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17
Supporting Information
Identification of GPI-anchored protein LYPD1 as an essential
factor for odontoblast differentiation in tooth development
Yao Fu, Kanako Miyazaki, Yuta Chiba, Keita Funada,
Tomomi Yuta, Tian Tian, Kanji Mizuta, Jumpei Kawahara, Ling Zhang, Daniel Martin,
Tsutomu Iwamoto, Ichiro Takahashi, Satoshi Fukumoto, Keigo Yoshizaki
List of Materials
Figure S1. The representative known marker genes for classification.
Figure S2. The representative known marker genes for classification using Monocle 3.
Figure S3. Silencing of Lypd1 expression using small interfering RNAs (siRNAs).
Figure S4. The result of qRT-PCR analysis and Western blotting on dental mesenchymal
differentiation marker genes in E15 tooth germs treated with zaragozic acid A or myriocin.
Figure S5. The result of Western blotting on dental mesenchymal differentiation marker
genes in mDP cells treated with MβCD.
Figure S6. Western blotting results of BMP2-induced Smad1/5/8 phosphorylation in
mDP cells transfected with mock-V5, LYPD1-FL-V5, or LYPD1-ΔGPI-V5.
Figure S7. The expression of LYPD family molecules.
Table S1. List of top genes from microarray analysis.
Table S2. scRNA-seq quality control.
Table S3. List of top 20 DEGs for each cluster.
Table S4. Lypd1 target sequences used for RNA interference.
Table S5. Primer sequences used in this study.
Table S6. Antibodies used in this study.
Dsred
Dlk1
Top2a
11
AC MSC llicle Pulp lium cyte cyte
ro
MT
uk
ith
l F enta
yth
Le
Ep
nta
Er
tal
De
De
Pr
-O
to
on
Log2 Exp
Log2 Exp
las
bla
st
te
AC MSC llicle Pulp lium cyt
cy
MT
ko thro
he
l F enta
eu
pit
De
nta
De
Aspn
Pr
do
-O
tob
las
AC MSC llicle Pulp lium cyte cyte
ro
MT
uk
ith
l F enta
yth
Le
Ep
nta
Er
tal
De
De
do
-O
nto
bla
st
AC MSC llicle Pulp lium cyte cyte
ro
MT
uk
ith
l F enta
yth
Le
Ep
nta
Er
tal
De
De
Log2 Exp
Log2 Exp
Log2 Exp
Log2 Exp
Pr
las
Pr
tob
Alas2
do
-O
AC MSC llicle Pulp lium cyte cyte
MT
he
uk rythr
lF
nta
pit
Le
nta
De tal E
De
De
Log2 Exp
AC MSC llicle Pulp lium cyte cyte
ro
MT
uk
ith
l F enta
yth
Le
Ep
nta
Er
tal
De
De
Tyrobp
las
Log2 Exp
las
tob
Log2 Exp
Log2 Exp
Pr
tob
do
-O
Krt14
do
-O
Mmp13
Pr
Log2 Exp
tob
Log2 Exp
Pr
10
do
-O
Log2 Exp
Log2 Exp
Log2 Exp
Log2 Exp
AC MSC llicle Pulp lium cyte cyte
MT
he
uk rythr
l F enta
pit
Le
nta
lE
De
De
Pr
do
-O
tob
las
AC MSC llicle Pulp lium cyte cyte
MT
he
uk rythr
l F enta
pit
Le
nta
lE
De
De
Figure S1. The representative known marker genes for classification. Expression of known marker
genes for dental cell types is projected onto the t-SNE plot (upper panel) and violin plot (lower panel).
Lypd1
Lypd1
Fgf3
Fgf3
Top2a
Top2a
10
Dlk1
Aspn
Mmp13
0 10
−5
−10
% Max
UMAP 2
100
−15 −5
75
Dlk1
Dlk1
Dlk1
−10
10
10
Mmp13
Mmp13
Mmp13
50
% Max
25 100
UMAP 2
−15
UMAP2
Aspn
Aspn
Aspn
75
Krt14
Tyrobp
Alas2
50
0 10
−5
−5
−10
−10
25
% Max
UMAP 2
100
−15 −5
−15
−10
−10
Krt14
Krt14
10
−10
Tyrobp 1
Tyrobp
UMAP
10
−10
Alas2
Alas2
75
10
50
10
25
−15
−10
10
−10
10
−10
10
UMAP 1
−5
−10
−15
−10
10
−10
10
−10
10
UMAP 1
UMAP1
Figure S2. The representative known marker genes for classification using Monocle 3.
