Widespread Expression of Sphingosine Kinases and Sphingosine 1-Phosphate (S1P) Lyase Suggests Diverse Functions in the Vertebrate Nervous SystemH. MENG and V.M. LEE
19.1. INTRODUCTION
Lysophospholipids such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are membrane-derived bioactive lipid mediators (1–3). S1P is derived from sphingosine, which is the backbone of most sphingolipids. S1P has been shown to play important roles in many cellular processes such as cell proliferation, differentiation, survival, and migration, and it exerts these diverse effects intracellularly and extracellularly. It can act as a second messenger inside a cell, or as a ligand for G protein-coupled receptors.
S1P is made up of sphingosine and a phosphate group. Sphingosine can be phosphorylated by sphingosine kinases to form S1P (4). The source of sphin-gosine is generated either by degradation of sphingolipids or by deacylation of ceramide. S1P can be dephosphorylatd by specific S1P phosphatases (S1PP) or by lipid phosphohydrolases with broader substrate specificity. Alternatively, S1P can be irreversibly degraded to ethanolamine phosphate and hexadecenal (long-chain aldehyde) by the action of S1P lyase.
Sphingolipids regulates cell growth and survival in many cell types. In contrast to S1P, S1P precursors, ceramide, and sphingosine are usually associated with cell death. Stressful stimuli or growth factor withdrawal can activate sphingomyelinase, which converts sphingomyelin to produce ceramide. Ceramide can regulate multiple events that led to stress response, growth arrest, or apoptosis. Ceramide is further metabolized by ceramidase to sphingosine,
420 SPHINGOSINE KINASES, S1P LYASE, AND THE VERTEBRATE NERVOUS SYSTEM
which can inhibit protein kinase C and induce apoptosis. Thus, the relative levels of S1P, ceramide, and sphingosine can regulate cell fate (5–7).
S1P has been shown to regulate diverse physiological processes such as differentiation and cell migration by interacting with their G protein-coupled receptors. To date, five S1P receptors have been characterized in mammals, S1P1–5 (reviewed in References 8–10). S1P1–5 can differentially regulate the small GTPases of the Rho family and have been shown to be important for cytoskeleton rearrangements and directed cell movement (1, 2, 11–13). S1P also plays a role in embryonic cell migration. During heart development, cardiac precursor cells are formed bilaterally in the anterior lateral plate mesoderm and then migrate to the midline to from a single heart tube. In the zebrafish mutant Miles apart, cardiac precursor cells fail to merge and those embryos develop two separate heart structures. Miles apart encodes the zebraf-ish homolog of S1P2; when expressed in Jurkat cells, it was able to induce calcium mobilization and mitogen-activated protein kinase (MAPK) activa-tion in response to S1P. Miles apart/s1p2 can also facilitate migration by stimu-lating assembly of extracellular matrix molecules, thereby promoting cell–matrix interactions. Consistent with this notion, Miles apart mutants also display blistering of their tails, suggesting that there is a defect in epithelial integrity and perhaps abnormal integrin-mediated interactions (14). Addi-tional evidence to support a role for S1P in migration comes from the S1P1 knockout mice. These mutant mice died before birth due to vascular abnor-malities that were caused by defective migration of vascular smooth muscle cells around the newly formed blood vessels (15, 16).
Lysophospholipid levels regulated by their metabolic enzymes can influ-ence cell migration or cell fate decisions. The sphingosine 1-phosphate lyase (sgpl) gene was identified in yeast as a suppressor of growth inhibition induced by D-erythro-sphingosine (17). In mutant yeast strain that was deficient in sgpl, endogenous S1P was much higher than wild-type strains under normal condi-tions, and comparable with the S1P level that is found in wild type after heat stress. Indeed, sgpl yeast mutants are much more heat resistant to their wild-type counterparts, suggesting that S1P may play a role in stress response. Sgpl was identified in slime mold from a genetic screen for increased resistance to cisplatin, an alkylating agent (18, 19). Besides the ability to resist cell death induced by cisplatin, sgpl mutants have developmental abnormalities, such as elongated morphology, tendency to form large aggregates, and increased sur-vival during stationary phase, as well as defects in directional migration. Inter-estingly, treatment of wild-type cells with S1P mimicked the sgpl mutant phenotypes. These data suggest that high levels of S1P accumulation due to sgpl depletion could counteract the toxic effects of cisplatin and promote cell proliferation.
