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Supplemental Figure 1

The generation of Spns2 conditional knockout mice. (A) Schematic representation of

the wild type Spns2 locus (Spns2+), the targeted allele, the floxed allele (Spns2f) and the

deleted allele (Spns2-). In the targeted allele, exon 2 of the Spns2 gene (yellow box) is

flanked by two loxP sites (green arrowheads). Together with the first loxP site, a

neomycin selection cassette, PKG-Neo-pA (gray box), flanked by two frt sites (orange

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arrowheads) has been inserted. Mice carrying the targeted Spns2 allele were crossed

with transgenic mice expressing Flp recombinase under the control of cytomegalovirus

early enhancer/chicken beta actin (CAG) promoter to remove the PKG-Neo-pA cassette,

resulting in the Spns2 floxed mice. To generate conventional Spns2 knockout mice, the

mice carrying a floxed Spns2 allele were crossed with transgenic mice expressing the

Cre recombinase under the control of cytomegalovirus (CMV) promoter. To inactivate

the Spns2 gene in endothelial cells, the Spns2 floxed mice were bred with Tie2-Cre

mice that carry the Cre recombinase driven by the Tie2 promoter. (B) Southern blot

analysis of AvrII-digested genomic DNA from tail biopsies of Spns2f/f, Spns2-/- and

Spns2+/+ mice were carried out using a probe indicated in A (blue line). As expected, 7.2

kb, 7.5 kb and 7.0 kb bands were detected in wild type (Spns2+/+), Spns2 floxed

(Spns2f/f) and Spns2 knockout (Spns2-/-) mice. (C) PCR analysis of the genomic DNA

from Spns2f/f, Spns2-/- and Spns2+/+ mice were performed using a primer set to amplify

the exon 2 as indicated in A (red arrows). 552-bp, 842-bp and 316-bp fragments were

amplified in wild type (Spns2+/+), Spns2 floxed (Spns2f/f) and Spns2 knockout (Spns2-/-)

mice. (D) Genomic PCR analysis of Spns2+/+, Spns2-/-, Spns2f/-, Spns2f/-;Tie2Cre,

Spns2f/f, Spns2f/f;Tie2Cre mice were performed using the primer sets to amplify the

exon 2 of Spns2 gene (upper panel) and Cre recombinase gene (lower panel).

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Supplemental Figure 2

Spns2 KO mice are functionally disrupted for Spns2. (A) Schematic representation of

the exon/intron structure of the wild type Spns2 allele and the corresponding wild type

mRNA (Wt mRNA). A mutant mRNA transcribed from the Spns2 deleted allele lacking

exon 2 (Mutant mRNA) is also shown at the bottom. Boxes and polylines represent

exons and introns, respectively. Blue and gray boxes correspond to the coding and

non-coding exons, respectively. Red boxes indicate exon 2. The size of exon is given

below each exon. The mRNA derived from the Spns2 deleted allele results in an

in-frame deletion of exon 2. (B) RT-PCR analyses of RNAs extracted from the lungs of

wild type (Spns2+/+) and Spns2 KO (Spns2-/-) mice were performed using two sets of

PCR primers as indicated in A (blue and green arrows). The size difference between the

two bands is consistent with the absence of exon 2 (66 bp) in the mutant mRNA.

Sequencing of the PCR products showed that Spns2 KO mice express a mutant mRNA

transcript lacking exon 2-derived sequence encoding aa 124-145 of wild type Spns2.

(C) HUVECs were transfected with the plasmid expressing either wild type (Wt) or

mutant (Mutant) Spns2 carboxy-terminally tagged GFP or with myristoylated

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GFP-encoding plasmid (Myr-GFP). GFP and phase contrast images were obtained using

an IX81 inverted microscope (Olympus). Note that mutant Spns2 protein failed to

localize at the plasma membrane. (D) HEK293 cells were transfected with the plasmid

expressing human Spns2 (hWt), mouse Spns2 (mWt) or mouse Spns2 mutant (mutant)

together with or without the Sphk1-expressing vector as indicated at the bottom. S1P

release from those cells was examined as described in Methods. Data are shown as

means ± S.D. (n=4). Note that Spns2 mutant lost the ability to export S1P.

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Supplemental Figure 3

Spns2 KO mice develop normally except for the symblepharon formation. (A-F) Body

and organ weights of 10-week-old wild type (Spns2+/+) and Spns2 KO (Spns2-/-) mice

were measured (body weight (A), heart (B), lung (C), liver (D), spleen (E), kidney (F)).

There were no differences in body and organ weights between wild type and Spns2 KO

mice. Data are shown as means ± S.D. (wild type, n=11; Spns2 KO, n=10). (G) Spns2

KO mice exhibit symblepharon.

