Fig. S1. TMEM88 isoform CRA_a is the isoform expressed in cardiomyocyte differentiation. (A) Schematic of TMEM88 isoforms. (B) PCR analysis of transcripts
from cDNA (top) and RNA (bottom) at day 5 of cardiac differentiation showing that TMEM88 isoform CRA_a is the dominant isoform expressed in heart development.
Fig. S2. Phylogenetic analysis of TMEM88 orthologs. (A) Phylogenetic analysis of protein sequences aligned for known
TMEM88 orthologs. Sequence homology is notated where red is 100% homology among aligned sequences. Putative protein
functional domains are notated in black bars. (B) Dendrogram showing phylogenetic relationship between species in which
TMEM88 is identified, namely vertebrate chordates.
Fig. S3. TMEM88 KD cells have hemogenic potential. (A) Schematic of differentiation protocol for assaying hemogenic
potential. (B,C) Representative flow cytometry profiles (B) and mean percentage (C) of CD43+/CD235a+ populations observed
in TMEM88 shRNA and control shRNA cells. n=3-7 biological replicates per group for all assays. Error bars represent s.e.m.
*P<0.05.
Table S1. Quantitative PCR primers for human genes
Gene name
Forward primer
Reverse primer
HPRT
TGACACTGGCAAAACAATGCA
GGTCCTTTTCACCAGCAAGCT
TMEM88
GCTGCCTTCAATCTTCTCCTG
ATAAAGGGCTCGGCTGTAGG
TNNT2
TTCACCAAAGATCTGCTCCTCGCT
TTATTACTGGTGTGGAGTGGGTGTGG
T
CAAATCCTCATCCTCAGTTTG
GTCAGAATAGGTTGGAGAATTG
Isl1
ATTTCCCTATGTGTTGGTTGC
CGTTCTTGCTGAAGCCGATG
MYH6
CAAGTTGGAAGACGAGTGCT
ATGGGCCTCTTGTAGAGCTT
MYL4
TCAAAGAGGCCTTTTCATTG
CGTCTCAAAGTCCAGCATCT
MYL7
TCCAACGTCTTTTCCATGTT
TCTGTCCCATTGAGCTTCTC
SCL
AAGGGCACAGCATCTGTAGTCA
AAGTCTTCAGCAGAGGGTCACGTA
HHEX
AATGCTGGATGATGACCACT
TAATTGAGCAGTGCACCAAA
OTX1
TATTGAAGAGAACCGCTTGG
TGTTGTTTTGTTCCCTGGTT
DKK1
AACAGCTATCCAAATGCAG
TCACAGGGGAGTTCCATAAA
WNT5a
TAGCAGCATCAGTCCACAAA
CAAAACACGGCATCTCTCTT
PDGFRα
CCGGCGTTCCTGGTCTTAG
GCTCACTTCACTCTCCCCAAAG
HAND2
AGATCAAGAAGACCGACGTG
TCCTTCTCCTCCTCCTCACT
HAND1
TCAAAGACGCACTCTTCCAC
GTGCAGCGACAAAAAGAAAA
MEF2c
GGACAAGGAATGGGAGGA
TGAGTAGAAGGCAGGGAGAG
NKX2.5
CCAAGGACCCTAGAGCCGAA
ATAGGCGGGGTAGGCGTTAT
TBX5
GAACCACAAGATCACGCAATTA
ACACCATTCTCACACTGGTAT
ATP2A2
ATGACAACCCACTGAGAAGAGAA
CGAAGGTCAGATTGGTCTCATATTT
RYR2
AGAACTTACACACACGCGACCTG
CATCTCTAACCGGACCATACTGC
NKX2-5
CAAGTGCTCTCCTGCTTTCC
GACAGGTACCGCTGTTGCTT
GATA4
ACACCCCAATCTCGATATGTTTG
GTTGCACAGATAGTGACCCGT
CD34
AAATCCTCTTCCTCTGAGGCTGGA
AAGAGGCAGCTGGTGATAAGGGTT
CD31
ATCATTTCTAGCGCATGGCCTGGT
ATTTGTGGAGGGCGAGGTCATAGA
SOX7
TGACAACTTGTTGCCAACTCCCTG
TTCAGCAGTGGAGGAAGAGCAGAA
VE Cadherin
TGGAGAAGTGGCATCAGTCAACAG