In Drosophila, disruption of the sgpl gene led to defects in cell survival and proliferation (20). Recently, Renault et al. showed that mutations in two lipid phosphate phosphohydrolyases (LPPs), wunen and wunen2, caused abnormal
RESULTS AND DIScUSSION 421
germ cell migration and survival (21), and that wunen and wunen2 negatively regulate germ cell migration. Wunen and wunen2 are capable of dephosphory-lating S1P and related phospholipids such as LPA, ceramide phosphate, and phosphatidic acid, and their enzymatic activities are necessary for its develop-mental functions. Because S1P and LPA signaling have been shown to be important in migration but Drosophila lack S1P or LPA receptors, it is possible that regulation of intracellular lysophospholipid levels is important in regulat-ing cell migration and survival during development.
19.2. RESULTS AND DISCUSSION
19.2.1. Sphingosine Kinases
Two Sphingosine kinases (Sphk1 and Sphk2) have been characterized in mammals and one has been identified in chicken thus far. In the chick embryo, SPHK1 transcript was detected as early as 4–5 somites (∼26–29 hours of devel-opment, stage 8) in the anterior neural fold (Fig. 19.1A) and its expression became more robust by 10–12 somites (embryonic day 1.5, stage 10; Fig. 19.1B). By E2.5 (stage 16, 26–28 somites), SPHK1 could be observed in the developing forebrain, otic vesicle, as well as somites (Fig. 19.1C). At E3.5–3.75 (37–42 somites, stage 19–20), SPHK1 was expressed in the forebrain, forming trigeminal ganglia, otic vesicle, olfactory epithelium in the head, and dorsal root ganglia in the trunk (Fig. 19.1D). More detailed analyses confirmed that from E3.5 to E5, SPHK1 was expressed in the trunk neural tube, hindbrain neuroepithelium, and neural crest cells that were coalescing to form the tri-geminal and dorsal root ganglia (Fig. 19.1E–K). In the mouse embryo, Sphk1 was expressed more diffusely but could be detected in the brain, spinal cord, and migrating neural crest cells (Fig. 19.2A–C; 22). At E8.5, Sphk1 transcript could be observed throughout the neural tube. Similar to chick embryos, Sphk1 expression was obvious in the developing trigeminal and dorsal root ganglia by E9.5–10.5 (Figs. 19.2B,C and 19.3). Sphk1 was also observed in the branchial arches and limb bud. Sphk2 expression overlapped with Sphk1 in the nervous system but Sphk2 level was lower than Sphk1 in general (Fig. 19.2D–F; 22).
