Stem Cell Reports
Repor tFive Transcription Factors and FGF Pathway Inhibition Efficiently Induce
Erythroid Differentiation in the Epiblast
Wei Weng1 and Guojun Sheng1,*
1Laboratory for Early Embryogenesis, RIKEN Center for Developmental Biology, Kobe, Hyogo 650-0047, Japan*Correspondence: sheng@cdb.riken.jp
http://dx.doi.org/10.1016/j.stemcr.2014.01.019
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

SUMMARY
Primitive erythropoiesis follows a stereotypic developmental program of mesoderm ventralization and internalization, hemangioblast
formation and migration, and erythroid lineage specification. Induction of erythropoiesis is inefficient in either ES/iPS cells in vitro or
nonhemangioblast cell populations in vivo. Using the chick model, we report that epiblast cells can be directly and efficiently differentiated into the erythroid lineage by expressing five hematopoietic transcription regulators (SCL+LMO2+GATA2+LDB1+E2A) and inhibiting the FGF pathway. We show that these five genes are expressed with temporal specificity during normal erythropoiesis. Initiation of
SCL and LMO2 expression requires FGF activity, whereas erythroid differentiation is enhanced by FGF inhibition. The lag between
hematopoiesis and erythropoiesis is attributed to sequential coregulator expression and hemangioblast migration. Globin gene transcription can be ectopically and prematurely induced by manipulating the availability of these factors and the FGF pathway activity.
We propose that similar approaches can be taken for efficient erythroid differentiation in vitro.

INTRODUCTIONRESULTS AND DISCUSSION

During primitive erythropoiesis in amniotes, hemangioblast cells are generated from the pluripotent epiblast
through gastrulation at the posterior primitive streak(ventral mesoderm precursors) and give rise to predominantly primitive erythrocytes. In both chick and mousemodels, a delay of approximately half a day has been reported between the onset of early hematopoietic markers
in newly ingressed extraembryonic mesoderm cells andthat of globin genes (McGrath and Palis, 2008; Minkoet al., 2003; Nakazawa et al., 2006; Silver and Palis, 1997).
A comparable delay is seen in Xenopus (Ciau-Uitz et al.,2010; Walmsley et al., 2008), zebrafish (Brownlie et al.,
2003; Ciau-Uitz et al., 2010; Patterson et al., 2007),and erythroid differentiation from embryonic stem cells/
induced pluripotent stem cells in vitro (Chang et al.,2006; Lu et al., 2008). The molecular nature of this delay
is unclear but has been hypothesized to reflect a requirement for combinatorial and sequentially expressed transcription factors, for extraembryonic endoderm contact,
or for the embryo to reach a conducive developmental
stage (Galloway et al., 2008; Gering et al., 2003; McGrathand Palis, 2008). Data from our lab and other labs suggested
that this delay may also reflect a requirement for hemangioblasts to downregulate fibroblast growth factor (FGF)
activities before the onset of erythropoiesis (Kumano andSmith, 2000; Nakazawa et al., 2006; Walmsley et al.,2008). In this work, we used the chick model and showedthat it was possible to bypass this requirement and achieveectopic and accelerated erythroid induction in postingression mesoderm and pluripotent epiblast cell populations.