Expression of known marker genes for dental cell types is projected onto the UMAP plot.
Lypd1
Dspp
0.8
0.6
***
***
0.4
*** ***
***
0.2
Relative expression
1.2
0.8
0.6
***
***
0.4
0.2
***
***
***
A NA
R iRN siR
s s 3
#1 #2 1 #
1 1 d
pd pd yp
Ly Ly L
LLy
yppd
d11
SSM ccoo
MA nntt
ARR rroo
T ll
Ly Tppoo ssiRiR
pd ooll NNA
Ly 1 # ssiRiR A
pd 1 NN
Ly 1 # siR AA
pd 2 NA
LLy 1 # siR
ypp 3 N
dd1 s A
1# iR
#44 N
ssiR A
iRN
NA
A A NA
R iRN siR
s s 3
#1 #2 1 #
1 1 d
pd pd yp
Ly Ly L
LLy
ypp
dd1
1S c c
SMM oo
AAR nntrtr
R oo
Ly TTppo l sl isRiR
p ooo NN
Ly d1 l sl isRi AA
p #1 RNN
Ly d1 siR AA
p #2 N
LyL d1 siR A
pydp #3 N
1d1 siR A
#4# N
4si A
RsiR
NAN
Relative expression
1.2
Figure S3. Silencing of Lypd1 expression using small interfering RNAs (siRNAs). (A) The relative levels
of Lypd1 mRNA in mDP cells transfected with control siRNA and Lypd1 siRNAs for 24 h. (B) The
relative levels of Dspp mRNA in mDP cells transfected with control siRNA and Lypd1 siRNAs for 72 h.
***, P <0.001, compared to the control siRNA group. Error bars represent the mean ± S.D.
0.2
0.4
0.4
0.2
DD
MM
M M SOSO
yry
iorio
cinci
Relative expression
**
0.8
**
0.6
0.4
0.2
DD
MM
M M SOSO
yry
iorio
cinci
Relative expression
0.6
PANX3
0.6
ALPL
0.4
45
100
71
71
DSPP
0.2
DM
SS
ZaZ O
ra
rg
ac aogzi
id …
Alpl
Alpl
GAPDH
30
Dspp
Dspp
1.2
0.8
0.6
0.4
25
1.2
1.2
0.8
***
0.2
Panx3
Panx3
1.2
0.4
LYPD1
0.8
DD
MM
Za OSO
Zra
ac raogzi
id …c
Lypd1
Lypd1
0.6
***
DD
MM
Za SO
Zraa O
gr o
ac agzi
id …
***
0.2
0.8
Relative expression
0.4
0.6
DD
MM
Za SO
Zraa O
gr o
ac a zi
id g…
0.6
0.8
cid
ic a
ag
DM
Zar (kDa)
1.2
***
0.2
Relative expression
0.8
Relative expression
Relative expression
1.2
1.2
Relative expression
Relative expression
1.2
Dspp
Dspp
Alpl
Panx3
Panx3
DM
LYPD1
0.8
PANX3
0.6
ALPL
0.4
0.2
DSPP
***
GAPDH
SO
in
rioc
My
(kDa)
25
45
100
71
71
30
DD
MM
M SOSO
yMr y
iorio
cinc
in
Lypd1
Lypd1
DD
MM
SS
MM O O
yyr
rioioci
…n
Figure S4. The result of qRT-PCR analysis and Western blotting on dental mesenchymal differentiation
marker genes in E15 tooth germs treated with zaragozic acid A or myriocin. (A and B, left) qRT-PCR
analysis of E15 tooth germs treated with DMSO, 40 μM zaragozic acid A, or 25 μM myriocin and
cultured for 7 days. The expressions of Lypd1, Panx3, Alpl, and Dspp were normalized to Gapdh mRNA
expression. *, P <0.05; **, P <0.01; ***, P <0.001. Error bars represent the mean ± S.D. (A and B, right)
Western blotting results of LYPD1, PANX3, ALPL, DSPP, and GAPDH in E15 tooth germs treated with
DMSO, 40 μM zaragozic acid A, or 25 μM myriocin after 7 days of culture. GAPDH was used as the
internal control.
mDP
MβCD
(kDa)
LYPD1
25
PANX3
45
ALPL
DSPP
GAPDH
100
71
71
30
Figure S5. The result of Western blotting on dental mesenchymal differentiation marker genes
in mDP cells treated with MβCD. Western blotting results of LYPD1, PANX3, ALPL, DSPP, and
GAPDH in mDP cells treated with 0 mM or 1 mM MβCD cultured for 48 h. GAPDH was used
as the internal control.
