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标题: 求助Development. 2011 Nov;138(21):4801-12.文献 [打印本页]

作者: chleiwu 时间: 2011-11-2 22:30 标题: 求助Development. 2011 Nov;138(21):4801-12.文献

Development. 2011 Nov;138(21):4801-12.3 Z! R. P( D. P9 W: M
ER71 directs mesodermal fate decisions during embryogenesis.
Rasmussen TL, Kweon J, Diekmann MA, Belema-Bedada F, Song Q, Bowlin K, Shi X, Ferdous A, Li T, Kyba M, Metzger JM, Koyano-Nakagawa N, Garry DJ.- @! @3 S* x) n' d
SourceLillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.

PMID: 21989919/ i, s3 ^; v: {1 L+ K7 a
http://www.ncbi.nlm.nih.gov/pubmed/21989919

作者: wang1234611 时间: 2011-11-4 09:39

1 ER71 directs mesodermal fate decisions during embryogenesis. 参考中文标题：点击查看机器翻译作者： Rasmussen TL. Kweon J. Diekmann MA. Belema-Bedada F. Song Q. Bowlin K. Shi X. Ferdous A. Li T. Kyba M. Metzger JM. Koyano-Nakagawa N. Garry DJ. 全文及相关链接：HighWire Press Swets Information Services 摘要： Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the EYFP(+) population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71 as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during embryogenesis. 参考中文摘要：Er71 nonviable突变和缺乏胚胎造血和内皮血统之中。进一步而言,需定义的功能作用在ER71细胞谱系的决定,我们产生转基因小鼠模型。我们策划的转基因小鼠模型Er71-EYFP通过熔断3.9 kb Er71赞助人EYFP记者的基因。使用FACS和转录的评价,我们仔细检查了EYFP(+)人口的细胞突变的、Er71野生littermates。在缺乏ER71,我们观察到一个数量的增长EYFP-expressing细胞,增加表达式的程序并减少心脏分子的表达hemato-endothelial程序,相比而言,野生littermate控制。我们也产生了一个新颖的Er71-Cre转基因小鼠模型使用相同的3.9 kb Er71推销员。研究表明,遗传fate-mapping ER71-expressing产生造血细胞和内皮血统的野生的背景。在缺乏ER71,这些细胞群体选择了mesodermal血统,包括心脏血统。延长这些分析中,我们使用了一个诱导胚胎干细胞/混合而且发现生命的性体系统ER71压抑的cardiogenesis品系。在一起,这些研究确定ER71作为一个至关重要的调节作用mesodermal命运决定说明造血和内皮血统的代价心脏血统之中。这加强我们对mesodermal决策机制控制命运的早期胚胎。出处： Development (Cambridge, England). 2011年138卷21期4801-12页相关链接： PubMed Google学术搜索 Google引文外部引文相关文献引文追踪(Google)

作者: tmdabc 时间: 2011-11-6 13:17

回复 chleiwu 的帖子% w$ f9 @2 C- P2 ^( m

4801 RESEARCH ARTICLE4 }: }- Q) d- \  g6 r' M3 z8 L) j* h
INTRODUCTION! v1 [4 O3 ~( Y$ V2 y* \
Transcriptional networks, signaling cascades, microRNAs and
other factors coordinately regulate stem and progenitor cell' Y/ L. Q: w, K% g$ q2 N6 F! R
populations to give rise to the mesodermal lineages (Davis and Zur& A1 {5 u3 K6 X* H
Nieden, 2008; Ivey et al., 2008; Omelyanchuk et al., 2009; Park et
al., 1998). During embryogenesis, a set of anterior progenitors1 {) D5 U! c; k- A) ]
coalesce to form the heart. These progenitors, along with the blood4 s' L, O0 B: g! p- d
and vasculature, comprise the circulatory system, which is the first
organ system to develop in mouse and human. Disruption or, x. c/ _" J) {* B
elimination of any one of these lineages results in early embryonic5 U  D% E5 a* x5 d
lethality. However, the factors that regulate the co-emergence of
these lineages from the mesodermal progenitor pools are/ C' ~9 T6 o. L3 [" I; X$ I1 c
incompletely defined.
Recent studies have identified a multipotent progenitor cell' m8 S# u) e% b, k! @
population that is capable of giving rise to the cardiomyocyte and
endothelial lineages during embryogenesis (Kattman et al., 2006;3 a9 E/ S6 f/ U% a! g: a3 c
Moretti et al., 2006; Wu et al., 2006). Although these multipotent
progenitors have been identified based on the expression of Flk1
(also known as Kdr), Nkx2-5 and Isl1, the transcriptional networks9 t9 A5 Q, _+ q- i+ e
that govern the fate of these progenitors are unknown. These and& X$ f0 n) B$ ]9 H
other studies emphasized the plasticity and context dependence of
regulatory cascades in the progenitor cell populations. For example,& e* ]0 j. Y' @! v* O4 ]
our previous studies demonstrated that Nkx2-5 has a dual4 d# K* R9 c# \! v5 I
transcriptional regulatory role in the promotion of the cardiac
lineage and suppression of the hematopoietic lineage (Caprioli et' O- Y7 p% v* s: e1 ^4 C
al., 2011). In this fashion, we proposed that the stoichiometry of
regulatory transcription factors and interacting factors could  q9 |. U* B- F
dynamically regulate the fate of progenitors. Definition of
additional networks and signaling pathways that promote and# f) ^4 `6 V7 q6 V
suppress the fate of distinct lineages that arise from multipotent
progenitors will enhance our mechanistic understanding of their. H, S+ ]8 ?5 B
developmental potential.
Ets-related protein 71 (ER71; Etv2) is a member of the Ets+ G  s; A1 A( v; R- w
transcription factor family that we and others have shown to be
essential for formation of the endothelial and hematopoietic
lineages (De Val et al., 2008; Ferdous et al., 2009; Lee et al., 2008).
Er71 can be induced by BMP, Wnt and Notch signals to promote
hematopoiesis (Lee et al., 2008) and synergizes with FoxC2 to. q8 W3 ?* F! c/ u$ Y
regulate the endothelial program by directly targeting Scl (also( N( ~: {2 j" L6 s* B; m
known as Tal1), Notch4, Cdh5 and Tie2 (also known as Tek) (De
Val et al., 2008; Ferdous et al., 2009; Lee et al., 2008). In Er71
mutant mouse embryos, there is a complete lack of hematopoietic4 c6 ?6 X' H2 L2 a
and endothelial lineages and the embryos are nonviable by
embryonic day (E) 9.5 (Ferdous et al., 2009; Lee et al., 2008).; n* G( ]; w# |6 |+ k& j; R9 b; O: d
Analysis of the Er71 mutant embryos revealed no differences in
cellular proliferation or cellular apoptosis compared with age-
matched wild-type littermates (Ferdous et al., 2009). These results
raised the question of whether the hematopoietic and endothelial6 d+ j  G% u& S4 v- L; p
precursors are still present but unable to differentiate to hemato-7 T: u- [% [  i6 z8 f$ \
endothelial lineages, whether they had been redirected towards
other lineages or whether these cells never arose during9 _6 ^8 \  _& R6 B+ l4 W" g" S
development.2 h- s/ c! \4 S6 x2 L: k) G
Development 138, 4801-4812 (2011) doi:10.1242/dev.070912
© 2011. Published by The Company of Biologists Ltd/ P  ?5 \! S2 S
1Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.
2Department of Integrative Biology & Physiology, Medical School, University of
Minnesota, Minneapolis, MN 55455, USA. ' m0 A6 D. {& n; I: m+ U
3Biolead.org Research Group, LC
Sciences, Houston, TX 77054, USA. 9 I7 Z0 f) J3 v+ ?3 R3 ^
4University of Texas Southwestern Medical$ L3 f, {  x. P2 ]8 g4 u* n
Center, Dallas, TX 75244, USA.
*Author for correspondence (garry@umn.edu)& J- A& t' T: |9 g5 I+ j. ^
Accepted 5 September 2011
SUMMARY& B+ G2 L. f! _( \+ p: k8 L/ B
Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for+ ~6 D# D5 q# \8 P! w
ER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse6 ?& }/ I' d, P) _  g: H/ w
model by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the; c" V2 X8 o6 h
EYFP+ population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the+ I, O% H2 B* O
number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-
endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse* E7 R* y+ F0 i) h* q5 l* p
model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the& _0 L& W, @) q1 J1 ^
hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed
to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic- T& |& ~# `( z0 V; A8 S, X. i
stem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71
as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense
of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during0 b+ B$ q5 T5 f( g1 C- m
embryogenesis.