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Supplemental Figure 4

The biochemical examination of the blood of Spns2 KO mice. Plasma levels of total

protein (TP) (A), total bilirubin (TBIL) (B), aspartate aminotransferase (AST) (C),

alanine aminotransferase (ALT) (D), triglycerol (TG) (E), glucose (GLU) (F), blood

urea nitrogen (BUN) (G) and albumin (ALB) (H) in 8-week-old wild type (Spns2+/+)

and Spns2 KO (Spns2-/-) mice. There were no differences in blood biochemical

parameters between wild type and Spns2 KO mice. Data are shown as means ± S.D.

(n=4).

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Supplemental Figure 5

The hematological profile of Spns2 KO mice. Blood was collected from 8-week-old

wild type (Spns2+/+) and Spns2 KO (Spns2-/-) mice, and analyzed for white blood cells

(WBC) (A), red blood cells (RBC) (B), platelets (C), hemoglobin (HBG) (D),

hematocrit (HCT) (E), mean corpuscular volume (MCV) (F), mean corpuscular

hemoglobin (MCH) (G) and mean corpuscular hemoglobin concentration (MCHC) (H).

Spns2 KO mice exhibited a decreased count of white blood cells compared with wild

type mice. Data are shown as means ± S.D. (n=4).

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Supplemental Figure 6

A representative flow cytometric analysis of peripheral blood cells from control

(Spns2+/+) and global Spns2 KO (Spns2-/-) mice. (A) The numbers represent the

percentage of CD4 and CD8 single-positive (SP) T cells in total lymphocytes. (B) The

numbers represent percentages of IgD+CD23+ cells in CD19+ peripheral blood

lymphocytes (mature recirculating B cells; Mature rec. B) in the gate on the plots.

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Supplemental Figure 7

Histological analyses of the thymi and peripheral lymph nodes (LN) of Spns2 KO mice.

Representative hematoxylin and eosin stained sections of thymus and peripheral LN

from wild type (Spns2+/+) and Spns2 KO (Spns2-/-) mice. No striking structural

differences of thymus and peripheral LNl between Spns2 KO and control mice were

detected.

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Supplemental Figure 8

Increased proportion of fully mature SP T cells and decreased proportion of

semi-mature SP T cells in the thymi of Spns2 KO mice. (A) Frequencies (left) and

numbers (right) of semi-mature (CD69+CD62Llo/-) and fully mature (CD69lo/-CD62L+)

CD4 SP T cells in the thymi of control (Spns2+/+) or Spns2 KO (Spns2-/-) mice (n=8).

(B) Frequencies (left) and numbers (right) of semi-mature (CD69+CD62Llo/-) and fully

mature (CD69lo/-CD62L+) CD8 SP T cells in thymi of control (Spns2+/+) or Spns2 KO

(Spns2-/-) mice (n=8). (C) Flow cytometric analysis for the expression of CD69 on fully

mature CD62L+ CD4 (left) and CD8 (right) SP T cells in the thymus of control (black

line) or Spns2 KO (orange line) mice. Control antibody staining is shown shaded in grey.

Data are representative for eight pairs of mice.

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Supplemental Figure 9

Reduced numbers of mature recirculating B (Mature rec. B) cells in the bone marrow of

Spns2 KO mice. Representative flow cytometric analysis of bone marrow derived B

cells from control (Spns2+/+) or Spns2 KO (Spns2-/-) mice. (A) The numbers indicate the

percentage of IgM and IgD expressing CD19+ B cells. Mature recirculating B (Mature

rec. B) cells and immature B cells were identified as CD19+IgM+IgD+ and

CD19+IgM+IgD-, respectively. (B) Frequencies (left) and numbers (right) of mature

recirculating B cells and immature B cells are shown (n=11). Bars and circles indicate

averages and values for individual mice, respectively.

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Supplemental Figure 10

Amount of plasma S1P bound to HDL and albumin in control (Spns2+/+) or Spns2 KO

(Spns2-/-) mice. (A) Representative gel filtration profiles of plasma S1P (bars) and

protein (solid line) from control (top: Spns2+/+) and Spns2 KO (bottom: Spns2-/-) mice.

100 l of plasma was chromatographed on a Superose 12 column. Fractions were

collected every 2 min (0.5 ml). For each fraction, S1P concentration was determined.

Slashed and gray bars indicate the amount of S1P bound to HDL and albumin,

respectively. (B) Amount of S1P associated with HDL and albumin in 100 l of plasma

from control (Spns2+/+) or Spns2 KO (Spns2-/-) mice (n=2).

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Supplemental Figure 11

PCR analysis of the genomic DNA isolated from the bone marrow of wild type mouse

(Spns2+/+), Spns2 KO mouse (Spns2-/-) and 13 Spns2 KO mice reconstituted with wild

type bone marrow (#1~#13) was performed as described in the legend of Supplementary

Figure 1C. 552-bp and 316-bp PCR products represent the wild type and Spns2-deleted

alleles, respectively. Note that the bone marrow of Spns2 KO mice was successfully

reconstituted by that of wild type mice.