BMP2 (200 ng/mL)
mock
min
P-Smad1/5/8
Smad1
5 15 30
LYPD1-FL
5 15 30
LYPD1- ΔGPI
15 30
(kDa)
71
71
Id1
17
LYPD1
25
GAPDH
30
Figure S6. Western blotting results of BMP2-induced Smad1/5/8 phosphorylation in mDP cells
transfected with mock-V5, LYPD1-FL-V5, or LYPD1-ΔGPI-V5. After 48 h of transfection with
mock-V5, LYPD1-FL-V5, or LYPD1-ΔGPI-V5, mDP cells were stimulated with 200 ng/mL
BMP2 for 0, 5, 15, and 30 min. Protein extractions were analyzed by western blotting using antiphospho-Smad1/5/8, anti-Smad1, anti-Id1, anti-LYPD1, and anti-GAPDH antibodies. GAPDH
was used as the internal control.
Log2 Exp
Lypd1
Lypd2
Lypd3
Lypd4
Lypd5
Lypd6
Lypd6a
Lypd8
Figure S7. The expression of LYPD family molecules. Expression of the LYPD family projected
onto the t-SNE plot.
Table S1. List of top genes from microarray analysis.
dental papilla specific genes
dental follicle specific genes
Gene
log2 FC
Gene
log2 FC
Egr3
3.4294799
scl0001849.1_2273
-2.1597871
Dlx1as
3.41258171
Ifitm5
-2.1806923
Crym
3.21380327
F13a1
-2.2228545
Gldn
3.16331449
Col17a1
-2.237549
Nptx2
3.02911848
Meis2
-2.2483591
Egr2
2.86945201
Gdf10
-2.2636838
Etv4
2.86150295
2610016A17Rik
-2.3458507
Amn
2.61352441
Chodl
-2.4108479
Pde1b
2.53347538
Cntn6
-2.4782101
Hs3st6
2.50566058
Dmrta2
-2.5275365
Enpp1
2.46353513
Krt15
-2.6430633
Syn1
2.41664882
Dlx3
2.37899683
Hs3st3b1
2.22015159
Rspo2
2.17785033
Spp1
2.16550735
S100a8
2.11296218
Meox1
2.11119714
Igfbp3
2.08298436
Thy1
2.08089496
3830431G21Rik
2.07782256
Fibcd1
2.06959112
Lypd1
2.06182423
LOC669420
1.99785314
Egr1
1.93691849
Sp6
1.93566062
Table S2. scRNA-seq quality control.
E16 molar
Number of cells captured
1,905
Mean reads per cell
110,782
Mean genes per cell
2,903
Sequencing saturation(%)
76.4
Table S3. List of top 20 DEGs for each cluster.