KEY WORDS: Transgenesis, Er71 (Etv2) knockout, Mouse! ?& h/ t0 m4 W( b* \# f  n
ER71 directs mesodermal fate decisions during! t$ k3 R- L. }* h: L/ A
embryogenesis
Tara L. Rasmussen1, Junghun Kweon1, Mackenzie A. Diekmann1, Fikru Belema-Bedada1,2, Qingfeng Song3,
Kathy Bowlin1, Xiaozhong Shi/ C; ?& V6 p( e
1, Anwarul Ferdous4, Tongbin Li
3, Michael Kyba1, Joseph M. Metzger1,2, : u" Q$ v- a9 Y) V0 q
Naoko Koyano-Nakagawa1 and Daniel J. Garry1,
*
DEVELOPMENT4802. a* \* p# }% h' \; @
In the present study, we have dissected ER71-mediated; J. p3 k. P3 ^8 S. O* m
mechanisms that govern fate determination during murine
embryogenesis. We have generated a novel transgenic mouse
model in which the 3.9 kb Er71 promoter drives the EYFP reporter) R- l) {& |' ]. y! Z
(Er71-EYFP), and crossed it into the Er71 wild-type and mutant
backgrounds. This strategy facilitates the isolation and) u- x0 D# ^# K, S2 D% P4 r# F# \1 l
characterization of cells that would normally express ER71 from% z' p2 z: v6 N9 [! u! P4 E
Er71 mutant embryos. In the absence of ER71, we observed the
conversion of cells that would normally give rise to lateral plate% H6 E3 b- \3 g
mesoderm into cells that produce paraxial and cardiac mesoderm.
Furthermore, ER71 overexpression in vitro using an inducible
embryonic stem cell/embryoid body system demonstrated
decreased cardiogenic potential. Collectively, these data: C' `, X3 r) u9 }: W/ ?; w( K- l
complement and extend our understanding of the functional role of; e. ^+ k3 G" [4 F  n* t
ER71 to differentially promote mesodermal fate decisions during7 k, V0 r% [& }5 M* q# K
embryogenesis., x8 H" D$ x% @% {# ~
MATERIALS AND METHODS
Generation of transgenic mice# P0 c+ ~1 Y3 K/ v
The 3.9 kb  Er71 promoter (Ferdous et al., 2009), which harbors the0 v) ~+ i, f7 Q( S
modules necessary to direct the temporal and spatial expression of ER71,
was cloned into the pEYFP-1 vector (BD Biosciences) and into a
promoterless pBS-Cre-pA vector to generate the ER71-EYFP and ER71-8 s5 U; \! \7 P, T8 n- {
Cre constructs, respectively. Transgenic animals were generated at the8 T: e1 }; j& s. R6 s# N8 W
University of Minnesota Mouse Genetics Laboratory using standard
methods. Transgenic mice were screened for DNA integration by PCR.
Expression analysis of ER71-EYFP lines was performed by examining' O) C" z0 M4 `
E7.0-9.5 embryos resulting from timed matings to wild-type CD1 mice4 K8 v1 G  g  d0 I* n% e
(Charles River). We also analyzed E11.0 and postnatal day (P) 3 offspring8 ]6 t1 w; i8 s
of timed matings of the ER71-Cre lines and the Rosa-EYFP line (Jackson; S! R+ \$ h4 I9 O6 V! A' }( L
Labs 006148). Founder mouse lines were crossed to the  Er71
heterozygotes previously described (Ferdous et al., 2009). In all cases, at
least three transgenic lines were examined to confirm similar temporal and3 ^9 A2 [: g' m! Y5 F6 B
spatial expression patterns. All mice were maintained at the University of
Minnesota under protocols approved by the Institutional Animal Care and
Use Committee and Research Animal Resources.
FACS analysis/ S7 H" |9 F% N$ u7 q
Embryos from timed pregnant females were harvested at specified stages.
Embryos were separated from yolk sacs, which were used for genotyping,
and imaged on a Zeiss Axio Observer Z1 inverted microscope prior to
dissociation. Embryos between E7.5 and E9.5 were digested in 30-50 l
0.25% trypsin plus EDTA at 37°C. Digestion was arrested with 500 l3 J: V; X5 h# E6 ]4 T  L
DMEM containing 10% fetal bovine serum (FBS). Cells were pelleted at% j3 R6 f( w' b
4°C (1600  g), resuspended in PBS and passed through a 70  m cell+ ?( H0 ?. t% B
strainer. Cells were incubated with antibody cocktails for 30 minutes,+ F" P# A& e7 U7 o  s
washed, and resuspended in PBS. Cells were analyzed or sorted using a/ ?, m1 L$ X/ R6 Y7 _
FACSAria (BD Biosciences). Various combinations of antibodies (1:1000)
were used, including Flk1-APC (eBiosciences 17-5821), Pdgfra-PE
(eBiosciences 12-1401), CD31-PECy7 (eBiosciences 25-0311), CD34-PE
(BD Pharmingen 551387), cKit-APC (eBiosciences 17-1171), CD41-# |7 Z/ S9 _" j! [( e+ J# t
PECy7 (eBiosciences 25-0411), CD45-PE (BD Pharmingen 553081),
CD45-PECy7 (eBiosciences 25-0451-81), and Ter119-APC (BD
Pharmingen 561033) antibodies.
Transcriptome analysis% U& Y7 A, E7 {) k
Total RNA was isolated from 1000-5000 FACS sorted cells in TRIzol
(Invitrogen) using the PureLink Mini RNA system (Ambion). RNA was6 S2 f; K) n/ K3 T, w; i
subjected to two rounds of amplification using the MessageAmp II aRNA
Amplification Kit (Ambion) and then labeled using the MessageAmp II-
Biotin Enhanced Kit (Ambion) according to manufacturer’s protocols.
Amplified RNAs were hybridized to Affymetrix mouse 430 2.0 full genome& s1 Z% b2 d8 u5 B* L" F
array chips at the BioMedical Genomics Center of the University of8 ^, X: V# ]% P6 N( \
Minnesota. The CEL data files produced from Affymetrix array experiments2 K8 z& j  U% {; W8 _, F
were processed using the affy package included in Bioconductor. The robust  Q; T% a# ~, _. S  d
multi-array (RMA) method (Irizarry et al., 2003) was used to perform data( `1 R/ N* m2 N; ]- @
normalization, background correction and expression quantification. The
limma package (Smyth, 2005) was used to identify differentially expressed* M/ f9 z) W6 ^8 U
genes by ANOVA analysis, and false discovery rate (FDR) values were
calculated using the Benjamini-Hochberg method (Benjamini and Hochberg,: f8 Q0 G/ E+ ?: y2 I  j$ E( Y" R9 x
1995). Average linkage hierarchical clustering analysis was performed using
the cluster program (de Hoon et al., 2004), with uncentered Pearson’s" g1 n  e0 h# N$ X9 G
correlation coefficient used to define pairwise similarity in gene expression.
Genes annotated with the gene ontology (GO) term ‘heart development’7 ]/ g( o1 w& E4 [
(GO:0007507) were downloaded with AmiGO tool (Carbon et al., 2009) and) l; N) p7 S/ e- C( T) [
used in producing the heatmaps. Microarray data is accessible in Gene4 |6 G! L7 b/ A& j0 C
Expression Omnibus (series GSE32223)." Y  i, N5 T) H. O" ~5 x; q% ~: D
Quantitative RT-PCR
Total RNA was isolated from 1000-5000 FACS sorted cells or embryoid3 J* x, E8 E1 k; `2 ~
body cells in TRIzol (Invitrogen) using the PureLink Mini RNA system.
RNA from FACS sorted cells was subjected to two rounds of amplification
as described above, but left unlabeled. cDNA was made using random1 d% }, t" J, i3 X) P. i8 w/ E2 o
hexamers and transcript levels were determined using VIC-labeled (Gapdh,
4352339E) or FAM-labeled (Er71, mm01176581_g1;  Scl,
mm01187033_m1; Cdh5, mm00486938_m1; Gata4, mm00484689_m1;8 p- L  ?1 f: H% o
Tbx5, mm00803518_m1; Tnnt2, mm00441922_m1) TaqMan probe sets) ?  \; t' Y$ W8 E: o
(Applied Biosystems).$ s* x* o) O- @: g- a$ [8 x: Y) t! |
Embryoid body differentiation and ER71 overexpression
Doxycycline-dependent ER71-overexpressing mouse embryonic stem& ?8 _( B- e# D* U
(mES) cells were generated using an inducible cassette exchange strategy
(Iacovino et al., 2009). In this system, ER71 tagged with a C-terminal HA5 [# f' c6 E" F7 t
epitope was overexpressed in response to 0.5 g doxycycline. To mimic
early embryonic development, the mES cells were differentiated into
embryoid bodies (EBs) using mesodermal differentiation media [1.5% FBS2 S; e& a8 `9 s$ ]
(Stem Cell Technology), 1 penicillin/streptomycin, 1 GlutaMAX
(Gibco), 100 g/ml Fe-saturated transferrin, 450 mM monothioglycerol, 50% P% {; O( z& r4 z9 E( Z6 r% u
g/ml ascorbic acid in IMDM (Invitrogen)] (Kennedy et al., 1997). ER71
was induced on day 3 after initiating differentiation and maintained through5 R) B4 L# F: r; O4 \
day 10 by supplementing the differentiation media with doxycycline. On
day 10, we fixed the EBs with 4% paraformaldehyde (PFA) and prepared# H8 Y7 v" Z4 D/ f: F
cryosections. We stained adjacent sections with Hematoxylin and Eosin8 k( M- f7 D% [/ J, A0 D, ^! }, v
using a standard protocol and performed immunostaining with mouse anti-  n! |' Z4 H+ F
TnT serum (1:200, DSHB clone CT3). We assayed the TnT-expressing area
of each EB using ImageJ software (NIH) and calculated the ratio of the
positive area to the total area from 33 sections of uninduced EBs and 42
sections of induced EBs. EBs were placed on laminin-coated coverslips' R3 @( c$ T  E$ O1 p# B
and stimulated at 1 Hz. Edge detection (Ionoptix, Milton, MA, USA) was- c7 m; Z7 f0 p5 l+ R
used to measure contractility of intact EBs.