Deleting either Sphk1 or Sphk2 did not have any significant impact on embryonic development, and Sphk1 or Sphk2 null animals were viable and fertile (22, 23). In contrast, Sphk1/Sphk2 double-knockout embryos did not survive beyond E13.5 due to severe bleeding (22). A subpopulation of Sphk1−/−;Sphk2−/− embryos displayed exencephaly where the cranial neural tube failed to close. Histological analyses revealed that neuroepithelium was thin and irregularly shaped in the brain, regardless of exencephaly. Cell death and proliferation assays showed that ablating Sphk1 and Sphk2, hence the lack of S1P, resulted in an increase of dying cells and decrease in dividing cells,
422 SPHINGOSINE KINASES, S1P LYASE, AND THE VERTEBRATE NERVOUS SYSTEM
Figure 19.1. Sphingosine kinase 1 (SPHK1) expression in the developing chick embryo. (A–D) SPHK1 expression (purple) pattern analyzed by whole mount in situ hybridiza-tion. (A, B) At stage 8 (st.8, four somites, ∼E1.25) and stage 9 (st.9, seven somites, ∼E1.5), SPHK1 was expressed in the anterior neural fold (*). (C) By stage 16 (st.16, 26–28 somites, E2.5), SPHK1 transcript could be observed in the developing telen-cephalon (TE) and in the somites (arrow). (D) Specific SPHK1 staining was detected in the TE, trigeminal ganglia (Trig), and otic vesicle (OV) in the head and dorsal root ganglia (arrowhead, DRG) in the trunk at stage 20 (st.20, 41–42 somites, E3.5). (E–K) SPHK1 section in situ hybridization (grayish black) combined with HNK-1 (turquoise) or Tuj-1 (magenta) immunostaining. (E–G, K) At stage 19 (st.19, 37–40 somites, E3.5), SPHK1 was detected in the neural tube and neural crest cells (NC, turquoise) that were condensing to form the DRG. By stage 25 (st.25, E4), DRG were conspicuous, and they expressed SPHK1 (grayish black), HNK-1 (turquoise), and Tuj-1 (magenta). This expression persisted in stage 27 (K, st.27, E5). (I, J) In the head, SPHK1 was observed in the hindbrain neuroepithelium (HB) and Trig at stage 19 (st.19, 37–40 somites, E3.5). SPHK1 continued to be expressed by the trigeminal (Trig) and the VIIth (VII) and VIIIth (VIII) cranial ganglia. (See color insert.)
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especially in the telencephalon. These phenotypes were attributed, in part, to the signaling of S1P via S1P1 because S1P1 was highly expressed in the telen-cephalon, and similar apoptotic and proliferative defects were also observed in S1P1 null embryos (22).
We recently studied the possible functional roles of Sphk1/Sphk2 in the developing peripheral nervous system (24). Majority of cells in the peripheral nervous system are derived from the neural crest. Neural crest cells are mul-tipotent stem cells that exist transiently in the embryo and migrate extensively to form and contribute to a plethora of tissues such as the sensory ganglia (e.g., trigeminal and dorsal root ganglia), sympathetic ganglia as well as pigment cells and craniofacial skeleton (25). Based on our expression analyses, neural crest cells and their derivatives expressed Sphk1 and very low levels of Sphk2
Figure 19.2. Sphk1 and Sphk2 expression in the developing mouse embryo. (A, D) Sphk1 and Sphk2 were expressed throughout the neural tube at E8.5. (B) At E9.5, Sphk1 was detected in the brain and in the developing trigeminal ganglion (dotted area, arrowhead). Sphk2 was present at a lower level in the brain and trigeminal gan-glion (E, dotted area, arrowhead). (C) By E10.5, Sphk1 expression was enriched in the trigeminal ganglion (dotted area, arrowhead) and trunk neural crest cells (arrow). The trigeminal ganglion (dotted area, arrowhead) also expressed Sphk2, albeit at lower levels (F). (See color insert.)
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424 SPHINGOSINE KINASES, S1P LYASE, AND THE VERTEBRATE NERVOUS SYSTEM
Figure 19.3. Sphk1 expression in E9.5 trigeminal ganglion. Section in situ for Sphk1 was performed, followed by Tuj-1 immunostaining. (A) Sphk1 transcript (grayish black) could be readily detected in the trigeminal ganglion at E9.5. (B) The same section was processed for Tuj-1 staining (green) to identify neurons. (C) Sphk1 in situ signal was converted to red in PhotoShop. Overlaying Sphk1 (red) and Tuj-1 (green) signals revealed that non-neuronal cells in the trigeminal ganglion expressed Sphk1 at this stage. (See color insert.)