Primitive Erythropoiesis Follows a HemangioblastIntrinsic Program
During chicken primitive erythropoiesis, SCL and LMO2
are first detected in nascent extraembryonic mesoderm at
Hamburger and Hamilton stage 4 (HH4), the full streak
stage (Figure 1A) (Minko et al., 2003; Nakazawa et al.,2006). By HH5, migrating hemangioblasts (Weng et al.,2007) express these genes robustly as revealed by RNAin situ hybridization analysis (Figures 1B and 1C).
Erythroid lineage differentiation, marked by embryonicrho globin (HGB1 gene) expression, is initiated only after
HH7 (zero to one somite) (Figures 1D and 1E). As development proceeds, primitive erythropoiesis intensifies (Figure 1F) and generates differentiated primitive red blood
cells before initiation of cardiac activity at HH11 and of circulation at HH12. To understand molecular mechanisms
controlling the progression from hematopoiesis (HH4) toerythropoiesis (HH7), we first investigated whether avian
primitive erythropoiesis follows a hemangioblast intrinsic
or extrinsic developmental program by graft analysis.
Hemangioblasts from HH6 embryos (before erythropoiesis)
were grafted isotopically to HH6 host, and host embryos
were incubated postgraft to HH8 (Figure 1G). All graft
hemangioblasts (circles) (15/15 grafts) initiated rho expression as strongly as in the host (arrowheads). HH6 hemangioblasts grafted to HH4 host, grown to HH6+ postgraft(Figure 1H), were also rho positive (circle and inset) (11/11 grafts) despite the host still being rho negative, and thedifferentiation potential of HH6 hemangioblast graft in
HH4 host was not dependent on graft location (Figures

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Figure 1. Developmental Delay from
Hematopoiesis to Erythropoiesis and Hemangioblast-Intrinsic Program of Primitive Erythropoiesis during Early Chicken
Development(A) Schematic diagram of developmental
landmarks. Early hematopoietic markers
begin their expression at HH4. Erythroid
lineage differentiation, marked by globin
gene expression, starts at HH7. Cardiac
activity is initiated at HH11 and circulation
is established at HH12–13.
(B) SCL is expressed robustly at HH4+ inmigrating hemangioblast cells.
(C) LMO2 expression is strong at HH5 in
migrating hemangioblasts.
(D) rho globin is not expressed at HH7.
(E) rho globin is weakly expressed at HH7+.
(F) rho globin is strongly expressed at
HH9. Expression analyses of SCL, LMO2,
and rho (B–F) were performed using thewhole-mount RNA in situ hybridization
method.
(G) HH6 donor hemangioblasts were graftedisotopically to HH6 host embryo. The hostwas further cultured to HH8 and stained forrho mRNA expression by in situ hybridization. rho expression was detected robustly
in both graft (circles) and host (whitearrowheads) hemangioblasts.
(H) HH6 donor hemangioblasts were graftedisotopically to HH4 host embryo. The hostwas further cultured to HH6+ and stained
for rho. rho expression was detected onlyin graft (circle and inset) tissues. The hosthemangioblasts were still negative for rho
at this stage.
(I) HH4 donor hemangioblast precursors inposterior primitive streak were grafted to
HH6 host embryo. The host was further
cultured to HH8 and stained for rho. rho was detected only in host hemangioblasts (white arrowheads). The graft tissue (circle and inset)
remained negative because graft cells followed their own timing of erythropoiesis. Scale bars, 500 mm.
S2A–S2C available online). In contrast, when HH4 posterior streak cells (hemangioblast precursors) were graftedto HH6 host, cultured postgraft to HH8+ (Figure 1I), erythropoiesis in host embryos was marked by strong rho expression (white arrowheads), whereas graft cells remained rho
negative (circle and inset) (zero out of nine grafts). Avian
primitive erythropoiesis therefore follows a hemangioblast-intrinsic developmental program.
SCL and LMO2 Initiate Erythropoiesis in a TimeDependent, but Not Stage-Dependent, Manner
To test whether this program could be initiated or accelerated by exogenously introduced SCL and LMO2, two key

early hematopoietic regulators, we cloned these two genes
into pCAGGS expression vector (Figure 2A, top). When
the SCL+LMO2-expressing construct was electroporated
into mesoderm precursors at HH4 and host embryos were
cultured postelectroporation to HH6+ (Figure 2B), neither
host hemangioblasts nor electroporated mesoderm cells(Figure 2B, brown) expressed rho (zero out of six embryos).
When similarly electroporated embryos were incubated to
HH8–10 (Figure 2C), exogenous SCL+LMO2 induced
ectopic rho in lateral somite and medial lateral plate territories normally negative for globins (boxed area, section
in inset; magnified view in Figures S1A and S1B) (80/91
embryos). The induction was not optimal because only a

