the S1P2 gene (Fig. 1A). For the S1P3 targeting vector, a 2-kb neomycin-resistant cassette (
      • Capecchi M.R.
      ) was inserted between an upstream intronic BglII site and a NcoI site in the coding region in the same orientation of the S1P3 gene, which resulted in a 1.5-kb genomic deletion (Fig. 1C). Gene targeting in TC1 embryonic stem (ES) cells and the generation of chimeric and heterozygous mice was accomplished as described previously (
      • Liu Y.
      • Wada R.
      • Kawai H.
      • Sango K.
      • Deng C.
      • Tai T.
      • McDonald M.P.
      • Araujo K.
      • Crawley J.N.
      • Bierfreund U.
      • Sandhoff K.
      • Suzuki K.
      • Proia R.L.
      ). The null mice were maintained on a mixed C57Bl/6, 129/Sv background. The S1P2 null mice were genotyped by Southern blotting using probe A and by PCR using the following three primers (Fig. 1A): primer 1, 5′-GCAGTGACAAAAGCTGCCGAATGCTGATG-3′; primer 2, 5′-AGATGGTGACCACGCAGAGCACGTAGTG-3′; and primer 3, 5′-TGACCGCTTCCTCGTGCTTTACGGTATCG-3′. Primers 1 and 2 detected the wild-type S1P2 allele and amplified an ∼170-bp fragment. Primers 1 and 3 detected the targeted S1P2 allele and amplified an ∼220-bp fragment (Fig. 1B, bottom). Forty cycles of 94 °C (1 min), 55 °C (1 min), and 72 °C (1 min) were used.
      Figure thumbnail gr1
      Fig. 1Gene targeting of the S1P2 and S1P3 genes. A, schematic representation of the S1P2 gene targeting strategy. The structure of the mouse S1P2 locus is shown at the top, the structure of the S1P2 targeting vector is depicted in the middle, and the predicted structure of the homologously recombined locus is shown on the bottom. Probe A and PCR primers (P1, P2, and P3) were used for genotyping. X, XbaI; EV, EcoRV; K, KpnI; TK, thymidine kinase gene; Neo, neomycin-resistance gene. B, top, S1P2 genotyping of ES cells by Southern blot analysis. The wild-type (WT) ES cell and targeted (Rec; recombined S1P2) ES cell genomic DNA, after XbaI digestion, yielded both 12- and 7.5-kbp bands. B, bottom, S1P2 genotyping of mice by PCR. The wid-type and targeted S1P2 alleles were detected as ∼170and 220-bp PCR fragments, respectively. WT, wild-type; HT, heterozygous; KO, knock-out. C, schematic representation of the S1P3 gene targeting strategy. The structure of the mouse S1P3 locus is shown at the top, the structure of the S1P targeting vector is depicted in the middle, and the predicted structure of the homologously recombined locus is shown on the bottom. Probe B and PCR primers (P4, P5, and P6) were used for genotyping. B, BamHI; Bg, BglII; N, NcoI; TK, thymidine kinase gene; Neo, neomycin-resistance gene. D, top, S1P3 genotyping of ES cells by Southern blot analysis. The wild-type (WT) ES cell and the targeted (Rec) ES cell genomic DNA, after BamHI digestion, yielded both 13- and 3-kbp bands. D, bottom, genotyping of mice by PCR. The wild-type S1P3 and the targeted S1P3 3 alleles were detected as ∼130- and 380-bp PCR fragments, respectively. WT, wild-type; HT, heterozygous; KO, knock-out.
      The S1P3 genotypes were determined by Southern blot analysis using probe B and by PCR using the following three primers (Fig. 1C): primer 4, 5′-TCAGTATCTTCACCGCCATT-3′; primer 5, 5′-AATCACTACGGTCCGCAGAA-3′; and primer 6, 5′-GTGCAATCCATCTTGTTCAAT-3′. Primers 4 and 5 detected the wild-type S1P3 allele and amplified an ∼130-bp fragment. Primers 5 and 6 detected the targeted S1P3 allele and amplified an ∼380-bp fragment (Fig. 1D, bottom). PCR reaction conditions were the same as for S1P2 genotyping.