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prior to E12.5. Because Sphk1 single mutant did not have any neural crest defects, Sphk2 must compensate for most, if not all of Sphk1’s functions in its absence. Consistent with previous reports, embryos lacking one to three alleles of Sphk did not display any obvious defects in the formation or development of the trigeminal and sympathetic ganglia. Neural crest formation and migra-tion did not appear to be greatly affected in Sphk1;Sphk2 double-null embryos
RESULTS AND DIScUSSION 425
but they showed acute deficits in the sensory ganglia. The number of neurons and neural crest progenitor cells in Sphk1−/−;Sphk2−/− embryos was severely depleted in both trigeminal and dorsal root ganglia due to a combination of proliferation defects and an increase in cell death. Because Sphk1−/−;Sphk2−/− embryos died by E12.5–13.5, this precluded further analyses of these enzymes’ functions in ganglionic development during later stages (24). Sphk1 and Sphk2 expression persisted in the dorsal root ganglia after E13.5 (Fig. 19.4), and we previously demonstrated that several S1P receptors were present in E16.5 sensory ganglia (26). Although cells in the sensory ganglia express Sphk1 and Sphk2, it is unclear if S1P is produced and whether it serves essential functions. Neural crest conditional Sphk1/Sphk2 double mutants had been generated by crossing Wnt1-Cre and Sphk1f/f and Sphk2−/− mouse lines. Pericytes (a cell type with neural crest origin) from Wnt1-Cre:Sphk1f/f;Sphk2−/− mice showed that S1P made by pericytes was required for thymocyte egression, demonstrating a role for locally secreted S1P (27). Wnt1-Cre:Sphk1f/f;Sphk2−/− animals were viable and displayed no gross abnormalities; however, the nervous system was not studied in those animals. It will be of interest to examine the neural crest conditional Sphk1/Sphk2 double mutant to evaluate if S1P signaling may continue to be necessary for cell survival in the sensory ganglia or that S1P may regulate other processes such as axonal pathfinding and/or glial cell differentiation.
Figure 19.4. Sphk1 and Sphk2 expression in E16.5 trigeminal and dorsal root ganglia. E16.5 mouse embryos were processed for Sphk1 or Sphk2 section in situ hybridization (grayish black, A, B, C, D) followed by Tuj-1 immunostaining (white, A′, B′, C′, D′). Sphk1 and Sphk2 transcript were observed in trigeminal ganglia (Tuj-1+, white, A′, C′) at E16.5. In the trunk, Sphk1 and Sphk2 expression persisted in the neural tube (NT) and dorsal root ganglia (DRG) at this age (B, B′ and D, D′).
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426 SPHINGOSINE KINASES, S1P LYASE, AND THE VERTEBRATE NERVOUS SYSTEM
19.2.2. S1P Lyase
S1P lyase (Sgpl1) breaks down S1P irreversibly into ethanolamine phosphate and hexadecenal; thus, it is an important enzyme for controlling the levels of S1P and sphingosine. In vertebrates, only one Sgpl1 has been identified thus far. In the chick embryo, SGPL1 transcript could first be observed at the tip of the neural fold at 3–5 somites (stage 8, E1.25), and by 8–9 somites (stage 9, E1.5), SGPL1 was expressed specifically in the hindbrain level of the neural tube (Fig. 19.5A, B). SGPL1 transcript could be detected in the developing trigeminal and dorsal root ganglia beginning at E2.5 (stage 16, 26–28 somites; Fig. 19.5C), and expression in the olfactory region was obvious by E3.5–3.75 (stage 20, 41–42 somites; Fig. 19.5D). Section in situ combined with a marker for neural crest cells HNK-1, showed that cranial ganglia clearly expressed SGPL1 by E3.5, and neural crest cells condensing to form the dorsal root ganglia were beginning to express SGPL1 at the same age (Fig. 19.5E, F). Similar to SPHK1, both cranial and trunk sensory ganglia continued to express SGPL1 at later stages (Fig. 19.5G–I).