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Figure 2. Induced Erythroid Lineage Differentiation in the Mesoderm by SCL+LMO2 and FGF Pathway Inhibition(A) Schematic diagrams of expression constructs and of transcriptional regulatory complex for erythroid differentiation containing SCL,
LMO2, LDB1, E2A, and GATA. Details are found in Experimental Procedures.
(B) SCL+LMO2 expression construct was electroporated into the posterior streak at HH4. Electroporated embryos were grown to HH6+ andstained for rho mRNA and GFP protein. Neither the endogenous hemangioblasts nor electroporated cells were positive for rho.
(C) SCL+LMO2 expression construct was electroporated into the posterior streak at HH4. Electroporated embryos were grown to HH10 andstained for rho mRNA and GFP protein. Endogenous erythropoiesis was robust (strong rho expression in extraembryonic mesoderm).
SCL+LMO2 induced ectopic rho expression in cells located in lateral somites and medial lateral plate (white rectangle with section shown in
inset; magnified views in Figures S1A and S1B).
(D) SCL+LMO2 expression construct was electroporated into the posterior streak at HH2/3, about 6 hr prior to endogenous SCL and LMO2
expression. Electroporated embryos were grown to HH5 and stained for rho mRNA and GFP protein (bottom left: rho only; bottom right:
rho+GFP). Electroporated cells initiated rho expression at HH5 because they had been under SCL+LMO2 control for approximately 12 hr.
(E) SCL+LMO2 expression construct was electroporated into the posterior primitive streak at HH2/3 and electroporated embryos weregrown to the equivalent of HH5 in the presence of SU5402 and stained for rho mRNA and GFP protein expression (bottom left: rho; bottom
right: rho+GFP). Combination of SCL+LMO2 and SU5402 induced strong rho expression in the mesoderm. The induction was widespread andnot limited to the endogenous hemangioblast territory. Scale bars, 500 mm.

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fraction of SCL+LMO2-expressing cells in each embryo
were rho positive. When electroporation was performed at
HH2/3 (about 6 hr before the endogenous SCL and LMO2
expression) and embryos grown postelectroporation to
HH5 (Figure 2D), SCL+LMO2-expressing cells were able toinitiate rho expression, albeit still inefficiently, in a population of otherwise rho-negative extraembryonic mesoderm
cells (bottom left: rho; bottom right: rho+GFP) (10/38
embryos). These results suggested that SCL and LMO2 areable to initiate, although not optimally, erythroid differentiation in a time-dependent, but not stage-dependent,
manner.
FGF Inhibition Enhances Erythroid-Inducing
Potentials of SCL and LMO2 in the Mesoderm
To achieve optimal induction, we analyzed the role of
FGF pathway in early erythropoiesis. We have previously
reported that FGF activity enhances endothelial and inhibits primitive blood differentiation (Nakazawa et al.,2006). Treatment of embryos from HH4+/5 to HH10 with
SU5402, a potent FGF inhibitor, led to a medial expansionof globin-positive territory (Figures S1C and S1D, arrows)(control: zero out of five embryos; SU5402: six out of six
embryos; Nakazawa et al., 2006) without hindering overall
development (Figures S1C, S1C0 , S1D, and S1D0 ). SU5402
treatment from HH2/3 caused growth arrest and failed toinduce rho (Figures S1E and S1E0 ) (0/14 embryos). In combination with exogenous SCL+LMO2, however, SU5402
treatment from HH2/3 promoted robust ectopic rho expression in the mesoderm (Figure 2E) (18/22 embryos). These
embryos were nevertheless still growth arrested, suggestingthat failure to enhance erythropoiesis by SU5402-only
treatment from HH2/3 is independent of its influence on
growth. We therefore analyzed the effect of SU5402 on
endogenous SCL and LMO2 expression. SCL or LMO2 intensity was not altered by SU5402 treatment after HH5,
whereas both genes failed to be expressed when embryos
were treated with SU5402 from HH3 (Figures 3A and 3B)(LMO2 control treatment: five out of five embryos; LMO2
SU5402 treatment: 0/12 embryos; SCL control treatment:eight out of eight embryos; SCL SU5402 treatment: zeroout of nine embryos). These data suggested that FGF
signaling is required for the initiation of SCL and LMO2
and, when supplied with exogenous SCL and LMO2, FGF
inhibition enhances their erythropoietic potentials.
When HH3 embryos were grafted with control-electroporated HH3 posterior streak mesoderm (Figure 3C, arrow)or SCL+LMO2-electroporated HH3 posterior streak mesoderm (Figure 3C, arrowhead) and the host embryos were
cultured postgraft in the presence of SU5402 to HH6 equivalent, SCL+LMO2-electroporated graft showed strong rho
positivity (Figure 3C, arrowhead), whereas control electroporated graft remained rho negative (Figure 3C, arrow).

