Fig. S1. Expression of haematopoietic and endothelial genes in DLP adult haemangioblasts. (A) In situ hybridisation showing
ETS TFs not expressed in the DLP. Black arrows indicate the position of the DLP. All embryos were hybridised as whole mounts and
are shown in lateral view, with anterior to the left and dorsal to the top. All embryos are shown at stage 26. (B) In situ hybridisation
analysis showing the expression of blood and endothelial genes not expressed in adult haemangioblasts (Fig. 1) but expressed in
developing blood vessels or developing blood cells. All embryos were hybridised as whole mounts and are shown in lateral view, with
anterior to the left and dorsal to the top. All embryos are shown at stage 34. (C) Expression analysis in sectioned control and Etv6
morphants showing co-expression of Lmo2 and VegfA in the DLP. In control embryos, dark blue staining (VegfA) masks the turquoise
staining (Lmo2) in the DLP (red arrow) where both transcripts are expressed. By contrast, in Etv6 morphants, VegfA is no longer
expressed in the DLP and therefore Lmo2 staining can be visualised (red arrow). Sections are in transverse orientation with dorsal to
the top. Sections are taken from stage 26 embryos. DA, dorsal aorta; Hyp, hypochord; Lpm, lateral plate mesoderm; n, notochord; s,
somites; Thromb, thrombocytes; VitV, vitelline vessels.
Fig. S2. SCL is required for embryonic erythrocyte differentiation. (A) Diagram showing the sequence targeted by the antisense
MO (red nucleotides) blocking the translation of the two X. laevis SCL alleles. Initiation ATG is in purple. Sequences targeted by Scl
MO2 and Scl MO3 are also indicated. (B) Western blot showing depletion of SCL protein in embryos injected with SCL MO at the
two-cell stage. 2xHA-SCL, present only in stage 10 sample, indicates HA-tagged protein generated from exogenous mRNA. Note that
no endogenous SCL protein is detected at stage 10. ACTIN was used as a loading control. (C) In situ hybridisation analysis showing
the absence of embryonic erythrocytes, as indicated by expression of embryonic Globin and Gfi1b (arrows), in SCL morphants.
Embryos were hybridised as whole mounts and are shown in lateral view, with anterior to the left and dorsal to the top. All embryos
are shown at stage 34. Numbers of embryos represented by each panel, out of the number analysed, are indicated in the top right
corner.
Fig. S3. Etv2 is not required for Flk1 expression in adult haemangioblasts. (A) Expression analysis showing that Flk1 expression
in Etv2-deficient embryos is normal in adult haemangioblasts (stage 24 and 26, arrows) but absent in post-adult haemangioblast
stages, stage 35. (B) Expression analysis showing the absence of differentiated endothelial markers, Erg1 and Tie2, in post-adult
haemangioblast Etv2 morphants, indicating a complete lack of blood vessels in these embryos. Embryos were hybridised as whole
mounts and are shown in lateral view, with anterior to the left and dorsal to the top. Numbers of embryos represented by each panel,
out of the number analysed, are indicated in the top right corner.
Fig. S4. Fli1 and Gata2 are required for Etv2 expression in the DLP. (A) Expression analysis showing that Etv2 expression in
the DLP is not initiated in Fli1 morphants. (B) Expression analysis showing that Etv2 expression is not initiated in the DLP of Gata2
morphants. Embryos were hybridised as whole mounts and are shown in lateral view, with anterior to the left and dorsal to the top.
The DLP is indicated by arrows. The arrowhead in Fli1 MO indicates Etv2 ectopic expression in the somites. Numbers of embryos
represented by each panel, out of the number analysed, are indicated in the top right corner.
Fig. S5. Adult haemangioblast programming in Fli1 morphants is not rescued by exogenous Gata2 mRNA. Fli1 MO was coinjected with a hormone-inducible form of Gata2, Gata2FL-GR-AH, at the two-cell stage and grown to stage 20 when Gata2FL-GRHA activity was induced with 10 mM dexamethasone (Dex). Embryos were then further cultured and collected at the stages indicated.
Expression of Flk1 at stage 22, Etv2 at stage 24 and Scl at stage 26 was not rescued in the DLP (arrows). Nevertheless, Scl expression
in developing erythrocytes in the ventral blood island was significantly rescued (red arrowheads). Black arrowheads indicate Etv2
ectopic expression in the somites. Embryos were hybridised as whole mounts and are shown in lateral view, with anterior to the left
and dorsal to the top. Numbers of embryos represented by each panel, out of the number analysed, are indicated in the top right corner.
Fig. S6. qPCR analyses on excised DLPs from Fli1 and Gata2 morphants. (A-C) Embryos injected at the two-cell stage with Fli1
MO (A), Gata2 MO (B) or Etv2 MO (C) were grown to stage 26; DLPs were then dissected, total RNA purified and SYBR green
qPCR analysis carried out. Note that Etv2 expression in Fli1 morphants appears greatly reduced by in situ hybridisation but a less
significant reduction is obtained by qPCR; this indicates that DLP samples used were contaminated with somitic tissue where Etv2 is
ectopically expressed (see Fig. 4C, supplementary material Fig. S4A and Fig. S5). Error bars indicate s.e.m.