Gene
Cluster
Log2 FC
p-value
Gene
Cluster
Log2 FC
p-value
Fgf3
Preodontoblast
8.0
4.24E-52
Mfap5
DMSC
5.7
5.04E-20
Smpd3
Preodontoblast
6.2
1.18E-54
Clec3b
DMSC
5.1
2.88E-31
Dlx6os1
Preodontoblast
5.8
1.81E-49
Dpt
DMSC
5.0
1.04E-29
Lmo1
Preodontoblast
5.3
3.40E-41
Kera
DMSC
4.6
3.62E-17
Plppr1
Preodontoblast
5.2
8.91E-39
Agtr2
DMSC
4.5
1.73E-33
Lypd1
Preodontoblast
5.0
3.55E-27
Col14a1
DMSC
4.1
4.48E-25
Dlx4
Preodontoblast
5.0
3.55E-27
Gdf10
DMSC
4.0
4.48E-25
Dkk1
Preodontoblast
4.9
3.90E-19
Angptl1
DMSC
4.0
2.49E-27
Tac1
Preodontoblast
4.9
3.41E-14
Fibin
DMSC
3.9
1.38E-13
Hs3st6
Preodontoblast
4.9
1.46E-32
Dlk1
DMSC
3.8
1.38E-13
Plac8
Preodontoblast
4.8
1.52E-16
AW551984
DMSC
3.7
1.28E-18
Rspo2
Preodontoblast
4.7
8.43E-29
Adam33
DMSC
3.6
7.74E-22
Wif1
Preodontoblast
4.5
8.43E-29
Eln
DMSC
3.6
1.38E-13
Vegfd
Preodontoblast
4.4
1.30E-21
Nr2f1
DMSC
3.6
1.09E-18
Sp9
Preodontoblast
4.4
7.67E-23
Osr1
DMSC
3.5
7.74E-22
C1ql1
Preodontoblast
4.1
5.63E-20
Mgp
DMSC
3.4
1.38E-13
Wnt10a
Preodontoblast
4.0
8.89E-18
Htra1
DMSC
3.4
1.89E-17
Bmp4
Preodontoblast
3.9
1.21E-22
1500015O10Rik
DMSC
3.4
8.09E-12
Tril
Preodontoblast
3.7
9.83E-14
Meox2
DMSC
3.3
1.41E-13
Dlx3
Preodontoblast
3.7
1.25E-17
9430076C15Rik
DMSC
3.3
1.09E-18
Mxd3
MTAC
3.8
1.43E-15
Pappa2
Dental Follicle
3.0
2.05E-10
Kif23
MTAC
3.7
1.67E-15
Cntn6
Dental Follicle
2.9
1.34E-08
Cdkn2c
MTAC
3.6
5.66E-15
Omd
Dental Follicle
2.9
3.98E-08
Ckap2l
MTAC
3.4
3.86E-14
Chodl
Dental Follicle
2.9
2.57E-10
Spc25
MTAC
3.4
2.89E-14
Calca
Dental Follicle
2.7
1.37E-04
Prc1
MTAC
3.3
2.98E-13
Sema3a
Dental Follicle
2.7
1.08E-08
Ckap2
MTAC
3.3
3.12E-13
Alcam
Dental Follicle
2.7
3.29E-09
Hmmr
MTAC
3.3
2.96E-12
Aspn
Dental Follicle
2.5
2.16E-07
Top2a
MTAC
3.2
3.65E-13
Dkk2
Dental Follicle
2.5
5.23E-07
Aurkb
MTAC
3.2
7.47E-12
Crlf1
Dental Follicle
2.5
8.47E-08
Mis18bp1
MTAC
3.1
1.20E-11
Lum
Dental Follicle
2.4
1.12E-07
Fam64a
MTAC
3.1
4.34E-11
C1qtnf3
Dental Follicle
2.4
2.60E-06
Ccna2
MTAC
3.0
1.11E-11
Ibsp
Dental Follicle
2.4
3.15E-02
Tk1
MTAC
3.0
1.94E-11
Fbln2
Dental Follicle
2.3
4.61E-07
Hjurp
MTAC
3.0
1.64E-11
Rspo3
Dental Follicle
2.3
1.01E-05
Aurka
MTAC
3.0
5.31E-10
Apod
Dental Follicle
2.3
5.65E-06
Racgap1
MTAC
3.0
4.84E-11
Tfap2b
Dental Follicle
2.3
3.49E-06
Nusap1
MTAC
3.0
1.12E-10
Fosl2
Dental Follicle
2.3
3.76E-06
Cenpf
MTAC
2.9
6.48E-10
Col8a1
Dental Follicle
2.2
1.94E-05
Ccnb1
MTAC
2.9
1.47E-09
Matn4
Dental Follicle
2.1
4.15E-04
Table S3. continued
Gene
Cluster
Log2 FC
p-value
Gene
Cluster
Log2 FC
p-value
Mmp13
Dental Pulp
5.1
3.35E-24
Myog
Leukocyte
9.7
3.32E-22
Pdzrn4
Dental Pulp