Taken together, these data indicated that erythroid differentiation in the mesoderm can be efficiently induced by
a combined FGF inhibition and exogenous SCL+LMO2.
Combination of Five Transcription Factors,
SCL+LMO2+GATA2+LDB1+E2A, and FGF Inhibition
Directly Induces Erythroid Differentiation in the
Epiblast
SCL+LMO2 overexpression, however, was insufficient todifferentiate epiblast-located cells (fated for neural andnonneural ectoderm tissues) into the erythroid lineage.
We reasoned that additional hemangioblast regulators
may be required to initiate globin gene expression in theectoderm. We analyzed our transcriptomic data sets (Nakazawa et al., 2009) of extraembryonic tissues from stages
HH4–9 for genes encoding transcription factors withexpression profiles similar to those of SCL and LMO2. We
uncovered two such genes, LDB1 and E2A, as potential
regulators of primitive erythropoiesis. Both have beenreported to play a role in mammalian hematopoiesis (Liet al., 2013; Mylona et al., 2013; Semerad et al., 2009;
Song et al., 2007; Wadman et al., 1997). In situ hybridization analyses confirmed that chicken LDB1 and E2A are
expressed in the extraembryonic mesoderm, with LDB1
initiating its expression at HH5 and E2A at HH7 (Figures
3D and 3E, arrows). Unlike SCL or LMO2, however, both
LDB1 and E2A are also strongly expressed in the neural
ectoderm (Figures 3D and 3E).
LDB1 and E2A have been proposed to form a functional
complex with SCL, LMO2, and GATA proteins to regulate
erythroid differentiation in vitro (Figure 2A) (Song et al.,2007; Wadman et al., 1997). Hemangioblast expression of
GATA genes has been reported previously (Minko et al.,
2003; Nakazawa et al., 2006; Sheng and Stern, 1999). To
test whether this complex is sufficient to initiate primitive
erythropoiesis in nonmesoderm cells, we generated
expression constructs for LDB1 and E2A and for GATA2(Figure 2A). Electroporation of SCL+LMO2+GATA2+
LDB1+E2A at HH2/3, followed by growth to HH5/6, however, was still unable to induce rho in the epiblast (0/20
embryos). In the mesoderm, they induced rho only slightly
better than with SCL+LMO2, suggesting that SCL+LMO2+
GATA2+LDB1+E2A were not able to bypass the requirement for FGF inhibition at these stages. However, when
SCL+LMO2+GATA2+LDB1+E2A electroporated embryos
were treated with SU5402, robust induction of rho was
observed in the epiblast (37/37 embryos) (Figure 3F). Moreover, FECH (Ferrochelatase), a gene essential for heme
biosynthesis and upregulated during primitive erythropoiesis (Dailey and Meissner, 2013; Nakazawa et al., 2009), wasalso induced strongly in the epiblast after the same treatment (13/13 embryos) (Figures S2D and S2E). Induced cells
were unable to mature into functional erythrocytes owing