      Mice with a global S1P1 deletion (
      • Liu Y.
      • Wada R.
      • Yamashita T.
      • Mi Y.
      • Deng C.X.
      • Hobson J.P.
      • Rosenfeldt H.M.
      • Nava V.E.
      • Chae S.S.
      • Lee M.J.
      • Liu C.H.
      • Hla T.
      • Spiegel S.
      • Proia R.L.
      ) and an endothelial cell-specific S1P1 deletion (S1P1loxP/Ko Tie2-Cre) (
      • Allende M.L.
      • Yamashita T.
      • Proia R.L.
      ) have been described previously and were maintained on mixed C57Bl/6, 129/Sv backgrounds.
      Embryos were obtained from timed pregnant females with the morning of the vaginal plug considered to be embryonic day (E) 0.5 (E0.5). Time-mated female S1P1+/S1P2/– mice were relatively inefficient in producing litters. To obtain sufficient numbers of embryos for genotyping we also time mated female S1P1+/S1P2+/– mice with male S1P1+/S1P2/– mice (Table IV).
      Table IVAnalysis of offspring from S1P1+/- S1P2+/- (female) × S1P1+/- S1P2-/- (male) crosses
      S1P1+/+ S1P2+/-S1P1+/+ S1P2-/-S1P1+/- S1P2+/-S1P1+/- S1P2-/-S1P1-/- S1P2+/-S1P1-/- S1P2-/-
      E9.5972119 (1)11 (1)6
      E10.519 (3)1435 (3)23 (1)13 (2)20 (6)
      E11.51014 (1)18 (2)28 (2)9 (1)8 (2)
      E12.516 (1)11 (2)30 (1)248 (2)10 (9)
      E13.52110203 (3)
      Northern Analysis—Total RNA was prepared from mouse brain, heart, and lung using TRIzol reagent (Invitrogen). Lung RNA was further purified using the RNeasy Mini kit (Qiagen). Total RNA (20 μg) was fractionated on denaturing formaldehyde-agarose gels (1%) and then transferred to positively charged nylon membranes (GeneScreen Plus; PerkinElmer Life Sciences). The membranes were prehybridized in PerfectHyb Plus (Sigma) with 0.1 mg/ml denatured salmon sperm DNA at 68 °C for 1 h. Hybridization was performed overnight at 68 °C with 32P-labeled DNA probes prepared by the random priming method. The coding region of mouse S1P1, rat S1P2, and human S1P3 cDNAs were used as probes. The membranes were washed in 2× SSC 0.1% SDS at 25 °C for 5 min and in 0.2× SSC 0.1% SDS at 50 °C for 5 min, 55 °C for 5 min, 60 °C for 5 min, and 65 °C for 5 min.
      Histological Analysis—Embryos at E9.5–E18.5 were obtained after timed mating. The embryos were fixed and embedded in paraffin. Serial sections (5-μm thick) were stained with hematoxylin and eosin. For immunostaining, the paraffin sections were deparaffinized and rehydrated. Antigen retrieval was accomplished by a 30-min incubation at 95 °C in Target Retrieval Solution (DAKO Corp.). Endogenous peroxidase activity was quenched by incubation with 0.3% hydrogen peroxide in methanol for 5 min. The specimens were incubated with anti-smooth muscle α-actin (U7033; DAKO Corp.) at room temperature for 1 h. After washing with PBS, the peroxidase reaction was visualized with diaminobenzidine/hydrogen peroxide.