In the mouse embryo, Sgpl1 was expressed throughout the neural tube at E8.5 with the most intense staining in the rostral and caudal regions (Fig. 19.6A). Diffuse staining could be observed throughout the embryo by E9.5 (Fig. 19.6B), and at E10.5, more enriched Sgpl1 expression was detected in the forebrain, brachial arches, and trigeminal ganglia (Fig. 19.6C). We used section in situ hybridization and immunostaining to further examine Sgpl1 expression in the nervous system (Fig. 19.7). At E10.5, Sgpl1 transcript was seen in the brain and trunk neural tube. The developing trigeminal and dorsal root ganglia were beginning to express Sgpl1 although the in situ signal was similar to the surrounding low level in the mesenchyme at this stage (Fig. 19.7A-A′, F-F′). By E11.5, Sgpl1 expression in the neural tube, dorsal root, and trigeminal ganglia was clearly above background (Fig. 19.7B-B′, G-G′), and this pattern persisted from E12.5 to E16.5 (Fig. 19.7C-C′–J-J′). Although we could detect the presence of Sgpl1 in the sensory ganglia, we were not able to determine which cell types expressed this enzyme at the present resolution. It will be very helpful to have antibodies to SGPL1 in the future to perform double labeling experiments and discern the identity of the SGPL1 expressing cells.
From our whole mount in situ hybridization data, we noticed that Sphk1 and Sgpl1 seemed to have specific expression in the developing olfactory system. When we examined the forebrain and olfactory region at E12.5, we observed robust Sgpl1 staining that overlapped with neurons (as shown by Tuj-1 staining; Fig. 19.8A), indicating Sgpl1 was expressed in the olfactory epithelium; however, it was unclear if all cells or which type of cells were Sgpl1+. More detailed analyses at E16.5 revealed that a subpopulation of cells in the olfactory epithelium were Sgpl1+ and some of them were dually labeled with Tuj-1, suggesting that a subset of olfactory sensory neurons expressed Sgpl1 (Fig. 19.8B–D). It will be interesting to determine if Sphk1 and/or Sphk2 was also expressed in olfactory neurons. Given the expression of Sgpl1 in
RESULTS AND DIScUSSION 427
Figure 19.5. Sphingosine 1-phosphate lyase expression in the chick embryo. (A, B) At stage 8 (st.8, four somites, ∼E1.25) and stage 9 (st.9, seven somites, ∼E1.5), SGPL1 (purple) was expressed in the hindbrain region of neural fold (*). (C) By stage 16 (st.16, 26–28 somites, E2.5), SGPL1 transcript could be observed in the trigeminal ganglia (arrow). (D) SGPL1 (purple) was expressed in the trigeminal ganglia (arrow), olfactory pit (*, OP), and otic vesicle (OV) in the head and forming dorsal root ganglia (arrow-heads) in the trunk at stage 20 (D, st.20, 41–42 somites, E3.5). (E–I) SGPL1 section in situ hybridization followed by HNK-1 (turquoise) immunostaining. (E, F) At stage 19 (st.19, 37–40 somites, E3.5), SGPL1 (grayish black signal) was present in the trigeminal (Trig), other cranial ganglia (arrowheads), branchial arches (BA), and otic vesicle (OV; E) as well as the neural tube (N) and dorsal root ganglia (DRG; F). (G) SGPL1 expres-sion appeared to increase from stage 19 to stage 25 (st.25, E4) in the DRG; N continued to express SGPL1. (H, I) SGPL1 transcript could be observed in the cranial ganglia (Trig, VII, VIII) and DRG at stage 27 (st.27, E5). (See color insert.)
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428 SPHINGOSINE KINASES, S1P LYASE, AND THE VERTEBRATE NERVOUS SYSTEM
Figure 19.6. Sphingosine 1-phosphate lyase expression in the mouse embryo. (A) Sgpl1 was expressed throughout the neural tube (NT) at E8.5. (B) By E9.5, Sgpl1 staining could be seen in the forebrain (FB), midbrain (MB), and trigeminal ganglion (dotted area). (C) Diffuse Sgpl1 signal was detected in the entire embryo at E10.5 but its expression was enriched in the FB, MB, trigeminal ganglion (dotted area), branchial arches (BA), as well as forelimb (FL) and hind limb (HL). (See color insert.)