TCTACAATCCCTTGCAGTGTGAG
Table S2. Accession numbers used for phylogenetic analysis of TMEM88 protein
sequences
Organism
Accession number
Anolis carolinensis (predicted)
XP_003229095.1
Bos taurus
NP_001091848.1
Callithrix jacchus (predicted)
XP_002747990.1
Canis familiaris (predicted)
XP_849909.1
Cavia porcellus (predicted)
XP_003466294.1
Danio rerio
NP_991233.1
Equus caballus (predicted)
XP_001504841.1
Heterocephalus glaber
EHB04502.1
Homo sapiens
NP_981956.1
Loxodonta africana
XP_003416951.1
Macaca mulatta
AFI34399.1
Monodelphis domestica (predicted)
XP_001369281.2
Mus musculus
NP_080191.2
Mustela putorius furo
AES08479.1
Nomascus leucogenys (predicted)
XP_003274682.1
Oreochromis niloticus (predicted)
XP_003457690.1
Ornithorhynchus anatinus (predicted)
XP_001517691.1
Oryctolagus cuniculus (predicted)
XP_002718829.1
Osmerus mordax
ACO09352.1
Pan troglodytes (predicted)
XP_001174852.1
Pongo abelii (predicted)
XP_002827028.1
Rattus norvegicus
NP_001121627.1
Sus scrofa (predicted)
XP_003132006.3
Xenopus (Silurana) tropicalis (predicted)
XP_002938658.1
Gallus gallus (predicted)
XP_417575.1
Table S3. All genes differentially regulated in TMEM88 KD samples compared with
controls
Upregulated
Fold
Downregulated
Fold
genes
change
genes
change
ROR1
FOLR1
LOC440925
RDM1
ITGA5
SEL1L3
KCNMB4
GPR177
GAS6
SMAGP
LOC730323
ANGPTL4
SAT1
CXXC5
ARHGAP28
POU5F1P1
EBF2
DLL1
ROR2
HHEX
LPPR1
MN1
PAPPA
SOX17
ITGB1BP3
4.000856
4.002246
4.005118
4.00784
4.0098
4.015974
4.016968
4.01699
4.02602
4.034592
4.03515
4.056518
4.062624
4.082572
4.083304
4.095174
4.096026
4.104322
4.111032
4.11168
4.114308
4.115172
4.116734
4.121296
4.123276
H19
HAPLN1
TXNIP
HBE1
DNAH2
SLC30A3
CBLN2
LOC728473
HOXB2
MYL4
RGS4
MEIS2
ACTA2
SMYD1
RGL1
HEY1
MEIS1
ALDH1A2
MYL7
SC5DL
TNC
HAND2
PCDH7
CXCR7
C20ORF75
-23.7498
-21.7221
-21.5633
-19.6078
-15.786
-15.6059
-15.3096
-14.9607
-14.1374
-13.3881
-12.8877
-12.3562
-11.4269
-10.8851
-10.2268
-9.78813
-9.70816
-9.51812
-9.48374
-9.21226
-9.17969
-8.99436
-8.97882
-8.92092
-8.81652
LOC100132934
ANKRD57
PYCR1
FOXC1
HLA-H
FAM176B
ID3
FAM65B
MOBP
RPS29
SNORD3D
PPP2R5A
C6ORF192
ALPL
FAM84B
SCD5
TGIF1
PPP2R2B
ACTN3
ARHGAP24
SNAI2
CXXC5
EFNB1
LOC645682
IFITM1
COCH
PCDH10
N4BP2L1
FGF8
RRAS
4.124376
4.126792
4.131604
4.145212
4.152056
4.16622
4.166842
4.179798
4.191176
4.196696
4.20808
4.221888
4.230414
4.240526
4.257236
4.2647
4.271488
4.290952
4.299924
4.318084
4.324106
4.324296
4.327936
4.328164
4.339566
4.343118
4.344406
4.354748
4.359748
4.368118
LYPD6B
PANX2
RFTN1
CYB5D1
RHOU
LOC100128893
