求助Development. 2011 Nov;138(21):4801-12.文献

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Development. 2011 Nov;138(21):4801-12.
- ^' e0 I5 c) ^0 e) S. Y3 M% \ER71 directs mesodermal fate decisions during embryogenesis.( D. `0 d! k. }; U$ `$ G
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.5 R1 j! Y" c" r6 x* l& ~$ M/ r
SourceLillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA., Q5 K4 d9 u& }8 T2 V! T: s
; t( P/ K' Y7 k# h, c' {
PMID: 21989919' r5 Z* j: l! k9 d* z
http://www.ncbi.nlm.nih.gov/pubmed/21989919

wang1234611

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)

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; v6 S- g5 z! d8 z. a5 [4801 RESEARCH ARTICLE* k4 D2 W. l/ K1 E/ W& n
INTRODUCTION
% P- J+ P2 d. ^& FTranscriptional networks, signaling cascades, microRNAs and
! W4 S' t: J( F+ S5 g4 m+ E; a- Q9 wother factors coordinately regulate stem and progenitor cell
7 m- w* I" {" Ppopulations to give rise to the mesodermal lineages (Davis and Zur
! Y# d7 n! `! g+ A0 fNieden, 2008; Ivey et al., 2008; Omelyanchuk et al., 2009; Park et
1 l4 h" i; W4 }al., 1998). During embryogenesis, a set of anterior progenitors
& {3 o- @. b# o3 f- qcoalesce to form the heart. These progenitors, along with the blood
: v6 f/ Z* F% p& E( r0 l  Vand vasculature, comprise the circulatory system, which is the first' r  Y4 m* ]# N
organ system to develop in mouse and human. Disruption or7 O: z  j3 ]$ p  N
elimination of any one of these lineages results in early embryonic' S8 a6 u+ o# l
lethality. However, the factors that regulate the co-emergence of# L0 L. |+ D7 C6 Q' `$ P8 c/ t
these lineages from the mesodermal progenitor pools are2 R) u* l- E3 N
incompletely defined.3 P+ s3 m' g2 l' G8 g. {  t; v4 L
Recent studies have identified a multipotent progenitor cell
. D$ j- Q3 ?, B0 Z0 mpopulation that is capable of giving rise to the cardiomyocyte and
. f3 P( V9 a7 m, z5 _endothelial lineages during embryogenesis (Kattman et al., 2006;
; A$ ^$ u) I' h' c7 Z1 w3 v& ~Moretti et al., 2006; Wu et al., 2006). Although these multipotent1 E" n! n; y- B( a
progenitors have been identified based on the expression of Flk1
2 R* s7 g2 k' u(also known as Kdr), Nkx2-5 and Isl1, the transcriptional networks( X; U6 c( c  K$ R9 m, M) b
that govern the fate of these progenitors are unknown. These and
. T8 C8 X* E+ J8 d& D, {- Qother studies emphasized the plasticity and context dependence of% v' n( ~+ W. t) j  C
regulatory cascades in the progenitor cell populations. For example,& t3 `' W4 @1 F
our previous studies demonstrated that Nkx2-5 has a dual
" E3 |5 ?) a8 M9 O' ltranscriptional regulatory role in the promotion of the cardiac" x" a& g" t% B, u
lineage and suppression of the hematopoietic lineage (Caprioli et' P) A8 k4 D0 k/ z$ t% U
al., 2011). In this fashion, we proposed that the stoichiometry of
* ]$ H& m. h1 u6 H1 `! F) oregulatory transcription factors and interacting factors could, t8 P* O" [  ?) ^9 A" G
dynamically regulate the fate of progenitors. Definition of
+ I$ T$ y( r4 m( J+ ~additional networks and signaling pathways that promote and
' X, i& M0 k  W4 W9 J  esuppress the fate of distinct lineages that arise from multipotent
* V* K- d9 ?  I" m, cprogenitors will enhance our mechanistic understanding of their. q0 _- \, c. Y
developmental potential.