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Figure 19.7. Sphingosine 1-phosphate lyase expression in the mouse trigeminal and dorsal root ganglia. At E10.5, Sgpl1 was expressed in the neural tube (NT) but staining in the dorsal root ganglia (DRG) (*) was barely above background (A-A′). Robust Sgpl1 signal could be observed in the hindbrain epithelium (HB) and the trigeminal ganglion (Trig) was beginning to express Sgpl1 (F-F′). By E11.5, Sgpl1 transcript was readily detectable in dorsal root (DRG) and trigeminal ganglia (Trig; B-B′; G-G′). (C-C′–J-J′) Sgpl1 expression persisted in the NT, DRG, and Trig between E12.5 and E16.5. (See color insert.)
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RESULTS AND DIScUSSION 429
trigeminal and dorsal root ganglia as well as olfactory neurons, and that deleting Sphk1/2 resulted in severe sensory ganglia deficits, it is tempting to speculate that S1P signaling may have a unique role in sensory neuron development.
To date, no Sgpl1 knockout is available but an Sgpl1 gene trapped line has been generated from a screen for platelet-derived growth factor (PDGF) downstream effector genes (referred as Sgpl1−/−; 28). Consistent with PDGF signaling, Sgpl1 mutants displayed vascular and skeletal abnormalities, and cell migration was also affected. The nervous system was not examined, but the craniofacial phenotype suggested that neural crest cell development was dis-rupted. It is not clear which aspect of embryonic and nervous system develop-ment may be abnormal in the Sgpl1−/− mice. They were smaller in size, but they survived until after birth and died by 8 weeks of age. Our preliminary survey
Figure 19.8. Sphingosine 1-phosphate lyase expression in the mouse olfactory epithe-lium. Sgpl1 section in situ hybridization followed by Tuj-1 immunostaining. (A) Hori-zontal section from an E12.5 embryo showed that Sgpl1 (grayish black) was expressed in the forebrain (FB) and olfactory epithelium (OE); neurons in this section were identified by Tuj-1 staining (green). (B–D) A section from an E16.5 embryo through the OE revealed that a subpopulation of cells in the OE expressed Sgpl1 (B, grayish black signal). (C) Same section in B processed for Tuj-1 immunostaining (red) to iden-tify neurons in the OE at this stage. (D) Sgpl1 signal in B was inverted to green in PhotoShop. Overlaying Sgpl1 (green) and Tuj-1 (red) staining demonstrated that a subset of olfactory neurons expressed Sgpl1. (See color insert.)
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430 SPHINGOSINE KINASES, S1P LYASE, AND THE VERTEBRATE NERVOUS SYSTEM
of the Sgpl1−/− embryos did not reveal any significant defects in the central and peripheral nervous system (date not shown), but additional experiments will be required to analyze any subtle phenotypes. Because S1P can be converted to sphingosine by sphingosine phosphatases, it is possible that any defects that result from the lack of SGPL1 activity and overaccumulation of S1P can be neutralized by phosphatases. This underscores the contribution by these S1P enzymes, Sphk1/Sphk2, Sgpl1, and sphingosine phosphatases to maintain a fine balance of S1P that is available.
19.3. SUMMARY
Sphingosine kinases and S1P lyase play key roles in regulating S1P signaling, hence a variety of biological processes. We have shown here that these enzymes are expressed broadly in the developing central and peripheral nervous systems although their precise, specific roles are not entirely clear. The use of conditional knockouts and a combination of in vivo, in vitro, and biochemical approaches should be employed to dissect the functions of Sphk and Sgpl1 as well as their possible interactions with S1P receptors and their downstream pathways.