FRMD6
NPY
VIM
NRP1
DGKI
VIM
SYT4
VCAN
CALB2
TMEM88
PRICKLE1
NKX3-1
PBX3
SVEP1
TNC
HS.445581
LIX1
LYPD6B
LPPR5
LEF1
LEF1
SNCAIP
GUCY1A3
PAQR8
-8.59683
-8.42286
-8.39856
-8.27654
-8.25396
-8.15514
-7.67159
-7.65954
-7.55462
-7.55382
-7.42265
-7.39896
-7.31614
-7.30417
-7.28894
-7.26211
-7.21179
-7.02026
-6.93417
-6.89235
-6.75651
-6.59981
-6.54368
-6.49369
-6.49168
-6.38005
-6.33708
-6.05589
-5.98712
-5.98249
KIT
TFPI
SPRY4
PHF17
ANPEP
IGSF21
CCND1
COL4A1
LFNG
DCLK1
LRIG3
HLA-B
TFPI
TUBB2A
DMBX1
LOC145853
EGFLAM
COL23A1
KIF1A
EPCAM
TFPI
CLDN11
SLC29A1
ARHGAP28
SNORD3C
ETV5
CORO2A
SMAGP
CXCR4
NKX2-2
4.394254
4.398614
4.406344
4.419472
4.423604
4.429584
4.433466
4.439438
4.469064
4.47124
4.472938
4.47747
4.501152
4.509986
4.51896
4.612462
4.651042
4.684272
4.68949
4.719088
4.725026
4.725814
4.736928
4.758378
4.797232
4.833372
4.865368
4.9028
4.907758
4.908846
ACTC1
CCDC3
FLRT3
DIO2
COL22A1
ALG13
PDGFRA
FRMD6
TUBB6
EPHA4
SCMH1
BMPER
RGS2
SLC40A1
LOC442597
CNKSR3
ALDH1A2
CXCR7
SLC7A2
RDH10
DKK1
BAALC
CGNL1
SMOC1
ENO3
RGS5
LYPD6
TRIM55
ZNF503
NAALAD2
-5.92491
-5.6862
-5.67848
-5.57666
-5.56889
-5.56804
-5.47511
-5.45636
-5.39355
-5.34948
-5.32939
-5.24516
-5.17112
-5.15173
-5.1485
-5.12889
-5.121
-5.11811
-5.11356
-5.096
-5.08173
-5.05517
-5.04589
-5.04155
-5.03374
-5.02424
-5.01771
-4.95016
-4.91872
-4.89936
LOC100133999
TGIF1
DEFB1
LGALS1
FOXA2
NCCRP1
C18ORF54
SNORA12
SLC29A1
CBLN1
SAMD11
EBF3
LOC642559
SERPINE2
N4BP2L1
DLL3
LOC341230
ADM
SEMA5B
FZD2
OTX2
EDNRB
SAMD3
HS.491292
MYOZ3
SNORD3A
EDNRB
RARRES2
MOXD1
EGFLAM
4.955584
4.991502
5.007494
5.014458
5.022946
5.036634
5.041204
5.061892
5.079006
5.116424
5.195992
5.209442
5.21454
5.217924
5.230968
5.256732
5.264542
5.311564
5.340104
5.376066
5.377916
5.384218
5.395258
5.443796
5.500402
5.521002
5.537378
5.544022
5.722086
5.737284
LRRN4
LYSMD2
HSPH1
HS.570988
CREG1
SH3RF2
IMPAD1
NDFIP2
PDGFRA
TACC1
PHLDA1
TGFBI
SLC17A8
COLEC11
RBM20
KIFAP3
HAPLN1
WNT5A
PKP2
GNG11
NFKBIZ
CA2
TMEM2
RGS5
LOC644422
PTN
AFAP1L2
ARHGDIB
NOS2A
TRIM55
-4.8819
-4.86676
-4.86595
-4.8624
-4.85473
-4.84451
-4.83704
-4.81092
-4.80673
-4.80175
-4.79357
-4.74807
-4.73815
-4.73175
-4.70557
-4.68284
-4.65474
-4.61257
-4.59864
-4.57792
-4.56337
-4.56334
-4.53244
-4.52746
-4.52167
-4.50146
-4.50121
-4.50085
-4.49837
-4.49769
DSCR6
LOC100131138
SAMD3
PCDH10
DLK1
FLJ25404
CRABP1
ENO2
CXCR4
COL13A1
LOX
VWF
TTYH1
OTX1
LOC652097
FZD7
ZIC3
COL13A1
LOC392871
SHISA3
BCAR3
SHISA2
OLFML2A
DLK1
FOXI2
ASS1
ASS1
NRG1
SAMD3
PKDCC
5.76282
5.776898
5.793386
5.92822
5.956014
6.077796