Table S1. Probes used for in situ hybridisation
Gene
EST ID
Accession
number
Runx1
SalI
RNA
polymerase
T7
Gata2
XbaI
SP6
Walmsley et al., 1994
VegfA
BamHI
T7
Cleaver et al., 1997
XhoI
SP6
Ciau-Uitz et al., 2010b
NotI or SacI or SmaI
T7
Ciau-Uitz et al., 2010b
αT4-globin
EcoRI
SP6
Walmsley et al., 1994
Fli1
SmaI
T3
Meyer et al., 1993
SmaI
T7
Ciau-Uitz et al., 2010b
BamHI
T7
Jones et al., 1999
NotI
T7
This report
NcoI or SphI or AatII
SP6
This report
Scl
Flk1
Erg1
NIBB XL087o23
IMAGE 5512670
BJ092634
AAH73350
Hex
Fech
NIBB XL208g04
BJ640944
Fog
Restriction enzyme
Reference
Tracey et al., 1998
Eto2
NIBB XL185m19
BJ635188
NotI or BamHI
T7
This report
SpiB
IMAGE 5537169
AAH46671
SalI or EcoRI
T7
Ciau-Uitz et al., 2010b
Lmo2
IMAGE 4174203
AAH97502
SalI or SmaI or EcoRI
T7
Ciau-Uitz et al., 2010b
Gfi1a
IMAGE 8547327
EB645267
EcoRI or ClaI
T7
Ciau-Uitz et al., 2010b
Gfi1b
IMAGE 5506836
BC077878
SalI or SmaI or EcoRI
T7
This report
Etv2
NIBB XL052p03
BJ086393
NotI or SmaI
T7
This report
Lyl1
IMAGE 8077488
DT063654
SmaI
T7
This report
Elk3
IMAGE 4963943
BC056051
SalI or SmaI
T7
This report
Sox7
IMAGE 4740338
BG812919
SmaI
T7
This report
Ami
IMAGE 7204180
CK797755
EcoRV or EcoRI or SmaI
T7
This report
Tie2
NIBB XL064i22
BJ092234
NotI or SacI or SmaI
T7
Ciau-Uitz et al., 2010b
CD31
IMAGE 4173797
BG017659
SalI or SmaI
T7
This report
Vecad
IMAGE 7638101
CX408264
SalI or ClaI
T7
This report
AA4
IMAGE 5515354
CF290542
SalI or SmaI
T7
This report
VWF
IMAGE 4674248
BX846912
SalI or SmaI
T7
This report
Flt1
IMAGE 4959298
AAH56023
SalI or SmaI
T7
Ciau-Uitz et al., 2010b
Flt4
IMAGE 4970772
CF289873
SalI
T7
Ciau-Uitz et al., 2010b
Etv6
NIBB XL153n17
EU760352
NotI or SacI
T7
Ciau-Uitz et al., 2010b
Fli1-like
IMAGE 8548018
BC129781
SmaI or EcoRI or ClaI
CBFβ
IMAGE 3404921
BG513054
SmaI or EcoRI
T7
This report
Egfl7
IMAGE 5542405
BC044267
SalI or SmaI
T7
This report
Bra
SspI
T7
Gata1
XhoI
SP6
Bertwistle et al., 1996
This report
biKlf
NIBB XL198d17
BJ638367
NotI
T7
This report
Klf2
IMAGE 6640768
BU914281
RsrII or KpnI
T7
This report
BbvCI or BbsI
T7
This report
cMyb
Ika
IMAGE 7017596
BC089243
EcoRV or EcoRI
T7
This report
CD41
IMAGE 5511404
BC077891
SpeI or NdeI
T7
This report
CD45
IMAGE 4683776
BQ735201
SalI
T7
This report
Ets1
IMAGE 7011315
BC075161
SalI or SmaI
T7
This report
Ets2
IMAGE 6631306
BC077264
SalI or SmaI
T7
This report
Elf1
IMAGE 5505961
BC084214
SalI or SmaI or EcoRI
T7
This report
Elf2
IMAGE 5073096
BC084060
SmaI
T7
This report
Elf3
IMAGE 5543230
BQ736872
SalI or EcoRI
T7
This report
Etv7
IMAGE 8462984
EB476932
EcoRV
T7
This report
Elk4
IMAGE 7011856
BC074199
SalI or SmaI
T7
This report
Table S2. Primer sequences for cloning and qRT-PCR
Primers for SYBR Green
ODC
Forward
Reverse
GTCAATGATGGAGTGTATGGATC
TCCATTCCGCTCTCCTGAGCAC
www.xenbase.org
Scl
Forward
Reverse
CAAAAGTGGTCCGACGCATC
TGGTTGCCTTCTTCTTCCTGG
Ciau-Uitz et al., 2010b
Lmo2
Forward
Reverse
GCACTGTAACTGACAACACAGCTA
GGGGTTCTATGGGATCTTACTCTT
This report
Etv2
Forward
Reverse
GACTCTGGCTTTCTCCATGACTAT
CTGTTAGTGCTGGTAACAACGTCT
This report
Flk1
Forward
Reverse
ACATTCCTGTAGAGCCTGTGGT
GGACTGGTAGTCGCTAGTTTGG
Meadows et al., 2009
GGATCAGATCTCTCCTGCGATGGAAGTGGCC
GATCCAGATCTTCCCATAGCCGTCACCATGC
This report
Primers for PCR cloning
Gata FL
Forward
Reverse