4.4
6.47E-24
Cma1
Leukocyte
9.0
1.34E-05
Pcp4
Dental Pulp
3.5
6.75E-15
Ccl9
Leukocyte
8.8
9.20E-18
Gpx3
Dental Pulp
3.5
1.50E-17
Ctss
Leukocyte
8.6
7.19E-38
Aldh1a2
Dental Pulp
3.3
2.72E-18
Myod1
Leukocyte
8.6
3.48E-34
Tnc
Dental Pulp
3.2
3.15E-15
C1qa
Leukocyte
8.5
2.23E-29
Rasl11a
Dental Pulp
3.1
1.91E-14
Actc1
Leukocyte
8.4
1.44E-26
Socs3
Dental Pulp
3.0
1.42E-13
C1qc
Leukocyte
8.0
1.23E-28
9130024F11Rik
Dental Pulp
3.0
3.89E-15
Tyrobp
Leukocyte
7.9
4.88E-52
Mafb
Dental Pulp
2.9
5.23E-15
Ms4a6c
Leukocyte
7.9
1.35E-34
Cebpd
Dental Pulp
2.8
1.42E-13
C1qb
Leukocyte
7.7
3.00E-27
Kctd12
Dental Pulp
2.8
1.42E-13
Clec4a2
Leukocyte
7.7
1.05E-38
Clec11a
Dental Pulp
2.7
4.02E-14
Spi1
Leukocyte
7.7
1.19E-47
S100b
Dental Pulp
2.7
1.51E-11
Csf1r
Leukocyte
7.6
3.76E-34
Ncam1
Dental Pulp
2.6
1.42E-13
Fcer1g
Leukocyte
7.3
7.26E-44
Btg2
Dental Pulp
2.6
6.67E-12
Laptm5
Leukocyte
7.2
1.04E-41
Alpl
Dental Pulp
2.6
4.07E-12
Rac2
Leukocyte
7.2
1.79E-41
1190002N15Rik
Dental Pulp
2.5
3.82E-12
Cldn5
Leukocyte
7.0
5.87E-17
Maf
Dental Pulp
2.5
5.69E-12
Gmfg
Leukocyte
6.8
4.95E-38
Fgfr2
Dental Pulp
2.5
8.47E-11
Ccl21a
Leukocyte
6.5
2.26E-03
Igfbpl1
Dental Epithelium
9.8
7.26E-35
Alas2
Erythrocyte
7.5
2.10E-99
Shh
Dental Epithelium
9.8
1.17E-49
Hba-a2
Erythrocyte
7.3
7.21E-16
Dsc3
Dental Epithelium
8.8
4.68E-51
Hbb-bt
Erythrocyte
7.2
7.21E-16
Dmkn
Dental Epithelium
8.6
3.60E-09
Gypa
Erythrocyte
7.2
2.94E-59
4631405K08Rik
Dental Epithelium
8.6
1.58E-44
Hba-a1
Erythrocyte
7.1
7.21E-16
Nkx2-3
Dental Epithelium
8.2
2.20E-42
Hbb-bs
Erythrocyte
6.8
7.21E-16
Epcam
Dental Epithelium
8.1
3.08E-59
Snca
Erythrocyte
5.7
4.60E-61
Trp63
Dental Epithelium
8.0
8.82E-51
Slc25a37
Erythrocyte
4.9
4.88E-51
tg_DsRed
Dental Epithelium
7.7
4.14E-59
Mkrn1
Erythrocyte
4.6
1.10E-50
Krt14
Dental Epithelium
7.3
4.80E-48
Fam46c
Erythrocyte
4.6
8.64E-43
Vwa2
Dental Epithelium
7.1
2.69E-46
Pnpo
Erythrocyte
4.4
4.42E-39
Cyp26a1
Dental Epithelium
7.0
6.38E-33
Fech
Erythrocyte
4.2
4.92E-44
Krt17
Dental Epithelium
7.0
2.17E-44
Bpgm
Erythrocyte
3.7
4.04E-34
Dsp
Dental Epithelium
6.8
7.88E-32
Ube2l6
Erythrocyte
3.0
1.04E-24
Sp6
Dental Epithelium
6.7
3.45E-45
March2
Erythrocyte
2.2
3.87E-15
Sfn
Dental Epithelium
6.6
5.56E-24
Fam220a
Erythrocyte
2.0
4.56E-13
Krt5
Dental Epithelium
6.2
2.97E-23
Bnip3l
Erythrocyte
1.3
1.02E-05
Spint2
Dental Epithelium
6.0
1.45E-44
Blvrb
Erythrocyte
1.2
2.53E-05
Mpped1
Dental Epithelium
5.9
2.68E-37
Prdx2
Erythrocyte
0.8
5.14E-03
Perp
Dental Epithelium
5.9
6.25E-30
Car2
Erythrocyte
0.8
2.62E-02
Table S4. Lypd1 target sequences used for RNA interference.