6.165808
6.238136
6.262328
6.285558
6.403142
6.420946
6.603152
6.70591
6.762338
6.813618
6.835856
6.846948
6.878524
7.06514
7.083552
7.094018
7.125652
7.313424
7.608852
7.773288
7.87986
7.990308
8.180212
8.239256
HAS2
MGC16121
BMP5
CDK6
MEF2C
MYH6
P2RY11
LRRC32
FZD4
LCP1
BCLAF1
MT2A
SIPA1L2
DOK4
LAMA1
C10ORF58
FNDC5
SRI
SRI
SOX3
BAMBI
SYNPO2L
MDK
LUM
TNFRSF19
MLLT11
EMILIN2
PGA5
TNNT2
GOLPH4
-4.48869
-4.48392
-4.48315
-4.48066
-4.47273
-4.46645
-4.45628
-4.44189
-4.4303
-4.41486
-4.40251
-4.39994
-4.39438
-4.39015
-4.38902
-4.38793
-4.38508
-4.37386
-4.36926
-4.36481
-4.35529
-4.34715
-4.34451
-4.3321
-4.33127
-4.32501
-4.31271
-4.30584
-4.30137
-4.29369
RIPPLY1
LOC340274
HS.551128
SAMD3
CYP26A1
LOC91461
SHISA3
SP5
LEFTY2
CYP26A1
ZIC2
EOMES
MESP1
CST1
CYP26A1
8.384826
8.40227
8.528542
8.78224
8.8947
9.26789
9.403176
9.445178
10.26754
11.01831
13.76717
14.14138
15.05687
20.3828
31.20496
MAGEB3
MT1E
NHS
PPP1R13B
RASGRP3
LOC645367
SFRS6
MYLK3
ST7
SLC35F1
UNC13B
IGFBP3
LIME1
RARB
GPR128
ATF7IP2
HMGCS1
MAT2A
LPPR4
VPS41
RHOBTB3
LIMA1
ANXA1
NR0B1
LOC646750
-4.2883
-4.2878
-4.28724
-4.27543
-4.25711
-4.24337
-4.23391
-4.22642
-4.22199
-4.164
-4.16381
-4.1521
-4.14381
-4.11432
-4.09057
-4.07473
-4.07167
-4.06338
-4.06313
-4.0434
-4.02966
-4.01749
-4.01527
-4.01411
-4.00486
Table S4. Cardiac genes downregulated in TMEM88 KD cells
Gene
Gene name
ACTA2
actin, alpha 2,
smooth
muscle, aorta
Alpha cardiac
actin
aldehyde
dehydrogenase
1 family,
member A2
Bone
Morphogenetic
Protein 5
ACTC1
ALDH1A2
BMP5
Fold
change
Gene
Gene name
MYL7
myosin, light
polypeptide 7
MYLK3
myosin light
chain kinase 3
NK3 Homeobox
1
-11.3924
-9.51366
-5.9381
NKX3-1
-5.1337
HEY1
MEF2C
MEIS1
heart and
neural crest
derivatives
expressed
transcript 2
hairy/enhancerof-split related
with YRPW
motif 1
myocyte
enhancer factor
2C
Meis
homeobox 1
PDGFRA
PKP2
platelet-derived
growth factor
receptor, alpha
polypeptide
Plakophilin 2
-8.9383
Retinoic acid
receptor, beta
RBM20
RNA binding
motif protein 20
-4.11246
-4.46914
-4.69134
SMYD1
-9.71356
-5.46416
-4.5948
RARB
-9.78112
MEIS2
-4.22808
-7.01284
-4.46914
HAND2
Fold
change
SET and MYND
domain
containing 1
Sorcin
-10.8528
Meis
SRI
homeobox 2
-12.3805
-4.34694
MYH6
Myosin, heavy
TNNT2
Cardiac Troponin
polypeptide 6,
T
cardiac muscle,
alpha
-4.46914
-4.2871
MYL4
myosin, light
TRIM55
Tripartite motifpolypeptide 4
-13.3614
containing 55
-4.92458
Cardiac genes were selected on the basis that they have known roles in heart development
or the gene mutant has a cardiac phenotype.