      For whole-mount embryo immunostaining, embryos were fixed in 4% paraformaldehyde at 4 °C overnight. The embryos were dehydrated through a methanol series, and endogenous peroxidase activity was quenched by incubation with 5% hydrogen peroxide in methanol at room temperature for 4 h. The embryos were rehydrated through a methanol series to PBS and incubated in 4% BSA in PBS plus 0.1% Triton X-100 (PBST) at room temperature for 1 h twice. The embryos were incubated with rat anti-CD31 (PECAM-1) monoclonal antibody (clone MEC13.3; PharMingen) and diluted 1:10 in 4% BSA in PBST at 4 °C overnight. Embryos were washed with 4% BSA in PBST at 4 °C for 1 h five times and then incubated with peroxidase-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories) diluted 1:100 in 4% BSA in PBST at 4 °C overnight. After washing with 4% BSA in PBST at 4 °C for 1 h five times and 0.2% BSA in PBST at room temperature for 1 h, the peroxidase reaction was visualized with diaminobenzidine/hydrogen peroxide.
      For electron microscopy, E14.5 embryos were fixed with 1% glutaraldehyde/4% paraformaldehyde at 4 °C for 24 h. Selected regions were postfixed with 1% osmium tetrahydroxide and processed for embedding in plastic resin. Semithin sections were cut, stained with toluidine blue, and examined. Selected areas were further thin sectioned, stained with uranylacetate and lead citrate, and examined by electron microscopy (Philips EM 410).

      RESULTS

      Generation of S1P2 and S1P3 Receptor Null Mice—The S1P2 and S1P3 genes were each targeted by homologous recombination in TC1 ES cells (Fig. 1). In the targeted S1P2 ES cell line, the wild-type S1P2 allele and the recombined S1P2 allele were detected by Southern blot analysis as 12-kb and 7.5-kb bands, respectively (Fig. 1B, top). In the targeted S1P3 ES cell line, the wild-type S1P3 allele and the recombined S1P3 allele were identified as 13-kb and 3-kb bands, respectively, on Southern blots (Fig. 1D, top). These targeted ES cell lines were used to produce chimeric mice that transmitted the targeted alleles to their offspring, resulting in heterozygous mice. The heterozygotes were interbred to generate homozygous S1P2 and S1P3 null mice.
      To confirm that the gene targeting procedures caused inactivation of the targeted loci, Northern blot analysis was performed using total RNA purified from the adult brain, heart, and lung of homozygous S1P2 and S1P3 mutant mice (Fig. 2). In the tissues of the S1P2 mutant mice the normal S1P2 transcript was not detected, whereas the S1P1 and S1P3 transcripts were present at normal levels. Likewise, the S1P3 transcript was absent in tissues from the S1P3 mutant mice without a significant difference in the expression of the other widely expressed receptor genes, S1P1 and S1P2. These results indicate that the targeting procedures resulted in null alleles for S1P2 and S1P3.
      Figure thumbnail gr2
      Fig. 2Transcript analysis of S1P receptors in S1P2 and S1P3 null mice. Total RNA from the brain, heart, and lung of 2-month-old wild-type (WT), S1P2/–, and S1P3/– male mice was used for Northern blot analysis. Probes for S1P1 (A), S1P3 (B), and S1P2 (C) were used as indicated. Ribosomal 28 and 18 S RNAs stained with ethidium bromide are shown as loading controls.
      Analysis of S1P1 S1P2 Double Null, S1P1 S1P3 Double Null, and S1P1 S1P2 S1P3 Triple Null Embryos—Consistent with previous studies on independently generated lines of S1P2 and S1P3 null mice, we found no evidence of generalized embryonic lethality in either of our lines of null mice (
      • Ishii I.
      • Friedman B.
      • Ye X.
      • Kawamura S.
      • McGiffert C.
      • Contos J.J.
      • Kingsbury M.A.
      • Zhang G.
      • Brown J.H.
      • Chun J.