Immunohistochemical analysis
Stage-specific embryos were harvested from time-mated pregnant females.. K- {. t5 k" r  k7 }) g% N* t
For paraffin sectioning, embryos were fixed for 4-8 hours at 4°C in 4%
PFA and embedded. For cryosectioning, embryos were fixed for 1 hour at
4°C in 4% PFA and embedded in OCT compound (Sakura). Sections were
blocked with immunohistochemical diluent (2% normal donkey serum, 1%" k: a) {# v  O# t; L4 _
bovine serum albumin, 0.3% Triton X-100, 0.02% sodium azide in PBS,
pH 7.3) at room temperature and incubated overnight at 4°C with primary
antibodies, including chicken anti-GFP (1:500, Abcam ab13970), rabbit& G1 w  }! E, O# X) F; e
anti-desmin (1:200, Novus Biologicals NB120-15200), rat anti-Tie2 (1:100,! R$ W7 Z3 z5 u
eBiosciences 13-5987-81), rat anti-Pdgfra (1:200, eBiosciences 12-1401-
81), goat anti-Nkx2-5 (1:500, Santa Cruz SC-8697) and anti-CD41 (1:100,% l6 J6 }. D1 w9 t9 |
BD Pharmingen 550274), anti-Cdh5 (1:100, BD Pharmingen 55289), anti-
CD31 (1:200, BD Pharmingen 550274), anti-Gata1 (1:100, Santa Cruz SC
265X) and anti-troponin T (1:200, DSHB CT3) sera. Slides were washed
and incubated with combinations of secondary antibodies (1:200) including
anti-chicken Dylight 488, anti-rabbit Cy3, anti-mouse Cy3, anti-rat Cy35 F/ I4 d6 `2 `$ B$ q# U
and anti-goat Dylight 549 (Jackson ImmunoResearch Laboratories).
Results were imaged on a Zeiss Axio Imager M1 upright microscope or a
Zeiss LSM 510 Meta confocal microscope.
Statistical analysis
Data represent the average of at least three replicates and s.e.m.
Significance was tested by the Kruskal-Wallis test with the Dunn multiple
comparison test for more than two groups; for example, when comparing5 g& r& s+ a8 G4 o* C
RESEARCH ARTICLE Development 138 (21)
DEVELOPMENTwild type, heterozygous and homozygous mutants. Significance was tested. u& V( `6 w% n/ g. K) D
by the Mann-Whitney U test for two groups. Analyses were performed
using Prism5 software (GraphPad).* _. [2 ]- H5 V: Y" ~9 F
RESULTS7 o- k# O6 u$ {# k' O$ C
Expression analysis of the ER71-EYFP reporter
Initial analysis of ER71 expression in the developing mouse4 L' e( H0 z" e0 }9 i
embryo was reported previously (Ferdous et al., 2009; Lee et al.,
2008). To further examine its potential role in cardiovascular
development, we performed a detailed expression analysis of ER71
from E7.0 to E9.5, when Er71 mRNA is expressed (Ferdous et al.,
2009). None of the commercially available ER71 antibodies
detected the endogenous protein, so we utilized an Er71 transgenic
mouse driving an enhanced yellow fluorescent protein (EYFP)" f6 a( e$ @; L* p9 j7 \
reporter. The 3.9 kb region upstream of the Er71 transcription start# {, J4 Z& p0 M, o  W
site was fused to the  EYFP reporter (Fig. 1A). This promoter
fragment was previously shown to drive reporter expression that
recapitulated endogenous ER71 expression (Ferdous et al., 2009).9 Y  C1 f; p% S  D1 `/ w
Four founder lines were generated with offspring embryos that
showed the same expression pattern.
To examine the expression pattern of the EYFP reporter, we. `2 F6 A5 z4 D- \
compared its expression to that of the angiopoietin 1 receptor Tie2,9 P; a1 C4 M+ y5 f+ U6 }& y
which marks endothelial cells in mice and humans (Dumont et al.,: W+ D% @3 h+ u+ s+ Q1 e- Q) \
1992; Sarb et al., 2010; Schlaeger et al., 1997; Schnurch and Risau,& ?9 a1 V& P( {  {$ c
1993). The EYFP reporter (representing ER71 expression) was
expressed as early as the early to mid gastrulation stage (E7.0; Fig.$ v. H7 F6 v0 ]$ I% r+ ^
1B, bracket). At this stage, reporter expression was limited to the$ k  u& J9 _3 }( O' r
extra-embryonic mesoderm (Fig. 1C) and showed co-expression
4803 RESEARCH ARTICLE ER71 governs progenitor fates6 y$ h) ^5 K; i2 p, t
Fig. 1. The transgenic ER71-6 v  r" W" h( Y% Q4 n
EYFP reporter is expressed- F; g0 \4 _! g$ a* }! X  V
during embryogenesis.! k; n6 M1 _' i) |' h
(A)The transgenic Er71-EYFP" j8 k1 u1 {% N9 Y0 }; @
construct used in these analysis.
(B-Q)Expression analysis of mouse
embryos between E7.0 and E9.5.
EYFP protein fluorescence (green)6 B+ r$ e% D0 A. K
is shown in whole-mount images
(B,D,F,J,K,N). Sections were
stained with anti-GFP (green),# a* A8 s3 E( E, P" L1 u" g
anti-Tie2 (red), anti-Nkx2-5 (red)' n; ^% y4 {  ~% w) z
or anti-Pdgfra (red) antibody as
indicated. DAPI nuclear staining is
shown in blue. Yellow indicates
overlap of green and red
channels. (B-C) Early to mid
streak stage, (D-E) early bud8 A* L+ d% E/ D/ u: n  A
stage, (F-I) early head-fold stage,. a+ W. l3 q3 b1 K: ^) f
(J-M) E8.5, (N-Q) E9.5; embryos/ V: q1 W7 l: g# Z+ H
were staged according to Downs% ], W; U9 f& d; ~# W- m4 T
and Davies (Downs and Davies,
1993). Boxed regions are shown3 O$ D6 |; `1 Q/ g" o  m
at higher magnification; planes of+ r0 B  B* d* `# N5 j, _
sections are indicated. See first
section of the Results for a
description of arrows, arrowheads" Q" _& [4 s; b* W4 d) d$ K* F
and other labels. al, allantois; ba,
branchial arch; bi, blood island;" j5 a8 D) k4 \: C% B! g7 e
cc, cardiac crescent; cv, cardinal! D* ]' D  Z- z, x4 O& }5 j3 c
vein; da, dorsal artery; ec,
endocardium; h, heart; hf, head
fold; np, neural plate; nt, neural
tube; ph, pharynx; pm, paraxial
mesoderm; s, somite; ys, yolk sac.: i1 i$ L/ I+ y) u- e% s2 [
Scale bars: 200m; 50m in
enlargements.4 u0 r& [; @" b3 \9 r
DEVELOPMENT4804
with Tie2 in the primitive blood islands (Fig. 1C,C, arrows) (Ema
et al., 2006b). EYFP was also weakly expressed in the mesothelial
layer of the yolk sac, but Tie2 was absent in this non-endothelial) _# F1 M3 E5 Y7 C) E" R
cell population (Fig. 1C,C, arrowheads). At E7.5, continued
expression of the EYFP reporter was observed in the extra-
embryonic mesoderm (Fig. 1D, bracket). In addition, scattered  A2 W+ \' A) ^9 L3 O
EYFP+ cells were observed in the lateral plate mesoderm (Fig. 1D,
arrows). Transverse sections showed that these scattered cells co-8 X1 z# Y, K6 l" S1 c( y. g! A( P
expressed Tie2 and most likely represent endothelial progenitors; @  v9 F$ b- }8 G
(Fig. 1E,E,E), although Tie2 expression has also been identified
in a subset of cells committed to the hematopoietic lineage
(Kisanuki et al., 2001; Li et al., 2005).