7 C: p" G( {/ T, A. J( B* c: j- @Ets-related protein 71 (ER71; Etv2) is a member of the Ets+ x  X5 j) h! {, D
transcription factor family that we and others have shown to be
  J4 k3 J2 S& f' ]; Q1 K' Wessential for formation of the endothelial and hematopoietic
/ K, {1 {: U' z' {1 A. Llineages (De Val et al., 2008; Ferdous et al., 2009; Lee et al., 2008).
  d  L. I+ D8 ^' E/ C- O/ dEr71 can be induced by BMP, Wnt and Notch signals to promote/ p# U' t8 j7 R4 j% K5 g
hematopoiesis (Lee et al., 2008) and synergizes with FoxC2 to
/ h' `5 u2 x9 [, G+ jregulate the endothelial program by directly targeting Scl (also4 v7 }* r" N; ~$ T1 I0 w
known as Tal1), Notch4, Cdh5 and Tie2 (also known as Tek) (De2 U( E4 X0 F9 M( J
Val et al., 2008; Ferdous et al., 2009; Lee et al., 2008). In Er71! v; s; B% j1 b& B' [: l. v; b
mutant mouse embryos, there is a complete lack of hematopoietic9 l" T2 ^8 b) {! l; N
and endothelial lineages and the embryos are nonviable by$ h" r4 W8 K, u2 I2 j4 q  N. O# C
embryonic day (E) 9.5 (Ferdous et al., 2009; Lee et al., 2008).7 R1 j- c, K. [. K$ d
Analysis of the Er71 mutant embryos revealed no differences in
9 o' X4 d1 Y0 {& Z! ], [cellular proliferation or cellular apoptosis compared with age-' e0 s. }+ ]# g4 `
matched wild-type littermates (Ferdous et al., 2009). These results" [" I6 S- s. p$ a  R8 [
raised the question of whether the hematopoietic and endothelial
9 a4 g/ L2 L0 }6 D( K" H. e/ kprecursors are still present but unable to differentiate to hemato-
" Q- K4 ~0 I, w0 Y( r0 |8 ~8 Pendothelial lineages, whether they had been redirected towards3 ^) y: ^. K% S2 H8 S3 @. J( C
other lineages or whether these cells never arose during* r9 V7 Q5 q6 i
development.* X' C+ m( C5 f5 V' e4 ]8 m! J
Development 138, 4801-4812 (2011) doi:10.1242/dev.070912
' a$ X5 R# A9 k  E" N© 2011. Published by The Company of Biologists Ltd5 y- f6 n- `; k# T, E& e  G
1Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.
; P1 u( t8 X9 w3 B2Department of Integrative Biology & Physiology, Medical School, University of. ~, j( v6 ~2 r; ^9 E
Minnesota, Minneapolis, MN 55455, USA. 1 ^# w/ n" Z  R; r9 J8 x$ I' S
3Biolead.org Research Group, LC5 B' Q3 K, R) X! _$ }" F! S( q! q
Sciences, Houston, TX 77054, USA.   h; y8 i5 j+ Y2 T5 ?- N8 u+ }2 l& k
4University of Texas Southwestern Medical
" i! j" }) k- I! f% r% zCenter, Dallas, TX 75244, USA.* x, N4 `  M: w6 J5 O
*Author for correspondence (garry@umn.edu)5 Q& c- J+ v7 w% h- _. F
Accepted 5 September 2011
7 ^' b0 n1 G7 p$ f5 Y7 P+ X# P% XSUMMARY
. w. X7 |! a) p  WEr71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for
- d/ A* ]9 y; w" J( l6 |6 BER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse
+ {# ?4 }+ c! W. ~, pmodel by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the
& `8 L4 W0 ?, W! M/ J. f8 \EYFP+ population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the
7 h7 D8 H" x, A$ l5 N* `8 R8 @number of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-; R$ W; f$ S* W0 x) ]4 B' P2 u
endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse0 k2 V3 ]4 M7 t1 G. |+ P. g
model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the$ x' A+ L# @/ g7 S! O( m4 B
hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed2 g5 C# H- Q: A/ z! ?& Z6 P
to alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic
* W6 u& e' ^8 G/ y8 k, w4 Nstem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER71- F* b; ]( F/ s; K8 Y4 z
as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense3 k& L; }, U; ?! f1 w8 e/ l
of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during
1 z6 W( b1 T" x- gembryogenesis.% N  M, _# N4 c1 }+ z' J, l. q
KEY WORDS: Transgenesis, Er71 (Etv2) knockout, Mouse
7 D/ v/ v6 E* M+ n- q" eER71 directs mesodermal fate decisions during- O* `) o4 `/ k, s3 W  n
embryogenesis* \0 `0 h, ]# d8 v% T/ F
Tara L. Rasmussen1, Junghun Kweon1, Mackenzie A. Diekmann1, Fikru Belema-Bedada1,2, Qingfeng Song3, / [1 H# _# Y3 K$ L2 B' X$ f
Kathy Bowlin1, Xiaozhong Shi
& T! p6 K$ z: x, p8 w9 u% {; y1, Anwarul Ferdous4, Tongbin Li+ M2 v6 T$ _5 t  X) v9 g
3, Michael Kyba1, Joseph M. Metzger1,2, : L: r' i4 j( j7 d$ _( Q
Naoko Koyano-Nakagawa1 and Daniel J. Garry1,7 Y% b: G" H3 Q" M3 q, N
*+ P& r3 t+ s. }7 f3 W. |: ^- w
DEVELOPMENT4802
% ^8 D3 w, e0 P/ bIn the present study, we have dissected ER71-mediated$ x6 `% f5 Y# }% b3 ?
mechanisms that govern fate determination during murine
3 j+ B. ^4 n3 ]; Eembryogenesis. We have generated a novel transgenic mouse# X" E8 E/ V8 {, q( i$ s
model in which the 3.9 kb Er71 promoter drives the EYFP reporter' [6 L% p% P; U  I3 k
(Er71-EYFP), and crossed it into the Er71 wild-type and mutant4 V  x: G+ |1 ?
backgrounds. This strategy facilitates the isolation and; {: }. L1 E; m$ `& i& w$ H
characterization of cells that would normally express ER71 from
& f$ ?2 q+ X) z, Y; J6 ]) s) m  [9 fEr71 mutant embryos. In the absence of ER71, we observed the$ H# q" W/ d. J* u/ _
conversion of cells that would normally give rise to lateral plate; `" o8 {" H( f2 B8 {5 u
mesoderm into cells that produce paraxial and cardiac mesoderm.) ^/ |' C7 A! p2 k
Furthermore, ER71 overexpression in vitro using an inducible7 Z  _9 C+ l& N7 l8 |! k; y
embryonic stem cell/embryoid body system demonstrated- p8 v. M7 G6 n! |2 w$ {9 P
decreased cardiogenic potential. Collectively, these data
% q. Q2 k- H9 w6 m6 bcomplement and extend our understanding of the functional role of# ?; Z# j) g+ g- j
ER71 to differentially promote mesodermal fate decisions during
, t; V$ u& Q* G$ W5 Uembryogenesis.. J! M/ |- O; |0 z  [" I5 F
MATERIALS AND METHODS, S  w' s6 d$ [  k& k- q9 d5 a
Generation of transgenic mice
6 V3 c) ?+ ~2 E) I0 G( J5 u8 [: @The 3.9 kb  Er71 promoter (Ferdous et al., 2009), which harbors the. s! C3 ^1 T3 K1 c7 j/ X
modules necessary to direct the temporal and spatial expression of ER71,
3 u+ S2 ]+ q5 A& Nwas cloned into the pEYFP-1 vector (BD Biosciences) and into a8 P# `# M, V6 {- e  k" b
promoterless pBS-Cre-pA vector to generate the ER71-EYFP and ER71-2 [$ x( [, ~) O, W- @5 v# S
Cre constructs, respectively. Transgenic animals were generated at the
( ~  g# J  C3 ~4 ^, u" q' D9 wUniversity of Minnesota Mouse Genetics Laboratory using standard. W" w6 n. }8 O
methods. Transgenic mice were screened for DNA integration by PCR.