      ). We determined whether the additional deletion of the S1P2 gene or the S1P3 gene could influence the embryonic phenotype produced by the absence of the S1P1 gene by performing crosses with mice carrying mutations in multiple S1P receptors. As has been shown in the absence of S1P1 alone, E12.5 embryos show bleeding along the body and head due to a defect in vascular maturation (Figs. 3B and 4A), leading to their death by E13.5–E14.5 (Table II and Fig. 4B) (
      • Liu Y.
      • Wada R.
      • Yamashita T.
      • Mi Y.
      • Deng C.X.
      • Hobson J.P.
      • Rosenfeldt H.M.
      • Nava V.E.
      • Chae S.S.
      • Lee M.J.
      • Liu C.H.
      • Hla T.
      • Spiegel S.
      • Proia R.L.
      ). The limbs are also underdeveloped (
      • Chae S.S.
      • Paik J.H.
      • Allende M.L.
      • Proia R.L.
      • Hla T.
      ). The phenotype of the S1P1 S1P3 double null embryos appeared similar to embryos with a single S1P1 gene deletion or perhaps marginally more severe, with most of the S1P1 S1P3 double null embryos alive at E12.5 (Table V and Fig. 4B) showing bleeding along the body and head together with underdeveloped limbs (Figs. 3D and 4A). Most of these S1P1 S1P3 double null embryos expired slightly sooner than the S1P1 single null embryos. These results indicated that the additional S1P3 gene deletion has, at most, a minor influence on the S1P1 null phenotype.
      Figure thumbnail gr3
      Fig. 3The phenotype of S1P receptor null embryos. The following embryos were analyzed at E12.5: wild-type (WT) (A); S1P1/– (B); S1P1+/S1P3/– (C); S1P1–/– S1P3/– (D); S1P1+/+S1P2/– (E); and S1P1/S1P2/– (F). In panel B the S1P1/– embryo shows bleeding along the body and head (arrowheads) and underdeveloped limbs (arrow). The S1P1/S1P3/– embryo in panel D shows a phenotype similar to that of the S1P1/– embryo. In panel F, the S1P1–/– S1P2–/– embryo shows a more severe bleeding phenotype than does the single S1P1/– embryo.
      Figure thumbnail gr4
      Fig. 4Frequency of hemorrhage and death for S1P receptor null embryos. A, percentage of S1P receptor null genotypes showing abnormal embryonic bleeding. B, percentage of S1P receptor null genotypes showing embryonic death. Death was judged by the absence of a heartbeat. Embryos were obtained from crosses as described for Tables , , , , , . KO, knock-out; DKO double knock-out; TKO, triple knock-out.
      Table IIAnalysis of offspring from S1P1+/- intercrosses
      S1P1+/+S1P1+/-S1P1-/-
      E9.5202817
      E10.582717 (2)
      E11.591510
      E12.53153 (2)30 (3)
      E13.5113213 (2)
      E14.512219 (7)
      Table VAnalysis of offspring from S1P1+/- S1P3-/- intercrosses
      S1P1+/+ S1P3-/-S1P1+/- S1P3-/-S1P1-/- S1P3-/-
      E11.51122 (2)12 (1)
      E12.5823 (4)13 (2)
      E13.591810 (9)
      E14.58122 (2)
      In contrast, the phenotype of the S1P1 S1P2 double null mice appeared to be substantially more severe than that of either the S1P1 single or S1P1 S1P3 double null embryos (Tables III, IV, V). At E12.5 just two of fifteen embryos with deletions in both the S1P1 and the S1P2 genes were alive as judged by a beating heart (Tables III and IV and Fig. 4B). These double null embryos started to display hemorrhage as early as E10.5 (Figs. 3F and 4A).
      Table IIIAnalysis of offspring from S1P1+/- S1P2-/- intercrosses
      S1P1+/+ S1P2-/-S1P1+/- S1P2-/-S1P1-/- S1P2-/-
      E9.5614 (1)9 (1)
      E10.56 (1)15 (2)4 (1)
      E11.58 (2)19 (1)17 (7)
      E12.537 (1)5 (4)
      Finally, we were able to examine a limited number of embryos with all three genes, S1P1, S1P2, and S1P3, deleted. Only one of five triple null embryos was found to be alive at E11.5 (Table VI and Fig. 4B), and we did not find any survivors at E12.5 (data not shown). About 50% of the triple null embryos showed bleeding at E10.5 (Fig. 4A). This triple null phenotype was similar to or perhaps slightly more severe than the S1P1 S1P2 double null embryos.