At E7.75, the EYFP signal localized to discrete structures
including the cardiac crescent and the progenitors of the dorsal aortae& O, d4 u, P2 V( r) l$ e1 ]
(Fig. 1F, cc, arrows). We examined the expression in the cardiac
crescent and paraxial mesoderm in detail using Nkx2-5 and Pdgfra
antibodies (Fig. 1G-I). We observed that the EYFP signal overlapped5 K4 q" Y+ F2 ]9 v# ~8 l- r7 T
extensively with Tie2 within the cardiac crescent (Fig. 1G,G).
These EYFP+ cells are presumably endocardial progenitors (Misfeldt
et al., 2009). By contrast, EYFP+ cells either weakly expressed or did! J& |' G0 A, [/ S9 S' F# X
not express Nkx2-5 (Fig. 1H,H, arrows and arrowheads,
respectively). We did not observe strong co-expression of Nkx2-5
and EYFP. Pdgfra is strongly expressed in paraxial mesoderm and
cardiac mesoderm (Fig. 1I, pm and asterisk). Double
immunohistochemical labeling of EYFP and Pdgfra demonstrated; x$ A/ n( J* S: @
that most cells either did not co-express these markers or weakly
expressed Pdgfra (Fig. 1I,I,I, arrowheads and arrows, respectively),
which is consistent with previous reports that the hemogenic lineage
is segregated from the paraxial mesoderm early during gastrulation
(Kataoka et al., 1997; Takakura et al., 1997).
At E8.5 and E9.5, we observed localization of the EYFP signal to
vascular structures throughout the embryo proper (Fig. 1J,N; arrows2 m/ p2 g, c3 R2 c5 w
point to the dorsal aorta and arrowheads indicate intersomitic vessels)
and yolk sac (Fig. 1K). Transverse sections of the embryos (Fig./ F+ L; q8 C$ Z/ y) k
1L,M,O,P) showed localized expression in the vessel structures
including dorsal aorta, cardinal vein, intersomitic vessels: O* v6 i5 y' T' R) L5 o+ h% s
(arrowheads in Fig. 1M) and the endocardium (arrowheads in Fig.
1L). The expression of EYFP largely overlapped with that of Tie2,9 b( s3 W' K5 S( g  b
indicating that the  Er71 promoter is active in early embryonic8 z" i3 D* d4 v
endothelial and hematopoietic cells (Fig. 1O-P). We also observed
that EYFP was co-expressed with the hematopoietic transcription) @5 p: Y" X4 F" D3 i3 }: q9 ]
factor Gata1, the cell surface marker of hematopoietic progenitors
CD41 (also known as Itga2b), the marker of hematopoietic and
endothelial lineages Cdh5, and with the primarily endothelium-. i7 x9 t4 @! D" F( A! @( P7 k$ W+ Y6 E
specific cell surface marker CD31 (also known as Pecam1)
(supplementary material Fig. S1). By E9.5, Nkx2-5 was localized to2 a4 x  j+ a0 w6 y
the myocardial layer of the heart and did not overlap with EYFP. [+ u) G4 p8 I8 G( z
expression (Fig. 1Q, arrowheads; compare with 1O).
Reporter expression is mislocalized in the Er71
mutant- H( U6 K$ d% S) {( x) e& x! N& E
In the Er71 mutant, the hematopoietic and endothelial lineages are
absent. However, no changes were observed in cellular
proliferation or apoptosis in the Er71 mutant embryo (Ferdous et+ S2 ^8 a9 k; a& G7 E( N3 \
al., 2009). Therefore, we crossed the ER71-EYFP reporter line into
the Er71 mutant background to define and characterize the cells
that should give rise to these lineages (Fig. 2). In the E7.75 wild-* a. P  I3 b4 v, V
type embryo, EYFP expression was observed throughout the2 g" d$ D- k4 W0 J$ L+ y
cardiac crescent, the dorsal aortae and the extra-embryonic
mesoderm (Fig. 2A). By contrast, in the E7.75 mutant embryo the
expression pattern was less definitive. EYFP-positive cells were) p; Q8 z/ i: R* v
found in the extra-embryonic tissue; however, expression of EYFP
within the embryo proper was mislocalized (Fig. 2B, arrow). EYFP8 a( ]: i+ z& _1 h9 G3 s
expression appeared diffuse throughout the cardiac crescent (Fig.* c- G$ W. J1 \2 T8 w
2B, asterisk). In the wild-type embryo at E8.0, EYFP expression
was observed specifically in the dorsal aortae, but not on the
periphery of the embryo (Fig. 2C, arrowheads). EYFP expression" N9 {* r* J& B7 M5 k
was also observed in the presumptive endocardium within the) d/ ]8 O! |. h2 Y% M
linear heart tube (Fig. 2C). By contrast, the Er71 mutant embryos
expressed EYFP only at the periphery of the embryo, which is
likely to represent paraxial mesoderm (Fig. 2D).
Immunohistochemical analysis of ER71-EYFP and Pdgfra, a3 U' z, R6 o1 g5 V
marker of paraxial mesoderm, expression in E8.5 wild-type and  J, f. K+ j2 g" _  c3 K0 `7 a0 i* l
mutant embryos demonstrates that EYFP-positive cells are, a: C5 [/ T3 Q& @$ L. y( {3 B; Y
interspersed within the Pdgfra-positive mesoderm (supplementary
material Fig. S2). FACS profiling showed that the frequency of# i1 C$ y9 F! S3 h
EYFP+ cells was increased in the Er71 mutant compared with the7 K( _  V( t& I7 x! y+ ?  ^4 s
wild-type control at E8.0 (Fig. 2G-I), but not at E7.75 (Fig. 2E,F,I).
The apparent increase in the percentage of EYFP+ cells could be8 K4 r* }& p; Q: n1 k2 u
due to a reduction in the total number of cells without a change in
the number of EYFP+ cells, as cell viability can be reduced by a/ d, `$ [/ w) E. e: N5 \
lack of vascularization. However, we ruled out this possibility as
we also observed a doubling in the absolute number of EYFP+ cells
in E8.0  Er71 mutant embryos as compared with wild-type
littermates (Fig. 2J)." L+ m6 B+ X; ]
These analyses demonstrated that cells that expressed the ER714 X( [: C- ]) U/ C, e
reporter were not only present in the Er71 mutant embryo, but were
also expanded in number. Since the Er71 mutant embryo lacks" o7 r& H* h. U! }$ F$ L( C
differentiated endothelial and hematopoietic lineages, we examined
whether these EYFP+ cells were arrested progenitors or cells
redirected to other lineages. We used FACS analysis to examine the/ Y& C4 a* E! p! d; v9 Z* h
cell surface markers of the EYFP+ cells in the Er71 mutant and; d1 k, Y/ r( v1 ~. ]
wild-type backgrounds. Previous studies have established that
Pdgfra is expressed in the primitive streak at E7.0, but is restricted2 S, J) H; ~5 V/ E- A5 D2 i
to the paraxial mesoderm at the early head-fold stages (Takakura% b, I( |) B3 q* P( I
et al., 1997). Flk1 is expressed more broadly in mesodermal. p( n0 T) [8 E# |2 x- P
precursors (Ema et al., 2006a; Motoike et al., 2003), including the8 E5 ^! k! F) @+ S5 x) V
posterior portion of the primitive streak. Flk1+/Pdgfra–5 R0 C- o6 ?* J5 e6 [. g' E
cells mark2 G/ h# L5 m) ?