Fragment
Lypd1 target sequences
Lypd1 #1 siRNA
GGGAUCAUGUACCGGAAGU
Lypd1 #2 siRNA
GCUGAGAGGACACGUUGUA
Lypd1 #3 siRNA
CCUUUCAAGUAACGCAAGA
Lypd1 #4 siRNA
GCUCUGUGCUUGUCGGCUU
Table S5. Primer sequences used in this study.
Gene
Forward
Reverse
Product size
Purpose
Gapdh
5′-ggagcgagaccccactaacatc-3′
5′-ctcgtggttcacacccatcac-3′
181
qRT-PCR
Lypd1
5′-caaagtgctgggatcatgtacc-3′
5′-tggaagaacaggagagtggtg-3′
218
qRT-PCR
Panx3
5′-cttacaaccgttccatccgc-3′
5′-caggtaccgctctagcaagg-3′
140
qRT-PCR
Alpl
5′-tcaggatgagactcccagga-3′
5′-gtgtgtgtgtgtgtcctgtc-3′
101
qRT-PCR
Dspp
5′-catgaaacgacgcctcagag-3′
5′-catcctcctctaccccgttc-3′
133
qRT-PCR
Lypd1-FL
5'-caccatgtgggttctcggcatc-3'
5'- gcagtgtgccaagcagagggctaagtg -3'
Lypd1-ΔGPI
5'-caccatgtgggttctcggcatc-3'
5'- tctcttcttgggcctcggcccattgcaaa -3'
Construction of Expression Vector
Construction of Expression Vector
Table S6. Antibodies used in this study.
Antibody
Purpose
Dilution
Provider
Catalog Number
LS-C179169
Anti-LYPD1
Western blotting
1:500
LSBio
Anti-PANX3
Western blotting
1:500
Invitrogen
433270
Anti-DSPP
Western blotting
1:500
Kerafast
NH083-FP
Anti-ALPL
Western blotting &
Immunostaining
1:500
Bio-techne
AF2910
Anti-P-smad1/5/8
Western blotting
1:500
Cell Signaling Technology
9511
Anti-Smad1
Western blotting
1:500
Cell Signaling Technology
6944
Anti-P-Smad2
Western blotting
1:500
Cell Signaling Technology
3108
Anti-P-Smad3
Western blotting
1:500
Cell Signaling Technology
9520
Anti-Smad2
Western blotting
1:500
Cell Signaling Technology
5339P
Anti-Smad2/3
Western blotting
1:500
Cell Signaling Technology
8685
Anti-P-Akt
Western blotting
1:500
Cell Signaling Technology
2965
Anti-Akt
Western blotting
1:500
Cell Signaling Technology
4691
Anti-P-β-Catenin
Western blotting
1:500
Cell Signaling Technology
4176P
Anti-β-Catenin
Western blotting
1:500
Cell Signaling Technology
8480
Anti-Id1
Western blotting
1:500
Santa Cruz
sc-133104
Anti-GAPDH
Western blotting
1:500
Cell Signaling Technology
5174
Horseradish peroxidaseconjugated goat anti-rabbit IgG
Western blotting
1:1000
Cell Signaling Technology
81-1620
horseradish peroxidaseconjugated rabbit anti-goat IgG
Western blotting
1:1000
Invitrogen
7074P2
Anti -Vimentin
Immunostaining
1:500
Santa Cruz
sc-7557-R
Anti-GFP
Immunostaining
1:500
Cell Signaling Technology
2955S
Alexa fluor 488 donkey antirabbit IgG
Immunostaining
1:500
Invitrogen
A21206
Alexa fluor 488 donkey anti-goat
IgG
Immunostaining
1:500
Invitrogen
A11055
Alexa fluor 594 donkey antirabbit IgG
Immunostaining
1:500
Invitrogen
A21207
Phalloidin-iFlour 594 reagent
Immunostaining
1:1000
Abcam
ab176757
...
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