      Table VIAnalysis of offspring from S1P1+/- S1P2-/- S1P3-/- intercrosses
      S1P1+/+ S1P2-/- S1P3-/-S1P1+/- S1P2-/- S1P3-/-S1P1-/- S1P2-/- S1P3-/-
      E9.5232
      E10.56 (1)5 (1)4 (1)
      E11.5145 (4)
      Immature Vascular Network in S1P1 S1P2 Double Null, and S1P1 S1P2 S1P3 Triple Null Embryos—The early demise of the S1P1 S1P2 double null and S1P1 S1P2 S1P3 triple null embryos was coincident with a bleeding phenotype, suggesting that vascular abnormalities were a cause of death. We assessed vascular development by performing whole-mount immunohistochemical staining using a rat anti-CD31 (PECAM-1) monoclonal antibody. The staining indicated a substantial development of the vascular network in the double and triple null embryos at E10.5 (Fig. 5, A, C, E, and G). However, close examination of the head region revealed a less mature appearing vasculature. In this area in the double and triple null embryos, the capillary network was less developed and contained fewer branches when compared with the wild-type embryos and the S1P1 null embryos (Fig. 5, B, D, F, and H).
      Figure thumbnail gr5
      Fig. 5Vascular development in S1P receptor mutant embryos. Whole-mount immunohistochemical staining was performed with rat anti-CD31 (PECAM-1) monoclonal antibody on the following E10.5 embryos: wild-type (A and B); S1P1/– (C and D); S1P1 S1P2 double null (E and F); and S1P1 S1P2 S1P3 triple null (G and H). Panels A, C, E, and G offer whole body views. Panels B, D, F, and H show close-up views of head vessels. The white lines in panels A, C, E, and G indicate the head regions viewed in panels B, D, F, and H). Arrows indicate branch points in the vessels.
      Analysis of S1P2 S1P3 Double Null Mice—We obtained viable S1P2 S1P3 double null mice by interbreeding double heterozygous mice. However, it was difficult to obtain viable offspring through interbreeding these S1P2 S1P3 double null mice. Generally, the litters derived from these mice were spontaneously aborted late in pregnancy or, rarely, a small number of pups were delivered. These results are in basic agreement with those of Ishii et al. who described very low numbers of viable offspring and perinatal lethality after interbreeding S1P2/S1P3/– mice (
      • Ishii I.
      • Ye X.
      • Friedman B.
      • Kawamura S.
      • Contos J.J.
      • Kingsbury M.A.
      • Yang A.H.
      • Zhang G.
      • Brown J.H.
      • Chun J.
      ). We examined the genotypes of the viable offspring from S1P2+/S1P3/– intercrosses 4 weeks after birth and found that only 11% were of the genotype S1P2/S1P3/– instead of the expected 25% (Table VII). After timed matings we found a similarly depressed frequency of S1P2/S1P3/– embryos at E18.5, indicating that lethality was occurring prior to this time (Table VII). The expected frequency of the S1P2 S1P3 double null embryos was obtained between E13.5 and E16.5. However, substantially more of these double null embryos were found without a heartbeat as compared with the two other genotypes, S1P2+/+ S1P3/– and S1P2+/S1P3/–, present in the litters (Table VII). Furthermore, a substantial fraction of viable double null embryos showed a bleeding phenotype (Figs. 4A and 6), suggesting that a vascular defect was the cause of the embryonic lethality. Hemorrhage was not observed in the S1P3 single null embryos in these same litters. Moreover, after timed mating of the S1P2 single null mice we determined that S1P2 embryos did not show abnormal bleeding or death during this same gestational period (data not shown).