Table S5. Gene ontology analysis of GO terms downregulated in TMEM88 KD cells
Name
P Value
FDR
Benjamini
Genes
Tissue
0.0001
0.13
0.0086
WNT5A, HAPLN1, LAMA1, TNC,
morphogenesis
SMOC1, LUM, TGFBI, COL22A1, VCAN,
EMILIN2
Extracellular
0.0008
1.35
0.3395
matrix
Heart
HOXB2, LEF1, CA2, MYH6
0.0032
5.12
0.1894
morphogenesis
Cardiac
TNNT2, LAMA1, ALDH1A2, ACTC1,
TNNT2, ALDH1A2, ACTC1, HAND2,
MYH6
0.0146
17.49 0.2897
TNNT2, ACTC1, MYH6
0.00001
0.020 0.0047
SRI, TNNT2, ALDH1A2, ACTC1, NRP1,
muscle
Heart
development
PKP2, HAND2, NKX3-1, SMYD1, RARB,
MYH6
Muscle protein 0.000006 0.008 0.0008
TRIM55, TNNT2, MYL7, ACTC1, MYL4,
ACTA2, MYH6
Muscle
contraction
0.000001 0.002 0.0004
TNNT2, MYL7, ACTC1, ACTA2, MYH6
Table S6. Gene ontology analysis of GO terms upregulated in TMEM88 KD cells
Name
P Value
FDR
Benjamini
Genes
Notch signaling
0.00036
0.575 0.0501
DLL3, DLL1, FOXC1, NRG1
0.00124
1.621 0.2843
EGFLAM, DLL3, DLL1, DLK1, NRG1,
pathway
EGF
GAS6
Mesenchymal
0.00001
0.031 0.0048
cell development
Mesoderm
EDNRB, EFNB1, EOMES, CYP26A1,
FOXC1, NRG1
0.00126
1.968 0.0982
DLL3, EOMES, FOXC1, SNAI2
0.00825
10.05 0.1707
COL4A1, ANPEP, ARHGAP24,
development
Angiogenesis
ANGPTL4
Blood vessel
0.00529
8.027 0.1701
CXCR4, ANPEP, FOXC1, SOX17,
development
LOX, ARHGAP24, ANGPTL4
Anterior/posterior 0.000003 0.005 0.0015
HHEX, FOXA2, OTX1, DLL3, ROR2,
pattern formation
CYP26A1, DLL1, LFNG, ZIC3
Table S7. Genes involved in hematopoiesis that are twofold upregulated in
TMEM88 KD cells
Gene
Gene name
HHEX
hematopoietically
expressed
homeobox
OTX1
Orthodenticle
homeobox 1
Sprouty homolog
4
c-Kit (CD117)
SPRY4
KIT
Fold
change
Regulates the earliest stages of definitive
hematopoiesis. Hhex is expressed in the
developing blood islands at E7.0 in the
endothelium of the developing vasculature and
heart at E9.0-9.5, and in the ventral foregut
endoderm at E8.5-9.0; it has been postulated to
play a critical role in heart and vascular
4.08404 development
Coordinately expressed with SCL to specify
6.72716 erythroid differentiation
Required for normal HSC development
4.37716
4.37716
CXCR4
Chemokine
receptor 4
6.2333
RPS29
LGALS1
Ribosomal Protein
S29
lectin,
galactosidebinding, soluble, 1
4.16986
4.99332
SOX17
SRY-box
containing gene
17
Developmental functions
4.12128
Surface marker of hematopoietic stem cells and
is functionally required for maintenance of
hematopoiesis and is involved in endothelial cell
biology
Critical in maintaining HSPCs in a quiescent
state and retaining all subsets of hematopoietic
stem and progenitor cells and is highly expressed
in endothelial cells
Acts through p53 to regulate hematopoietic stem
cell development
Mediates interactions between hematopoietic
cells and the stromal microenvironment and
increased expression is seen in activated
endothelial cells
Sox17 is expressed in hemogenic endothelium
and emerging HSCs and is required for HSC
development