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Supplemental Figure 12

In situ hybridization for Spns2 mRNA. Antisense probe was hybridized to the sections

of heart, lung, hypothalamus, olfactory bulb and kidney (Spns2: purple). Serial sections

were also stained with anti-CD31 antibody (CD31: brown) to identify the endothelial

cells. The scale bars represent 20 m.

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Supplemental Figure 13

Plasma concentrations of S1P in control (Spns2f/f) or EC-Spns2 cKO (Spns2f/f;Tie2Cre)

mice. Data are shown as means ± S.D. (n=3).

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Supplemental Figure 14

Reduced number of mature T cells in peripheral lymph nodes of EC-Spns2 cKO mice.

Frequencies (left) and numbers (right) of CD4 SP (CD4) and CD8 SP (CD8) T cells in

peripheral lymph nodes from control (Spns2f/f) or EC-Spns2 cKO (Spns2f/f;Tie2Cre)

mice are shown (n=11). Bars and circles indicate averages and values for individual

mice, respectively.

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Supplemental Figure 15

Reconstitution of EC-Spns2 cKO mice with control bone marrow. (A) PCR analysis of

the genomic DNA isolated from the bone marrow of wild type mouse (Spns2+/+), Spns2

KO mouse (Spns2-/-), Spns2 floxed mouse (Spns2f/f), 8 Spns2 floxed mice reconstituted

with bone marrow from Spns2 floxed mice (Spns2f/f→Spns2f/f) and 9 EC-Spns2 cKO

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mice reconstituted with bone marrow from Spns2 floxed mice

(Spns2f/f→Spns2f/f,Tie2Cre) was performed as described in the legend of

Supplementary Figure 1C. 552-bp, 842-bp and 316-bp PCR products represent the wild

type, Spns2-floxed and Spns2-deleted alleles, respectively. Note that the bone marrow

of EC-Spns2 cKO was successfully reconstituted by that of Spns2 floxed mice.

(B, C) Flow cytometric analyses of Spns2 floxed mice reconstituted with bone marrow

from Spns2 floxed mice (Spns2f/f→Spns2f/f) and EC-Spns2 cKO mice reconstituted

with bone marrow from Spns2 floxed mice (Spns2f/f→Spns2f/f,Tie2Cre). (B)

Frequencies and total numbers of CD4 SP (CD4) (left) and CD8 SP (CD8) (right) T

cells in the thymus are shown (Spns2f/f→Spns2f/f, n=8; Spns2f/f→Spns2f/f,Tie2Cre, n=9).

(D) Frequencies and total numbers of CD4 SP (CD4) (left) and CD8 SP (CD8) (right) T

cells in the peripheral blood are shown (Spns2f/f→Spns2f/f, n=8;

Spns2f/f→Spns2f/f,Tie2Cre, n=9).

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Supplemental Figure 16

Reduced number of mature recirculating B cells in bone marrow of EC-Spns2 cKO

mice. Flow cytometric analyses of bone marrow derived B cells from control (Spns2f/f)

or EC-Spns2 cKO (Spns2f/f;Tie2Cre) mice. (A) The numbers indicate the percentage of

IgM and IgD expressing CD19+ B cells. Mature recirculating B (Mature rec. B) cells

and immature B cells were identified as CD19+IgM+IgD+ and CD19+IgM+IgD-,

respectively. (B), Frequencies (left) and numbers (right) of mature recirculating B cells

and immature B cells are shown in left and right panels, respectively (n=11). Bars and

circles indicate averages and values for individual mice, respectively.

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Supplemental Figure 17

Number of mature recirculating B cells in peripheral lymph nodes of EC-Spns2 cKO

mice. Frequencies (left) and numbers (right) of mature recirculating B cells

(CD19+CD23+IgD+) in peripheral lymph nodes from control (Spns2f/f) or EC-Spns2

cKO (Spns2f/f;Tie2Cre) mice are shown (n=11). Bars and circles indicate averages and

values for individual mice, respectively.

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Supplemental Figure 18

Flow cytometric analyses of Spns2 floxed mice reconstituted with bone marrow from

Spns2 floxed mice (Spns2f/f→Spns2f/f) and EC-Spns2 cKO mice reconstituted with

bone marrow from Spns2 floxed mice (Spns2f/f→Spns2f/f,Tie2Cre). Frequencies (left)

and total numbers (right) of mature recirculating B cells in the bone marrow (A) and

peripheral blood (B) are shown (Spns2f/f→Spns2f/f, n=8; Spns2f/f→Spns2f/f,Tie2Cre,

n=9).