      Table VIIAnalysis of offspring from S1P2+/- S1P3-/- intercrosses
      S1P2+/+ S1P3-/-S1P2+/- S1P3-/-S1P2-/- S1P3-/-
      E12.59176
      E13.515(1)3315 (2)
      E14.5620 (1)7 (2)
      E15.5-16.5162818 (1)
      E18.518296
      P2826388
      Figure thumbnail gr6
      Fig. 6Hemorrhage in the S1P2 S1P3 double null embryos. A, control littermate S1P2+/S1P3/– embryo at E13.5. B, S1P2 S1P3 double null embryo at E13.5. C, S1P2 S1P3 double null embryo at E14.5. D, S1P2 S1P3 double null embryo at E15.5.
      Abnormal Endothelial Cells in the S1P2 S1P3 Double Null Embryos—The S1P2 S1P3 double null embryos began to hemorrhage around E13.5. Transverse sections of E13.5 S1P2 S1P3 double null embryos showed free red blood cells and edema in subcutaneous areas (Fig. 7B). The aorta in the S1P2 S1P3 double null embryos appeared to be covered normally by vascular smooth muscle cells, unlike the defective coverage of the aortae in S1P1 null mice (
      • Liu Y.
      • Wada R.
      • Yamashita T.
      • Mi Y.
      • Deng C.X.
      • Hobson J.P.
      • Rosenfeldt H.M.
      • Nava V.E.
      • Chae S.S.
      • Lee M.J.
      • Liu C.H.
      • Hla T.
      • Spiegel S.
      • Proia R.L.
      ) and in mice with S1P1 deleted from endothelial cells (
      • Allende M.L.
      • Yamashita T.
      • Proia R.L.
      ) (Fig. 8, A–C). Electron microscopic analysis of microvessels from S1P2 S1P3 double null embryos revealed endothelial cells with abnormally thin cell bodies (Fig. 7C), which, in some cases, were broken (Fig. 7, C and D). However, cell-cell junctions between the endothelial cells appeared intact (data not shown).
      Figure thumbnail gr7
      Fig. 7Histological analysis of S1P2 S1P3 double null embryos. A E13.5 littermate control S1P1+/S1P3/–. B, S1P2 S1P3 double null embryos were sectioned transversely at the thoracic level and stained by hematoxylin and eosin. The S1P2 S1P3 double null embryo shows hemorrhage and edema in subcutaneous region (brackets in panels A and B). C and D, electron microscopic analysis of microvessels in E14.5 S1P2 S1P3 double null embryos. The S1P2 S1P3 double null endothelial cells were abnormally thin (arrowheads in panel C) and broken (arrows in panels C and D). Scale bars in panels A and B represent 200 μm; scale bars in panels C and D represent 1 μm.
      Figure thumbnail gr8
      Fig. 8Smooth muscle coverage of aorta from S1P2 S1P3 double null embryos. A, E13.5 S1P2+/S1P3/B, S1P2S1P3 double null. C, S1P1loxP/Ko Tie2-Cre embryos were sectioned transversely at the thoracic level and immunostained with anti-smooth muscle α-actin. S1P2+/S1P3/– and S1P2 S1P3 double null aortae were covered normally by vascular smooth muscle cells (brackets in panels A and B). However, smooth muscle cell coverage was discontinuous around the aorta in the S1P1loxP/Ko Tie2-Cre embryo (arrows in panel C). Scale bars, 50 μm.

      DISCUSSION

      In the embryo, the vasculature develops through the sequential processes of vasculogenesis and angiogenesis (
      • Conway E.M.
      • Collen D.
      • Carmeliet P.
      ,
      • Lindahl P.
      • Hellstrom M.
      • Kalen M.
      • Betsholtz C.
      ,
      • Allende M.L.
      • Proia R.L.