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Figure 3. Induced Erythroid Lineage Differentiation in the Epiblast by SCL+LMO2+GATA2+LDB1+E2A and FGF Pathway Inhibition(A) Embryos were cultured in the presence of control DMSO (left) or SU5402 (right) from HH3 to HH5 and stained for LMO2. SU5402
inhibited the initiation of endogenous LMO2 expression.
(B) Embryos were cultured in the presence of control DMSO (left) or SU5402 (right) from HH3 to HH5 and stained for SCL. SU5402 inhibitedthe initiation of endogenous SCL expression.
(C) Left: dark-field view; right: bright-field view. HH3 embryos were electroporated with either control or SCL+LMO2-expressing constructs
in the posterior streak. The electroporated posterior streak tissues (control: left circle/arrow; SCL+LMO2: right circle/arrowhead) weregrafted to the extraembryonic territory of an HH3 host embryo. The host was further grown in the presence of SU5402 to the equivalent of
HH6 and stained for rho expression. Both the host and control electroporated graft tissue were negative for rho. SCL+LMO2 electroporated
graft tissue expressed rho robustly (arrowheads, inset).
(legend continued on next page)
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to SU5402-induced growth arrest. However, these electroporated epiblast cells, when grafted to the area vasculosa
of an HH7 host embryo and grown postgraft to HH12/13(Figures S2F–S2P), were able to differentiate into cellswith erythrocyte-like morphology and strong rho expression (Figures S2S–S2U), whereas control epiblast cells remained as a compact group rho-negative cells (Figures
S2Q and S2R).
Although these five transcription factors constitute corecomponents of the transcriptional complex regulating
erythroid differentiation, it is unclear whether all five factors are required exogenously or whether some of themcan be substituted by other known hematopoietic regulators. When electroporated individually, none of these fivetranscription factors could induce rho in the epiblast(Table 1). Neither could GATA1 or EKLF, two hematopoietic
regulators not included in our initial analysis (Table 1). As
the earliest-expressing hematopoietic-specific transcription factors, SCL and LMO2 together induced rho in theepiblast only very weakly (Table 1). Progressive additionof more transcription factors, as a combination of three
to seven, all of which contained SCL and LMO2, led toa gradual increase in their inducing ability (Table 1). The
SCL+LMO2+GATA2+LDB1+E2A combination was among
the ones with the highest inducing abilities. These datasuggested that efficient erythroid induction of epiblast cells
requires the FGF pathway to be inhibited and hematopoietic regulators to function cumulatively and cooperatively.
Interestingly, the requirement of FGF pathway inhibition
appeared to be needed only because of the high endogenous FGF activities in the epiblast (Lunn et al., 2007).
When SCL+LMO2+LDB1+E2A+GATA2 electroporated
embryos were grown to later stages (HH10/11) without
SU5402 treatment, ectoderm cells in the forebrain and surface epidermis which have low endogenous FGF activities(Bailey et al., 2006; Chambers and Mason, 2000) wereable to express rho strongly (Figure 3G) (43/43 embryos).

The chicken epiblast loses its pluripotency at HH5 (Shinet al., 2011), when neural markers become strongly
expressed (Uchikawa et al., 2003). The late erythroid induction in the ectoderm therefore represents a process of transdifferentiation, the molecular mechanism of which awaits
further elucidation.
In summary, primitive erythropoiesis follows a stereotypic developmental progression in vivo (Figure 3H). We
showed in this work that the conversion of nascent mesoderm to the erythroid lineage requires FGF inhibition
together with two early hematopoietic transcription regulators, SCL and LMO2. In the epiblast, direct erythroid differentiation of pluripotent cells, without them undergoing
mesoderm induction, epithelial-mesenchymal transition,or migration, requires concerted action of five hematopoietic regulators (SCL, LMO2, GATA2, LDB1, and E2A) and
FGF inhibition. In cells with low endogenous FGF signaling
activities, these five factors alone are sufficient for erythroidtransdifferentiation. Whether induced erythroid cells canmature in vivo into functional erythrocytes and whether
the combination of five factors and FGF inhibition has
the same potent erythropoiesis-inducing ability in vitro
await future investigation.