19.4. MATERIALS AND METHODS
19.4.1. Whole Mount In Situ Hybridizations
Chicken and mouse embryos were processed for whole mount in situ hybrid-ization as previously described (29). Plasmids for chicken SPHK1 and SGPL were obtained from the BBSRC ChickEST Database (30, 31) via ARK-Genomics. Full-length mouse Sphk1, Sphk2, and Sgpl1 clones were purchased from OpenBiosystems (Huntsville, AL). Following linearization of DNA tem-plates, digoxigenin-labeled probes were generated using appropriate RNA polymerases (Promega, Madison, WI) and color reactions were developed with NBT/BCIP or BM purple (Roche, Indianapolis, IN). Whole mount and section in situ hybridizations for each probe were repeated at least three times or more to confirm our results.
19.4.2. Section In Situ Hybridization and Immunohistochemistry
For section in situ hybridizations, embryos were fixed with modified Carnoy’s solution, processed for paraffin sectioning, and in situ hybridization as described (32). For post in situ immunohistochemistry, slides were incubated in Tuj-1 (Covance, Emeryville, CA; 33) or HNK-1 (34) overnight at 4°C followed by three washes in phosphate buffer saline with 0.1% Tween 20. Fluorochrome conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA) were applied for 2 hours at room temperature in the dark.
REFERENcES 431
ACKNOWLEDGMENTS
Work described in this chapter was supported in part by a March of Dimes Basil O’Connor Starter Scholar award (VML).
REFERENCES
1. Spiegel S, English D, Milstien S. 2002. Sphingosine 1-phosphate signaling: providing cells with a sense of direction. Trends Cell Biol 12:236–242.
2. Saba JD. 2004. Lysophospholipids in development: miles apart and edging in. J Cell Biochem 92:967–992.
3. Hla T. 2004. Physiological and pathological actions of sphingosine 1-phosphate. Semin Cell Dev Biol 15:513–520.
4. Le Stunff H, Milstien S, Spiegel S. 2004. Generation and metabolism of bioactive sphingosine-1-phosphate. J Cell Biochem 92:882–899.
5. Saba JD, Hla T. 2004. Point-counterpoint of sphingosine 1-phosphate metabolism. Circ Res 94:724–734.
6. Reiss U, Oskouian B, Zhou J, Gupta V, Sooriyakumaran P, Kelly S, et al. 2004. Sphingosine-phosphate lyase enhances stress-induced ceramide generation and apoptosis. J Biol Chem 279:1281–1290.
7. Payne SG, Milstien S, Spiegel S. 2002. Sphingosine-1-phosphate: dual messenger functions. FEBS Lett 531:54–57.
8. Allende ML, Proia RL. 2002. Sphingosine-1-phosphate receptors and the develop-ment of the vascular system. Biochim Biophys Acta 1582:222–227.
9. Brinkmann V. 2007. Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology. Phar-macol Ther 115:84–105.
10. Choi JW, Lee CW, Chun J. 2008. Biological roles of lysophospholipid receptors revealed by genetic null mice: an update. Biochim Biophys Acta 1781:531–539.
11. Spiegel S, Milstien S. 2002. Sphingosine 1-phosphate, a key cell signaling molecule. J Biol Chem 277:25851–25854.
12. Spiegel S, Milstien S. 2003. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 4:397–407.
13. Ishii I, Fukushima N, Ye X, Chun J. 2004. Lysophospholipid receptors: signaling and biology. Annu Rev Biochem 73:321–354.
14. Kupperman E, An S, Osborne N, Waldron S, Stainier DY. 2000. A sphingosine-1-phosphate receptor regulates cell migration during vertebrate heart development. Nature 406:192–195.
15. Lee MJ, Thangada S, Claffey KP, Ancellin N, Liu CH, Kluk M, et al. 1999. Vascular endothelial cell adherens junction assembly and morphogenesis induced by sphingosine-1-phosphate. Cell 99:301–312.