      ). Beginning at around E8.5 in the mouse, vasculogenesis includes the differentiation of endothelial cells, their migration and proliferation, and the formation of the primitive vascular plexus. The subsequent process of angiogenesis, initiating at about E10.5, involves the sprouting, splitting, and remodeling of existing vessels to generate a more complex, tree-like network of highly branched small and large vessels. As part of the angiogenic process, maturation of vessels occurs as the endothelial tubes are invested with vascular smooth muscle cells to provide support and stability for the nascent vascular network. Maturation of vessels extends late into gestation.
      The S1P1 receptor within endothelial cells is required for the coverage of vessels by vascular smooth muscle cells (
      • Allende M.L.
      • Yamashita T.
      • Proia R.L.
      ). In the absence of the S1P1 receptor, lethality occurs at E12.5–E14.5 due to rupture of the unreinforced vessels (
      • Liu Y.
      • Wada R.
      • Yamashita T.
      • Mi Y.
      • Deng C.X.
      • Hobson J.P.
      • Rosenfeldt H.M.
      • Nava V.E.
      • Chae S.S.
      • Lee M.J.
      • Liu C.H.
      • Hla T.
      • Spiegel S.
      • Proia R.L.
      ). Unlike the embryonic lethality caused by the absence of the S1P1 receptor, mice null for either the S1P2 or the S1P3 receptor, the two other widely expressed S1P receptors, were viable and fertile and showed normal cage behavior (Table VIII). These findings are in basic accord with observations of independently generated S1P2 and S1P3 null mice that were reported by Ishii et al. (
      • Ishii I.
      • Friedman B.
      • Ye X.
      • Kawamura S.
      • McGiffert C.
      • Contos J.J.
      • Kingsbury M.A.
      • Zhang G.
      • Brown J.H.
      • Chun J.
      ,
      • Ishii I.
      • Ye X.
      • Friedman B.
      • Kawamura S.
      • Contos J.J.
      • Kingsbury M.A.
      • Yang A.H.
      • Zhang G.
      • Brown J.H.
      • Chun J.
      ). We did not observe the frequent spontaneous seizure behavior that has been described in the third S1P2 null line (
      • MacLennan A.J.
      • Carney P.R.
      • Zhu W.J.
      • Chaves A.H.
      • Garcia J.
      • Grimes J.R.
      • Anderson K.J.
      • Roper S.N.
      • Lee N.
      ), which may be related to strain differences or differences in targeting procedures.
      Table VIIIViability of S1P receptor null mice
      S1P1 nullEmbryonic lethal at E12.5-14.5
      S1P2 nullViable
      S1P3 nullViable
      S1P1/S1P2 nullEmbryonic lethal at E10.5-12.5
      S1P1/S1P3 nullEmbryonic lethal at E12.5-13.5
      S1P2/S1P3 nullPartial embryonic lethality after E13.5
      S1P1/S1P2/S1P3 nullEmbryonic lethal at E10.5-11.5
      Because the S1P2 and S1P3 receptors, like the S1P1 receptor, are expressed in embryonic endothelial cells (
      • McGiffert C.
      • Contos J.J.
      • Friedman B.
      • Chun J.
      ), we determined whether they might have a cooperative or a redundant function with the S1P1 receptor by crossbreeding the null mice. We found that an additional deletion of the S1P3 gene did not substantially alter the phenotype of the S1P1 single null embryos. By contrast, the phenotype of the S1P1 S1P2 double null embryos was distinctly more severe with bleeding and lethality occurring about 2 days earlier than in the S1P1 single null embryos. The phenotype of the S1P1 S1P2 S1P3 triple null embryos was similar to or slightly more severe than that of the S1P1 S1P2 double null embryos.