lateral plate mesoderm that will give rise to the endothelial and/ C0 T% y7 j% c# A* @1 L7 u
hematopoietic lineages and Flk1–
/Pdgfra+ cells mark the paraxial  v9 R" N0 q6 `5 f3 m$ c" k
mesoderm. Flk1 and Pdgfra are known to be co-expressed in the
cardiac crescent (Kataoka et al., 1997) and in cardiac progenitors4 B0 u4 ~2 W9 \, M% e' ~7 |
of embryoid bodies (EBs) (Bondue et al., 2011; Kattman et al.,  |% r- V) H# M' i
2006). In the present study, the frequency of Flk1+ cells within the0 \; L9 c" w+ `1 ~* c
EYFP+ population was significantly reduced at E7.75 and E8.0
(Fig. 3A,B,D,F). CD41 (Fig. 3D,E) and CD34 (Fig. 3E,F)( y% D8 M& @) N+ y( ~" s
expression was essentially absent at E8.0 in the EYFP+ cells of the+ e$ l! Y- y* C$ f
Er71 mutant. These results show that the lateral plate mesoderm6 h6 s2 v4 O7 a4 P5 s$ E0 S
lineage and its derivatives, i.e. the hematopoietic and endothelial
lineages, are reduced in the Er71 mutant.- n% I& M) o+ P* k4 P/ L9 M) r
In order to assess which lineages are expressing ER71-EYFP, we+ g1 ~7 w1 P+ I$ `# I
analyzed the EYFP+ cells using Flk1 and Pdgfra antibodies.: X9 I7 p2 N3 \. G9 q
Concurrent with the decrease of Flk1 expression at E7.75 and E8.0,, R+ T* V/ j" y0 F; M
we observed an increase in the frequency of EYFP+ cells that
express Pdgfra (Fig. 3A,B). This correlated with a significant2 i$ U* ~/ N2 V% p. s, J
increase of Pdgfra single-positive cells at E7.75 and a significant
increase of Flk1+/Pdgfra+ cells at E8.0 (Fig. 3A,C). There was also
a trend toward expansion of Pdgfra single-positive cells within the
EYFP+ population at E8.0 (Fig. 3A,C). To support these6 W; b0 O) {. }
observations, we used immunohistochemistry to analyze the EYFP; m' ?. b4 w# m4 H
expression patterns. In wild-type embryos, EYFP expression is* O( w/ T) _8 d/ u
distinct from Pdgfra+ paraxial mesoderm. A select number of
EYFP-positive cells are found within this region; however, these
cells are Pdgfra–- t. G& q- E, ?* ^+ Q
(supplementary material Fig. S2A,B). In mutant
RESEARCH ARTICLE Development 138 (21)! m$ d2 J/ \- {+ i9 v7 w
DEVELOPMENTembryos, many EYFP+ cells are found within the Pdgfra+ paraxial2 m0 x! [: R1 M8 R: C, A# F3 O
mesoderm. Some of these cells are double positive for EYFP and
Pdgfra (supplementary material Fig. S2C,D). Likewise, in wild-
type embryos, EYFP expression is distinct from troponin T (TnT)6 c: x/ h# u& D! e3 l4 U0 g5 B
+
cardiomyocytes (supplementary material Fig. S2E). However, in7 A* k1 {" m9 I
mutant embryos double-positive cells are found (supplementary
material Fig. S2F,G, arrows). One interpretation of these data is) E9 }. }, u0 K& r/ Z1 _' z9 R
that, in the absence of ER71, the EYFP+ progenitor cells, L8 j( T# b% d" t
differentiate towards the paraxial (Flk1–
/Pdgfra+) and cardiac! {& @& i& c, Y7 H  x* i7 v: a
mesodermal (Flk1+/Pdgfra+) lineages at the expense of the lateral
plate/hemangiogenic mesodermal (Flk1+/Pdgfra–% h' r/ c  V7 t
) lineage.
An alternative explanation of these results is that EYFP is
expressed ectopically in cardiac (Flk1+/Pdgfra+) and paraxial# ]& O7 |- C1 N" {, U
(Flk1–
/Pdgfra+) mesodermal populations in the absence of ER71.
If this were the case, we would expect the cardiac and paraxial
mesodermal populations within the EYFP–/ E+ p8 t  d. ?2 p; }, t( b
gate to decrease in
the mutant embryos because these cells expressed EYFP and
would be included in the EYFP+ gate. Furthermore, we would* \5 Z& Y6 O: F0 I: P: `7 N
expect no change in the overall representation of these markers
in the unfractionated (the sum of EYFP+ and EYFP–1 w; D# {  P+ y
) gate. To3 d3 ]: p( A) q* J' a4 l+ U: m
examine this possibility, we analyzed unfractionated cells from3 ~/ p/ [/ n5 @6 e1 w  R4 Q
the whole embryo as well as EYFP–7 i  N* E% G: l3 l
fractionated cells. In the$ A2 J: Y1 f' S! ]$ N
EYFP–
fraction no changes were observed in the mesodermal
populations at E7.75 (supplementary material Fig. S3C,D) and3 _$ Q+ F' i- o9 F. k( `0 |
E8.0 (supplementary material Fig. S3G,H). Analysis of the& Y$ }* P8 A+ Y, A, f
whole population (the sum of EYFP–
and EYFP+ gates) showed
no change in cardiac or paraxial populations at E7.75
(supplementary material Fig. S3A,B); but at E8.0, we observed& u' G, m$ d; \- Z
a small, but reproducible, increase in the cardiac mesoderm
(Flk1+/Pdgfra+) (supplementary material Fig. S3E,F). We predict4 c1 p+ A5 l( J% B' [
that the increase in cardiac mesoderm in the whole embryo is1 v& k8 ^' b( _# S% J
small because the EYFP+ population is a minor fraction of the8 F) z/ o7 p! b% w
entire embryo.% g: I  L! D3 n0 j  ^
Collectively, our results support the model that, in the Er71
mutant background, EYFP+ cells differentiate to the cardiac
mesoderm lineage and contribute to the increase in cardiac5 {; y/ Q; c# F; ?+ b% S
mesoderm (Flk1+/Pdgfra+) markers, rather than cardiomyocytes or% C$ q. k5 S( I* ^- k+ U( _
cardiac progenitors ectopically expressing EYFP.
4805 RESEARCH ARTICLE ER71 governs progenitor fates
Fig. 2. The ER71 reporter is misexpressed in Er71 mutant embryos. (A-H)EYFP expression in Er71 wild type (A,C,E,G) and mutant (B,D,F,H)' @# l( x0 N$ O
mouse embryos at E7.75 (A,B,E,F) and E8.0 (C,D,G,H). Asterisks indicate the cardiac crescent (A,B) or forming linear heart tube (C,D). Arrowheads- k8 a9 Z! I6 V" {# N$ P
indicate the bilateral dorsal aortae (C,D) or its progenitors (A,B). Arrows point to regions in which EYFP is misexpressed in the mutant (A-D).
Representative FACS profiles of individual embryos (E-H) show the percentage of EYFP positive cells per embryo. Scale bars: 200m. (I)The
percentage of EYFP+ cells for all experiments. (J)The average number of EYFP+ cells sorted per embryo. WT, wild type; HET, Er71 heterozygote; MT,$ ^& h. Z& \6 T/ \5 ?: `) M
homozygous Er71 mutant. ***, P<0.001. Error bars indicate s.e.m.: y& M" u: g" L2 x. F( k
DEVELOPMENT4806. S% K. m4 w5 J( W: I2 ~% }
Cardiac and skeletal muscle genes are
upregulated in ER71-EYFP+ cells in Er71 mutant
embryos7 A' N! h5 Z/ X' `: K& _
To further examine whether the EYFP+ cells in the absence of+ x# w6 T! M% R; M
ER71 were redirected to other mesodermal lineages, we performed
a transcriptome analysis. We used an Affymetrix microarray
platform and examined the EYFP-positive and EYFP-negative& h. r3 v+ w9 ]
cells from wild-type and Er71 mutant littermate embryos. Genes
that were significantly dysregulated were filtered based on the false; A/ w2 l& E/ z. j! x( F( @8 a7 s: j- l% S6 E
discovery rate (FDR<0.01 for EYFP+ versus EYFP–
; FDR<0.35 for& P2 C* K6 r9 b! U* U4 B- e
wild type versus mutant; FDR<0.01 for the interaction between  I( g! q  v! y3 l/ a$ @
these two variables). Clustering analysis was carried out
subsequently (Fig. 4A). Interestingly, there were no transcripts that
showed differential expression between EYFP–
populations in the$ f' E2 s6 A# q8 Z8 E! Z5 T2 }5 E
Er71 wild-type and mutant backgrounds. Also, the cluster analysis
was unable to resolve differences between the EYFP–
populations6 z1 t& m6 R0 l. J
RESEARCH ARTICLE Development 138 (21)
Fig. 3. ER71-EYFP+ cells in Er71 mutant mouse embryos lack markers of hematopoietic progenitors in exchange for those of other, P7 u) {! a; N6 R4 K8 ~
mesodermal lineages. (A)Using fluorophore-conjugated antibodies and FACS analysis, we observed an enrichment of Pdgfra+ cells in ER71-EYFP+
mutant cells at E7.75 and E8.0. (B,C)Summary of multiple experiments comparing individual fluorophores Flk1 and Pdgfra (B), and combinations of
Flk1 and Pdgfra (C). (D,F)Hematopoietic progenitor cell surface markers are not expressed in the embryo proper at E7.75, but there is a significant  |* b: \8 [- ]4 k
reduction in CD41 (D) and CD34 (F) at E8.0. (E)Summary of multiple experiments comparing hemogenic and endothelial markers CD41 and CD34.8 S, s( J5 C+ y
**, P<0.01; *, P<0.05. Error bars indicate s.e.m. LPM, lateral plate mesoderm; CM, cardiac mesoderm; PM, paraxial mesoderm.
DEVELOPMENT(Fig. 4A). These transcriptome results for the EYFP–1 R( O. Z$ T. Y
cells support
the notion that EYFP is not being expressed ectopically.