EXPERIMENTAL PROCEDURES
Hen’s eggs were purchased from Shiroyama Farm and incubated at
38.5 C to desired stages. SU5402 (#572630, Calbiochem) was dissolved in albumen at 85 mM. Standard protocols were followed
for new culture, grafting, SU5402 treatment, RNA in situ, electroporation, tissue section, and imaging analyses (Alev et al., 2013;
Nakazawa et al., 2006). DNA fragments for in situ hybridization
probes were generated by PCR, cloned into pGEM-T vector,
and confirmed by sequencing (LDB1: nucleotides 436–890 of
NM_205070.1; E2A: nucleotides 104–932 of NM_204486.2; SCL:
nucleotides 717–1,750 of NM_205352.1; LMO2: nucleotides
46–522 of AF468789.1; rho: nucleotides 31–465 of CHEST324a16)(D) Expression pattern of chicken LDB1 from HH4–8. Initiation of LDB1 in hemangioblasts starts at HH5 (arrows). Expression in neural
ectoderm is also prominent.
(E) Expression pattern of chicken E2A from HH4–8. Initiation of E2A in hemangioblasts starts at HH7 (arrows). Expression in neural
ectoderm is also prominent.
(F) SU5402 treatment in combination with ectopic expression of SCL+LMO2+GATA2+LDB1+E2A resulted in robust rho induction in theepiblast (arrowheads in lower panel). Top: whole mount (left: rho; right: rho+GFP); bottom: section. Level of section indicated in the top
left panel.
(G) Electroporation of SCL+LMO2+GATA2+LDB1+E2A at later stages, without SU5402 treatment, led to robust rho expression in neural(white arrowheads) and nonneural (black arrowheads) ectoderm tissues where endogenous FGF activities are low. Left: whole mount; right:
sections.
(H) A model for normal developmental progression from hematopoiesis to erythropoiesis. Hemangioblast precursor cells are located in theposterior primitive streak. They receive ventralizing (BMPs) and EMT (FGFs and Nodal) signals and are internalized to become migratory
mesenchymal-shaped mesoderm cells. Early hemangioblast regulators (SCL and LMO2) start to be expressed soon after internalization. This
process requires FGF activity. Commitment of hematopoietic cells to the erythroid lineage needs additional cofactors (LDB1, E2A, GATA,
EKLF, and possibly other factors regulated by core factors) and inhibition of the FGF pathway activity.
Scale bars, 500 mm except where indicated.

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Table 1. Efficiency of Erythroid Induction in the Epiblast by Hematopoietic Transcriptional Regulators Either Individually or in
Combination
Electroporation Combinationa

No Induction ()b

SCL

10/10 (100%)

LMO2

10/10 (100%)GATA2

10/10 (100%)LDB110/10 (100%)

E2A

13/13 (100%)GATA19/9 (100%)

EKLF

8/8 (100%)

GATA1+EKLF

9/9 (100%)

SCL+LMO2

3/16 (19%)

Very Weak
Induction (+/)c

Moderate
Induction (+)dStrongInduction (++)e

13/16 (81%)

SCL+LMO2+GATA2

4/4 (100%)

SCL+LMO2+LDB1

12/14 (86%)

2/14 (14%)

SCL+LMO2+E2A

4/9 (44%)

2/9 (22%)

3/9 (33%)

SCL+LMO2+GATA1

7/10 (70%)

3/10 (30%)

SCL+LMO2+EKLF

5/10 (50%)

5/10 (50%)

8/15 (53%)

3/15 (20%)

SCL+LMO2+GATA2+E2A

4/10 (40%)

6/10 (60%)

SCL+LMO2+GATA2+LDB1

3/9 (33%)

6/9 (67%)

5/9 (56%)

3/9 (33%)

9/37 (24%)

28/37 (76%)

3/9 (33%)

5/9 (56%)

2/10 (20%)

8/10 (80%)

SCL+LMO2+E2A+LDB1

4/15 (27%)

SCL+LMO2+GATA1+EKLF

1/9 (11%)

SCL+LMO2+GATA2+E2A+LDB1
SCL+LMO2+GATA1+EKLF+E2A+LDB1

1/9 (11%)