      In the triple null embryos, PECAM-1 staining indicated that the vascular system had substantially formed. This result shows that the S1P1, S1P2, and S1P3 receptors are largely dispensable for the process of embryonic vasculogenesis. However, vascular defects were apparent in the heads of S1P1 S1P2 S1P3 triple null and S1P1 S1P2 double null embryos. Here the vessels appeared less mature than in the S1P1 null or wild-type embryos, indicating that the S1P1 and S1P2 receptors were required for some aspects of vessel development during angiogenesis. Such a vascular defect could contribute to the more severe phenotype observed in the double and triple null mice.
      Perinatal lethality had been described previously for S1P2 S1P3 double null mice (
      • Ishii I.
      • Ye X.
      • Friedman B.
      • Kawamura S.
      • Contos J.J.
      • Kingsbury M.A.
      • Yang A.H.
      • Zhang G.
      • Brown J.H.
      • Chun J.
      ). We found that the S1P2 S1P3 double null mice were born at about one-half of the expected frequency with a higher rate of embryonic death, after E13.5, compared with the other genotypes in the same litters. Thus, the observed inability of the S1P2 S1P3 double null intercrosses to produce viable offspring may be the result of the high frequency of late stage embryonic lethality that could lead to the termination of the entire pregnancy. Hemorrhage was coincident with and may also be the cause of the lethality for S1P2 S1P3 double null embryos. Histological examination of the S1P2 S1P3 double null embryos revealed bleeding and edema in the subcutaneous regions. Ultrastructural analysis of microvessels in the S1P2 S1P3 double null mice showed that the endothelial cells appeared to be abnormal with thin cell bodies, suggesting that these vessels may be fragile and prone to rupture.
      The S1P1 receptor is believed to couple exclusively to a Gi signaling pathway, whereas the S1P2 and S1P3 receptors couple to Gi as well as to the Gq and G12/13 pathways (Table I). The overlap in these signaling pathways suggests that redundancy of function of the three S1P receptors is possible and may explain the generally increased phenotypic severity when multiple receptors were deleted. Alternatively, vessel development may require the integration of distinct cellular activities derived from individual S1P receptors. For instance, S1P1 and S1P3 receptors have been shown to stimulate Rac-coupled cortical actin assembly and Rho-coupled stress fiber formation, respectively (
      • Lee M.J.
      • Thangada S.
      • Claffey K.P.
      • Ancellin N.
      • Liu C.H.
      • Kluk M.
      • Volpi M.
      • Sha'afi R.I.
      • Hla T.
      ). S1P2 and S1P3 receptors both mediate sphingosine-1-phosphate stimulation of Rho, yet the S1P2 but not the S1P3 receptor mediates down-regulation of Rac activation, membrane ruffling, and cell migration (
      • Takuwa Y.
      ).
      A major physiologic function for S1P receptor signaling was revealed in the S1P1 null embryos, which demonstrated that the S1P1 receptor functions within endothelial cells to promote their interactions with smooth muscle cells. In addition to stabilizing the vasculature, endothelial smooth muscle cell interactions are required for vessel remodeling. Mice lacking endoglin and myocardin are deficient in smooth muscle cells and show defects in vascular remodeling, including the growth and sprouting of endothelial tubes (
      • Li D.Y.
      • Sorensen L.K.
      • Brooke B.S.
      • Urness L.D.
      • Davis E.C.
      • Taylor D.G.
      • Boak B.B.
      • Wendel D.P.
      ,
      • Li S.
      • Wang D.Z.
      • Wang Z.
      • Richardson J.A.
      • Olson E.N.
      ). The vessel defects seen in the double and triple null mice may be the result of a more severe block in endothelial cell smooth muscle cell interactions than that which occurs in the single null S1P1 embryos. Alternatively, a more direct effect on angiogenesis is possible. In either case, the vascular phenotypes of embryos with deleted S1P1, S1P2, and S1P3 genes (Table VIII) support the concept that these S1P receptors coordinate the transition from a leaky immature vascular network to a mature stable system in embryonic development and, possibly, in adults.

      Acknowledgments

      We thank Drs. Tim Hla and Sarah Spiegel for the S1P receptor cDNAs.

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