Furthermore, this indicated that there were no significant non-cell-  H- R0 \! D* B8 D; p8 l: q
autonomous effects on gene expression resulting from the lack of
ER71 at this time point." ~  F; `2 w( ^1 _& A# n5 t
Within the EYFP+ populations, the Er71 mutant and wild-type
cells were significantly different (Fig. 4A). A large number of- O$ E- S: P! s1 Y! R- L
hematopoietic and endothelial transcripts were decreased in
expression between  Er71 wild-type EYFP+ and  Er71 mutant
EYFP+ cells (Fig. 4C). Importantly, ER71 expression was restricted, k3 b$ E1 y" ?7 `6 ^# o2 [" Y: o
to the EYFP+ cells in the wild-type background (Fig. 4C, arrow),  ]) y- F/ i# s' M+ _
confirming that the  Er71-EYFP transgenic model reflects1 ~" C5 X  Z2 g, P  o  F
endogenous ER71 expression. Other transcripts that were
downregulated in the mutant EYFP+ cells compared with the Er71- b; E  d- l+ G7 D
wild-type EYFP+ cells included endothelial and hematopoietic+ b  }* u' o$ ~1 `  A& Y' O
transcripts such as Hbb-y, Fli1, Erg, Tal1, Cldn5, Cd93, Sox18,0 P% L, O9 |, q7 K
Cdh5, Sox7, Mmrn1, Eng and Nos3 (Fig. 4C). These data indicated
that the EYFP reporter-positive cells contributed to the endothelial# [3 S. X* V  m* s  M
and hematopoietic lineages and did not express markers of those  n5 B  r  O6 S  M
lineages in the absence of ER71, consistent with the previously
described phenotypes (Ferdous et al., 2009; Lee et al., 2008).+ O0 a" H5 A8 x) W  J5 f
Transcripts that were significantly upregulated in the Er71 mutant. }1 n0 j8 B$ K( f- D8 ]
EYFP+ cells compared with the Er71 wild-type EYFP+ cells were4 l2 g3 N" |* u7 l' v* p
associated with muscle lineages (Fig. 4B). These transcripts1 w+ s8 L+ x1 f- ?6 {. ]' ^1 d
included Smarcd3, Myl3,  Irx4, Tnni1, Myl2 and Tbx5 (cardiac-
restricted transcripts); Acta1, Myl1, Svep1, Dpf3 and Cdo1 (skeletal
muscle-expressed transcripts); and  Acta2 and  Myl9 (vascular+ B& @9 H: s4 n; w- }$ \
smooth muscle-specific transcripts). These results suggested that# B. R6 |" p# m- {* u  _
the EYFP+ cells gave rise to other mesodermal lineages in the
absence of ER71.
In order to determine whether the cardiac lineage was broadly
affected, we analyzed transcripts from the cardiac development8 [0 [  x: b. F! z: }
gene list in gene ontology (GO) terms and performed a hierarchical
clustering analysis. Similar to the results obtained from whole-4 S- f7 L" b3 @) \
genome analysis, wild-type EYFP+ cells segregated from the
mutant EYFP+ cells (and all EYFP–% M9 ^  d9 p" ^" N( w
cells) (supplementary material
Fig. S4A). This result demonstrated that the difference between
these two populations can also be detected based only on cardiac
development criteria. Some transcripts were decreased in EYFP+
Er71 mutant populations compared with EYFP+ wild-type cells,
including a number of transcripts that are important for endocardial
development (including  Eng,  Hhex,  Sox18,  Nfatc1,  Sox17)( f" Z7 d- L2 u
(supplementary material Fig. S4B). A number of transcripts were9 V) G+ w4 |/ q/ `
increased in expression in the EYFP+ Er71 mutant population' I1 G) @* ?1 \/ k/ d
compared with the other three groups (EYFP+ wild-type and both! n; g4 L8 \3 o  w/ m. T3 l1 k
EYFP–6 i0 H$ N& N+ Q4 k: w; ~
populations), including a number of cardiac transcription
factors (Myocd, Nkx2-5, Irx4, Isl1, Gata6, Tbx20, Tbx5, Gata4,. T  g- ^, `. B, W; |/ K) Q1 a7 d
Smyd1 and Srf) and structure-function transcripts (Myl2, Myh7,
Myl3, Tnnc1, Tnnt2, Tnni3) (supplementary material Fig. S4C). We2 f% n: J3 u" o
validated changes in selected transcripts by qPCR on independent$ t/ N9 V: Y* m* e, X1 _
samples and confirmed decreased expression of Er71, Tal1 and
Cdh5 and the overexpression of cardiac transcripts including Nkx2-, J/ q7 `- `% h" V7 L2 z! h
5, Gata4 and Tbx5 in Er71 mutant EYFP+ cells (Fig. 5).' D- F3 d" x! p  ?5 @+ B5 g
In summary, our transcriptome analysis indicated that cardiac
genes are over-represented in Er71 mutant EYFP+ cells, whereas
endothelial, endocardial and hematopoietic genes are under-, Y6 A/ }, f% V; _: r- l+ F' L
represented./ [% g' C. p6 U) r
A novel Er71-Cre transgene marks endothelial and& Z3 Y1 W1 j. |- A1 p4 u6 y6 l" D
hematopoietic lineages
In order to determine whether ER71-expressing cells give rise to
organs or programs other than the endothelial and hematopoietic7 k& q" a  F5 x" D4 k
lineages, we generated an Er71-Cre transgenic mouse model (Fig.
6A). We obtained a total of six founder lines, with embryos that4 P' I1 V  t+ {% o7 U
showed similar expression patterns after breeding with the Rosa-
4807 RESEARCH ARTICLE ER71 governs progenitor fates
Fig. 4. Transcriptome analysis reveals that hemato-endothelial) N* s; q* J! l( m' c
and cardiac lineages are dysregulated in the Er71 mutant
embryo. Transcriptome analysis was performed on EYFP+ and EYFP–
cells sorted from wild-type and Er71 mutant littermate mouse embryos.
(A)Clustering analysis shows that wild-type and mutant EYFP–
cells are
indistinguishable, whereas EYFP+ cells from wild-type and mutant0 [+ Q* E4 I* R) e1 c$ d. ?
embryos show distinct gene expression profiles. (B)Transcripts/ C& M. N0 ]  i
upregulated in the EYFP+ mutant progenitors include cardiac and
skeletal muscle genes. (C)Downregulated transcripts in the Er71
mutant background include hemato-endothelial transcripts, including6 i$ O9 D' _2 ?7 V) h* T6 X
Er71 (Etv2) (arrow).6 Z  J& ^( a4 L% }
DEVELOPMENT4808
lacZ reporter mice (supplementary material Fig. S5). Two
transgenic lines were crossed to the Rosa-EYFP reporter mice and+ t. u; o# ^6 I. @
used for immunohistochemical and FACS analyses. Our results5 F0 j" I4 Z' f( v9 Q
revealed that E11.5 embryos were marked by Rosa-EYFP or Rosa-2 X) d* @. f4 H
lacZ in the endocardium, cardiac cushions, vasculature,
mesenchyme and the fetal liver, which is the stage-appropriate site
of hematopoiesis (Fig. 6B; supplementary material Fig. S5G,H;& A4 s" s$ X" u
data not shown). Hearts of P3 neonates were marked by Rosa-6 J; ]% H7 u- m* g- `5 `" U
EYFP in the vascular structures (Fig. 6C). Co-staining for EYFP6 N& k/ K  a4 O; K& }, [3 d
and desmin, an early muscle-specific structural protein, showed
that the Rosa-EYFP reporter did not overlap with desmin staining: g1 e; A8 g! G# ~
in the heart at either time point (Fig. 6B,C). FACS analysis of
whole E11.5 embryos showed that 7-8% of cells in the E11.5
embryo were derived from ER71-expressing cells (Fig. 6D,E). Of: n# @* s! X) A% D! [. G
these EYFP+ cells, ~12% were endothelial (CD45–! S1 E9 A  G: h- d+ ^
CD41–. f. x3 Z. m" W, Q+ C- K
Tie2+;( k$ P) w% }) G8 g
Fig. 6F), 23% were non-erythroid hematopoietic cells (CD45+, also6 b9 P3 h+ Y/ C. y3 p' u
RESEARCH ARTICLE Development 138 (21), k. `8 ?4 L0 o$ h; [
Fig. 5. Representative transcripts from the cardiac program are overexpressed in the Er71 mutant. (A-F)qPCR analysis was performed on" ^+ J" _: O) ~5 E& B
RNA isolated from EYFP+ cells in the wild-type (embryos 1 and 2) and mutant (embryos 3 and 4) backgrounds. We observed the loss of Er71 (A),
Cdh5 (B) and Scl (C) expression and an increase in the expression of representative cardiac program transcripts including Nkx2-5 (D), Tbx5 (E), and. ?7 k( I4 ^# j
Gata4 (F). Error bars indicate s.e.m." t/ @& t1 d) u, P6 f  T9 p
Fig. 6. Genetic fate-mapping studies indicate that ER71-expressing cells give rise to the endothelial and hematopoietic lineages.