SCL+LMO2+GATA1+GATA2+EKLF+E2A+LDB1aElectroporated embryos are treated with SU5402 in all cases. HH2/3 embryos were electroporated in the epiblast with DNA constructs expressing theseregulators. Embryos were cultured after electroporation in the presence of SU5402 to the equivalent of HH6 and stained for rho mRNA and GFP protein
expression.
b() is defined as having no globin-positive signals at all in the epiblast.
c(+/) is defined as having weak globin-positive signals in less than 10% of electroporated, GFP-positive epiblast cells.
d(+) is defined as having moderate globin-positive signals in 10%–50% of electroporated, GFP-positive epiblast cells.
e(++) is defined as having strong globin-positive signals in more than 50% of electroporated, GFP-positive epiblast cells.

(Nakazawa et al., 2006; Shin et al., 2009). For electroporation,
expression vector pCAGGS-2AGFP was constructed by insertinga 2A sequence 50 -TGGGCCAGGATTCTCCTCCACATCACCGCATGTTAGCAGACTTCCTCTGCCCTCTCCACTGCC-30 into the 50
end of GFP. An additional 2A sequence was inserted between
chicken LMO2 (nucleotides 46–519 of AF468789.1) and murine
SCL (nucleotides 142–1,128 of NM_011527). LMO2-2A-SCL fragment was cloned into 50 end of 2AGFP for pCAGGS-LMO2-2ASCL-2AGFP. LDB1-2A-E2A was generated by introducing another
2A sequence 50 -CAATGCACAAACTACGCACTACTAAAACTAGCAGGAGACGTAGAGAGTAATCCAGGACCC-30 between chicken
LDB1 (nucleotides 61–1,293 of NM_205070.1) and chicken E2A(nucleotides 98–2,110 of NM_204486.2), which was then inserted
into the 50 end of 2AGFP of pCAGGS-2AGFP for pCAGGS-LDB12A-E2A-2AGFP. Chicken GATA2 (nucleotides 410–1,807 of
NM_001003797.1) was used to construct pCAGGS-GATA22AGFP. Chicken GATA1 (nucleotides 41–952 of NM_205464.1)
was used to construct pCAGGS-GATA1-2AGFP. Chicken EKLF
(nucleotides 133–1,350 of XM_422416.4) was used to constructpCAGGS-EKLF-2AGFP. Chicken EKLF is regarded phylogeneticallyas the ortholog of mammalian KLF17 (Antin et al., 2010), but itis the only erythroid-specific Krüppel-like factor reported in thechick so far (Antin et al., 2010; Chervenak et al., 2006) and isused in this study as the functional ortholog of mammalian

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EKLF (KLF1). All experiments with chicken embryos wereapproved by the Animal Care and Use Committee of RIKEN Center
for Developmental Biology.

Ciau-Uitz, A., Liu, F., and Patient, R. (2010). Genetic control ofhematopoietic development in Xenopus and zebrafish. Int. J.
Dev. Biol. 54, 1139–1149.

SUPPLEMENTAL INFORMATION

Dailey, H.A., and Meissner, P.N. (2013). Erythroid heme biosynthesis and its disorders. Cold Spring Harb. Perspect. Med. 3,
a011676.

Supplemental Information includes two figures and can be foundwith this article online at http://dx.doi.org/10.1016/j.stemcr.
2014.01.019.

AUTHOR CONTRIBUTIONS
W.W. and G.S. planned the experiments, W.W. performed the experiments, W.W. and G.S. analyzed the data, and G.S. and W.W.
wrote the paper.

ACKNOWLEDGMENTS
We would like to thank Ms. E. Sukowati (Lab for Early Embryogenesis, RIKEN CDB) for technical assistance during revision andDrs. M. Shin (former member of Lab for Early Embryogenesis,
RIKEN CDB) and C. Alev (Lab for Early Embryogenesis, RIKEN
CDB) for help with transcriptomic analysis. This work was supported by RIKEN.
Received: August 26, 2013
Revised: January 27, 2014
Accepted: January 29, 2014
Published: March 6, 2014

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