17. Saba JD, Nara F, Bielawska A, Garrett S, Hannun YA. 1997. The BST1 gene of Saccharomyces cerevisiae is the sphingosine-1-phosphate lyase. J Biol Chem 272:26087–26090.
432 SPHINGOSINE KINASES, S1P LYASE, AND THE VERTEBRATE NERVOUS SYSTEM
18. Li G, Alexander H, Schneider N, Alexander S. 2000. Molecular basis for resistance to the anticancer drug cisplatin in Dictyostelium. Microbiology 146(Pt 9):2219–2227.
19. Li G, Foote C, Alexander S, Alexander H. 2001. Sphingosine-1-phosphate lyase has a central role in the development of Dictyostelium discoideum. Development 128:3473–3483.
20. Herr DR, Fyrst H, Phan V, Heinecke K, Georges R, Harris GL, Saba JD. 2003. Sply regulation of sphingolipid signaling molecules is essential for Drosophila develop-ment. Development 130:2443–2453.
21. Renault AD, Sigal YJ, Morris AJ, Lehmann R. 2004. Soma-germ line competition for lipid phosphate uptake regulates germ cell migration and survival. Science 305:1963–1966.
22. Mizugishi K, Yamashita T, Olivera A, Miller GF, Spiegel S, Proia RL. 2005. Essential role for sphingosine kinases in neural and vascular development. Mol Cell Biol 25:11113–11121.
23. Allende ML, Sasaki T, Kawai H, Olivera A, Mi Y, van Echten-Deckert G, et al. 2004. Mice deficient in sphingosine kinase 1 are rendered lymphopenic by FTY720. J Biol Chem 279:52487–52492.
24. Meng H, Yuan Y, Lee VM. 2011. Loss of Sphingosine kinase 1/S1P signaling impairs cell growth and survival of neurons and progenitor cells in the developing sensory ganglia. PLoS ONE 6:e27150.
25. LeDouarin N, Kalcheim C. 1999. The Neural Crest, 2nd edition. New York: Cam-bridge University Press.
26. Meng H, Lee VM. 2009. Differential expression of sphingosine-1-phosphate recep-tors 1–5 in the developing nervous system. Dev Dyn 238:487–500.
27. Zachariah MA, Cyster JG. 2010. Neural crest-derived pericytes promote egress of mature thymocytes at the corticomedullary junction. Science 328:1129–1135.
28. Schmahl J, Raymond CS, Soriano P. 2007. PDGF signaling specificity is mediated through multiple immediate early genes. Nat Genet 39:52–60.
29. Wilkinson D. 1992. Whole mount in situ hybridization of vertebrate embryos. In In Situ Hybridization: A Practical Approach. D Wilkinson, ed. New York: Oxford University Press, pp. 75–83.
30. Boardman PE, Sanz-Ezquerro J, Overton IM, Burt DW, Bosch E, Fong WT, et al. 2002. A comprehensive collection of chicken cDNAs. Curr Biol 1:1965–1969.
31. Hubbard SJ, Grafham DV, Beattie KJ, Overton IM, McLaren SR, Croning MD, et al. 2005. Transcriptome analysis for the chicken based on 19,626 finished cDNA sequences and 485,337 expressed sequence tags. Genome Res 15:174–183.
32. Etchevers HC, Vincent C, Le Douarin NM, Couly GF. 2001. The cephalic neural crest provides pericytes and smooth muscle cells to all blood vessels of the face and forebrain. Development 128:1059–1068.
33. Lee MK, Tuttle JB, Rebhun LI, Cleveland DW, Frankfurter A. 1990. The expression and posttranslational modification of a neuron-specific beta-tublin isotype during chick embryogenesis. Cell Motil Cytoskeleton 17:118–132.
34. Tucker GC, Aoyama H, Lipinski M, Tursz T, Thiery JP. 1984. Identical reactivity of monoclonal antibodies HNK-1 and NC-1: conservation in vertebrates on cells derived from the neural primordium and on some leukocytes. Cell Differ 14:223–230.