(A)The transgenic Er71-Cre mouse model used for these studies. (B,C)Immunohistochemistry for GFP (green) and desmin (red) and DAPI staining
(blue) in E11.5 (B) and P3 (C) heart. a, atria; bw, body wall; cu, cardiac cushion; fl, fetal liver; v, ventricle. (D)A representative FACS profile showing7 ?% x4 |* _, r$ u
EYFP+ versus side scatter width (SSW). (E)

ercentage of Rosa-EYFP+ cells from all experiments (n9). (F-H)EYFP-positive cells were gated and
analyzed for lineage contribution. Representative FACS profiles are shown for (F) endothelial lineage (CD41–
CD45–
Tie2+) cells and (G) CD41+. M$ Y4 ?1 q1 c, ]
CD45+, (H) CD45+ Ter119–
and Ter119+ CD45–8 g: T; C+ Q" r. D
hematopoietic lineage cells. (I)

ercentage of Rosa-EYFP+ cells of each lineage from all experiments
(n9). Error bars indicate s.e.m. DEVELOPMENTknown as Ptprc; Fig. 6H), and 13% were erythroid (Ter119+; also
known as Ly76; Fig. 6H). Interestingly, the EYFP+ population
segregated into two populations of varying EYFP expression (Fig.
6D). Ter119 primarily marked the EYFP-low population, whereas+ G+ a" j, d- `& a7 g) `3 j' T1 i
endothelial and other hematopoietic markers primarily marked the& w$ k2 U! _  j, s
EYFP-high population (data not shown). This suggests that the) f! ^6 n  ]' ^  L6 C. R9 o
Rosa reporters are expressed weakly in erythroid cells and might& e) Q( @+ @' A# ]
therefore inefficiently mark this population./ J3 B; z4 ~! a' o6 W2 s  C
ER71-EYFP-expressing cells give rise to0 }* J1 c7 U2 V6 \) i% @8 @) k0 x
myocardium in the Er71 mutant
To determine whether ER71 reporter-positive cells could produce
alternative lineages in the Er71 mutant, we crossed the Er71-Cre
and  Rosa-EYFP alleles into the  Er71 mutant background. As, u1 z. ?8 Q: V: ]. g. b
observed with the ER71-EYFP reporter, Er71 mutants carrying the+ O0 [: {' K6 O5 y
Er71-Cre and  Rosa-EYFP alleles displayed altered EYFP. U& p: d8 u; W
expression patterns. Compared with the Er71 wild-type embryos at# G) ~- V* d% n
E8.0, in which Rosa-EYFP is expressed in the yolk sac, vessels and" V% |/ E: T' f; x6 H3 j
heart tube (Fig. 7A), the Er71 mutant embryos had robust EYFP/ z: G4 ?4 h" {
expression localized in the allantois, posterior mesoderm and
paraxial mesoderm (Fig. 7F). We speculate that the increased- ^* w7 ^0 g9 v
expression in the allantois and the posterior mesoderm is due to the4 z' r, H7 @4 E1 J) Z' v
progenitor cells differentiating to alternative mesodermal lineages,5 n  l2 |) v9 A: _  n4 c
perturbed migration patterns, or a combination of the two. Embryos
were analyzed using immunohistochemical techniques for co-5 _  x" s4 J6 v& W+ @; ]
expression of EYFP and Nkx2-5, a cardiac transcription factor,- P  E) `2 a9 w. Q  m: t+ A
EYFP and desmin, or EYFP and troponin T, a cardiac sarcomeric
protein. In wild-type hearts at E8.0, EYFP was expressed in the
endocardium, the major vessels such as the dorsal aortae and in1 x8 u7 \7 f( i: D
hematopoietic cells within the heart (Fig. 7B-E). There was no
overlap of EYFP expression with Nkx2-5, desmin or troponin T.+ {0 K# d% v' h
In the Er71 mutant embryo, the vessels and endocardium were
absent. However, EYFP expression persisted in the heart. In the7 [8 P' w$ ]# a
heart of the Er71 mutant embryo, all EYFP-expressing cells had an
Nkx2-5-positive nucleus (Fig. 7G,H). Some of the EYFP-
expressing cells were also positive for the structural proteins! D7 V2 g" b5 c7 v
desmin and troponin T (Fig. 7I,J, arrows). These results support the$ W/ k+ R) u0 u1 M
notion that the EYFP+ cells are capable of differentiating to the" Y  ?* ?# ?1 M  `4 [
myocardial lineage in the absence of ER71. It is likely that6 `" ]5 G# b: [' }0 m4 j" h
progenitor cell populations are also capable of differentiating# u6 q4 L# N; B
toward other lineages depending on the context of the spatial and
temporal cues that they are exposed to during embryogenesis.
However, the heart is one of the few organs present at E8.0 and( U/ T. ~; |! a
Er71 mutant embryos are nonviable by E9.5. Therefore, the
lethality of the  Er71 mutant limits our ability to evaluate the- q  }" S. P- r5 }1 |
contribution of EYFP+ progenitors to other lineages that develop5 e! Y5 U2 z) A& Y5 N
later during embryogenesis.
ER71 overexpression suppresses development of
the cardiac lineage
To complement our analysis of the  Er71 mutant embryo, we. n4 t) B) }5 W
overexpressed ER71 using a doxycycline-inducible embryonic
stem cell (ES)/EB system. An ES cell line in which expression of
ER71 can be induced by doxycycline treatment was generated by
a cassette exchange method (Iacovino et al., 2009). ES cells were
induced to form EBs in differentiation media and cultured for 10
days with or without 0.5 mg/ml doxycycline from day 3. In the. f' L- x6 U! B. K
ER71-overexpressing EBs, we observed a decrease in the
expression of troponin T, which we quantified by comparing the
positively stained area with the total area of the EB (Fig. 8A-E).3 r9 z# B" d1 o& f6 R8 d! l% G
We also observed a decrease in cardiac gene expression (Nkx2-5,( l4 G' j+ e% ]. @
Tbx5 and  Gata4), as quantified by qPCR analyses (Fig. 8F).! O. z. Z2 z% N  d: v. u9 m2 p/ e' B
Finally, we quantitated the ability of the EBs to beat with edge' }! J. }: S8 ]; t0 J/ g
detection. The doxycycline-induced EBs had a dramatic reduction
in contractility (Fig. 8G) and in percent shortening (Fig. 8H).
Collectively, these results further support the notion that ER71
suppresses the myocardial fate of mesodermal progenitors.3 q' H. ^: w1 G# i2 D
DISCUSSION
An array of signaling cascades and networks govern the fate of
progenitor cell populations and their contribution to mesodermal
lineages during embryogenesis. Recent studies have demonstrated% ~$ D$ H5 Y. t& [6 U8 ]
4809 RESEARCH ARTICLE ER71 governs progenitor fates5 P  q8 N, Q7 c" z* r0 C. n( X, U
Fig. 7. Genetic fate-mapping studies in the Er71 mutant show that the affected lineages give rise to myocardium in vivo. (A,F)Whole-1 F) ~' W& o* `5 q
mount images of Rosa-EYFP expression in Er71 wild-type (A) and mutant (F) mouse embryos. The red line indicates the plane of sectioning for the
corresponding panels. (B,C,G,H) Immunohistochemical analysis of EYFP and Nkx2-5 shown at low (B,G) and high (C,H) magnification. (D,E,I,J)+ u5 v1 c& p  a
Immunohistochemical analysis of EYFP and desmin (D,I) or troponin T (E,J) expression. ys, yolk sac; h, heart; al, allantois; da, dorsal aortae; ec,
endocardium; mc, myocardium. Arrowheads indicate EYFP colocalization with desmin or troponin T. Scale bars: 200m in A,F; 20m in B-E,G-J.6 A0 Z2 i  r( y4 l( |
DEVELOPMENT48109 n* `* H1 z7 _+ t2 ?* j6 _
that various progenitors give rise to multiple mesodermal lineages.
This broad developmental capacity is evident from the fact that4 b, U2 W  @2 q% J  M3 G1 Z6 b
multipotent progenitors have been shown to differentiate to diverse+ j* y: ~" T: C
lineages, including hematopoietic, endothelial, cardiomyocyte and
smooth muscle lineages. We have previously demonstrated that
ER71, a member of the Ets transcription factor family, is essential
for the development of mesodermal lineages. Er71 mutant embryos
lack hematopoietic and vascular lineages and are nonviable. Here,
we have further defined the role of ER71 during mesodermal
patterning and have made three new discoveries that advance our
mechanistic understanding of mesodermal fate decisions during1 w! c, L! _4 H& r0 d+ n: q
development.
First, we have defined the expression pattern of ER71 during- ?4 f5 G  M$ u/ |. q# O- p
embryogenesis using a newly generated transgenic reporter
mouse model. Using immunohistochemical techniques, we6 g& j: K8 O6 w; ?3 O; k1 G8 c: ~
demonstrated that ER71 is largely co-expressed with Tie2 in
endothelial and hematopoietic progenitors. In the absence of
ER71, we observed that these progenitors are expanded in
number but do not represent the hematopoietic and endothelial9 E" @: d/ [% R2 q) C2 l6 ^, y
lineages; rather, they are redirected to other mesodermal; ~& S0 I6 |2 m5 a& N+ K' V" y+ x
lineages, including the cardiac lineage. These data also show that
ER71 is not required for the initiation or maintenance of its own
expression. In the absence of ER71, this increase in the number' W. e4 Q+ Y# Q1 E3 T
of progenitors (EYFP+ cells) might represent a delay in cell( L  ]# x" @$ d  ]7 J2 {" Y
cycle withdrawal due to the lack of distinct mesodermal lineages
(i.e. hematopoietic and endothelial lineages) or, alternatively,
decreased programmed cell death involving these progenitors.* _# _- q% v) U* W3 l8 U" V% E
We did not observe an increase in apoptosis in the Er71 mutant
embryo compared with its wild-type littermate control (Ferdous' d0 C- R7 U( D6 [# N" e
et al., 2009). Thus, we hypothesize that the progenitor cell
expansion in the absence of ER71 is a result of the lack of
cellular differentiation to hemato-endothelial lineages.
Our second discovery defined the fate of the ER71 progenitors/ C, t: F: O2 [& c% \! U- X
in the presence and absence of ER71. Using genetic fate-mapping8 J% D) C7 ]8 J+ d9 b
strategies, FACS and whole-genome analysis, we demonstrated that1 @9 _+ C) j, P
ER71 progenitors give rise to hematopoietic, endothelial and: }" _8 `' u; n. i  N
mesenchymal lineages. In the  Er71 mutant background, these# d9 u/ I" a. _3 p$ h
progenitors (EYFP+ cells) produced other mesodermal lineages,
including the cardiomyocyte lineage (supplementary material Fig.2 M2 M. r" i: U: G5 g* X0 y9 n, d
S6). Moreover, our FACS and transcriptome data supported the
notion that paraxial mesodermal programs (transcripts enriched in
the skeletal muscle lineage) were increased in the Er71 mutant
progenitors. Accumulating evidence suggests that the cardiac and: U9 v$ B! [) j5 Z* D7 p
hematopoietic lineages develop from a common progenitor and that
the specification of these lineages is inversely regulated. We
recently demonstrated that Nkx2-5 has a dual transcriptional
regulatory role during embryogenesis as it represses Gata1-
mediated hematopoiesis and promotes cardiogenesis of the
multipotent mesodermal progenitors (Caprioli et al., 2011). The! [: D/ _8 b1 w; E! k! y3 m- l+ }! c
results of the present study further support the plasticity of the$ w- K! I$ g3 Z) b8 B0 R3 d- C
ER71 progenitors and their capacity to respond to cues and
contribute to other mesodermal lineages.  N% {  d2 E# E4 m$ p
The origin of endocardium relative to the myocardium has: m- J. ?& i& R. M+ @& @
been controversial (Harris and Black, 2010). Studies in zebrafish
and chick suggest that endocardial and myocardial lineages are; N8 K* p1 A0 Z/ K* u
distinct from the point of gastrulation, and there are no9 ^; x1 _' S& V( F) s, L1 c; E
multipotent progenitors (Cohen-Gould and Mikawa, 1996; Wei8 l  o1 u4 G. S9 B4 ^5 J
and Mikawa, 2000). By contrast, lineage-tracing studies in
mouse embryos and ES cells suggest that endocardial
progenitors are specified as multipotent progenitors in the7 G9 d2 W7 g' t
cardiac crescent and cell fate is later specified to either$ w3 q, p* ?9 C! h9 z7 S. W
endocardial, myocardial or vascular smooth muscle fates1 _+ U8 Y$ t5 d( i, r
(Kattman et al., 2006; Misfeldt et al., 2009). In our novel ER71-) d; y. i- ^5 Y& E
EYFP reporter mice, we observed weak co-expression of Nkx2-6 n/ C6 j7 M1 J
5 protein and EYFP in the cardiac crescent at E7.75. However,# W0 G' B3 W: {; r$ s2 s
lineage-tracing experiments showed that cells that expressed
ER71 during any point in their development did not become
cardiomyocytes. By contrast, Nkx2-5-Cre lineage-tracing' B5 E- s4 N$ f9 [5 \
experiments show that at least some of the endocardial lineage% H+ R" S. R6 M
derives from cells that expressed Nkx2-5 (Stanley et al., 2002).
Taken together, these observations favor a model in which
common progenitors of endocardial and myocardial lineages are
specified as Nkx2-5+ cells in the cardiac crescent, but those that
RESEARCH ARTICLE Development 138 (21)
Fig. 8. Overexpression of ER71 in differentiating EBs inhibits  E; z; D" o7 f/ X
cardiac development. (A-D)Sections of representative EBs either
untreated (A,C) or induced to overexpress ER71 (B,D) stained with
Hematoxylin and Eosin (C,D) or anti-TnT (A,B). (E)Quantitative analysis of! \, a6 ]0 B5 U& w2 j
the troponin T-positive area in 33 induced (Dox) and 42 untreated* N# ]% ?* w( ~4 z1 Q3 Q
sections. (F)qRT-PCR confirms that representative cardiac program/ v5 C& O9 s" o2 J" ]" P; e
transcripts (Nkx2-5, Tbx5, Gata4) are downregulated in ER71-
overexpressing EBs. (G,H)Extent of contraction and relaxation after 26 r. E* G2 w1 Y7 }, a' k4 m
seconds of stimulation (G) and percent shortening (H) are reduced in9 t0 P9 P6 _1 ^( A5 a
ER71-overexpressing EBs. ****, P<0.001; *, P<0.05. Error bars indicate4 s. P0 M: Z, h/ R
s.e.m.4 A, N8 [6 @& u( y" {
DEVELOPMENTwill become endocardium will downregulate myocardial( o+ ?; w0 }: w# \& Y4 _
potential, including the expression of Nkx2-5. Once ER71 is
expressed, the fate is restricted to endocardium, and not0 j2 x' s  m& n; G( b) C& _! L
myocardial lineages. Future studies are warranted to decipher the
mechanism that governs the specification of endocardial cells4 k0 q, m8 o' J0 q, E5 j( Q; l3 a* \
within the cardiac progenitor pool.7 K" O+ u+ ]' T1 H1 I
Our third finding is that ER71 overexpression repressed cardiac
potential. These results support the notion that ER71, like Nkx2-5,
has reciprocal and overlapping dual roles in the specification of  c/ u+ L/ f1 O6 j
mesodermal lineages. Our previous studies demonstrated that* T9 Y, B( v7 P( Q
Nkx2-5 overexpression suppressed hematopoiesis, but not the
endothelial program (Caprioli et al., 2011). We further defined that0 W/ _  H. s5 c2 m! V
Nkx2-5 could, in a context-dependent fashion, transcriptionally; Q& i6 g9 p# m
activate  Er71 and promote endocardial lineage development
(Ferdous et al., 2009). Collectively, our previous studies and recent
results support the notion that distinct transcriptional networks can,
in a context-dependent fashion (and dependent on the local
environment), activate a hematopoietic or endothelial program and& l& X' q) ~* ?' W
antagonize the cardiac program. These studies further emphasize* A" Y# P& |+ p% u2 h, h
the plasticity of progenitors and their contribution to distinct7 _4 t5 P3 g$ y
lineages.
In summary, our data support a dual role for ER71 in the
specification of the endothelial and hematopoietic lineages and the! I: Z: J! C2 Z( W$ s9 M
suppression of other lineages (i.e. myocardial and paraxial
mesodermal cell fates) (supplementary material Fig. S6). Our
studies further support the notion of multipotent mesodermal; B' M8 \5 n, D0 D
progenitors that are dependent on transcriptional cascades and other3 J" ~+ X: f8 M+ \
cues to direct them to populate one or more lineages. Moreover,9 Z+ q- p& t1 b" }
our studies reveal that global deletion of a single gene (i.e. Er71): L; k- L: G5 Y6 \: ~$ g+ n
aborts one or more lineages and redirects the cells to other lineages
that would not typically arise from those progenitors. These studies- Y7 z$ Z( G7 i9 D% ^" k6 r- a, `
further our understanding of the regulatory mechanisms that govern# C% t7 v' W% Y  t. W9 e, V3 h
mesodermal cell fate decisions and embryogenesis." d/ @9 P, ]" P8 O
Acknowledgements
We thank Jennifer L. Springsteen and Alicia M. Wallis for assistance with- m, {7 `) R1 |' N; ~
histological analyses and animal care.3 H1 ]8 c) }7 s0 [. G2 m6 I3 h
Funding
Funding support was obtained from the National Institutes of Health [U01
HL100407 and R01 HL085729 to D.J.G.]; and the American Heart Association
[Jon Holden DeHaan Foundation 0970499 to D.J.G.]. Deposited in PMC for6 z" F8 U' ~: g! N7 }" m
release after 12 months.. h. {5 f- S3 e- s# @
Competing interests statement
The authors declare no competing financial interests.
Supplementary material$ u% R6 E4 _! u0 O8 r2 r; e
Supplementary material available online at
http://dev.biologists.org/lookup/suppl/doi:10.1242/dev.070912/-/DC1- |1 o! A" a) L* _9 ^% {. F3 y
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RESEARCH ARTICLE Development 138 (21)
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