: V, h& X8 }0 uExpression analysis of ER71-EYFP lines was performed by examining
5 F! u" u" ]3 w* U8 P8 qE7.0-9.5 embryos resulting from timed matings to wild-type CD1 mice( S, D. t' u" h, y! N7 P# f) p
(Charles River). We also analyzed E11.0 and postnatal day (P) 3 offspring
1 z% C* s0 E; [& |% H) |of timed matings of the ER71-Cre lines and the Rosa-EYFP line (Jackson
# v% u1 w8 h3 ]: o+ ~- QLabs 006148). Founder mouse lines were crossed to the  Er71
) T. C: Y& g* ^6 j4 _heterozygotes previously described (Ferdous et al., 2009). In all cases, at% D( R* O* x& s  F* Z3 ^
least three transgenic lines were examined to confirm similar temporal and( @& [4 U' ~+ |' y- p4 o' a! e
spatial expression patterns. All mice were maintained at the University of" S3 K1 E- k  _- ]2 P" a- |% G  J% J
Minnesota under protocols approved by the Institutional Animal Care and
8 T; V: q% y4 l7 f6 ^- U  G; \Use Committee and Research Animal Resources.# h( X' v( v0 |7 i8 A
FACS analysis
# n$ f3 s/ z# nEmbryos from timed pregnant females were harvested at specified stages.1 \2 [4 q2 s$ j, m
Embryos were separated from yolk sacs, which were used for genotyping,0 v7 y& u1 B, @) |. M
and imaged on a Zeiss Axio Observer Z1 inverted microscope prior to
3 W+ {9 @# S- \, j+ T4 |6 y6 odissociation. Embryos between E7.5 and E9.5 were digested in 30-50 l2 N0 A6 J( l: I* O. O4 L) l5 e, l
0.25% trypsin plus EDTA at 37°C. Digestion was arrested with 500 l
$ R: C- X6 B4 d5 dDMEM containing 10% fetal bovine serum (FBS). Cells were pelleted at+ L% A  y2 S$ r4 s+ q0 F. H
4°C (1600  g), resuspended in PBS and passed through a 70  m cell
/ a% S: z$ v' j5 W" d" lstrainer. Cells were incubated with antibody cocktails for 30 minutes,
3 s9 G$ S4 v6 Z' _9 j- f4 zwashed, and resuspended in PBS. Cells were analyzed or sorted using a
2 _$ j1 ]7 E. J' AFACSAria (BD Biosciences). Various combinations of antibodies (1:1000)& ~% D. M4 F! P3 S
were used, including Flk1-APC (eBiosciences 17-5821), Pdgfra-PE! |8 b0 Y' m2 J. @" w+ F: X
(eBiosciences 12-1401), CD31-PECy7 (eBiosciences 25-0311), CD34-PE8 C2 e' ]. z6 v0 N
(BD Pharmingen 551387), cKit-APC (eBiosciences 17-1171), CD41-
) s1 t" T2 j7 p. G. APECy7 (eBiosciences 25-0411), CD45-PE (BD Pharmingen 553081),  q# I( c$ f- K& U
CD45-PECy7 (eBiosciences 25-0451-81), and Ter119-APC (BD
, U3 M8 G4 |9 ^Pharmingen 561033) antibodies.
7 x; [5 u! P+ t# ]3 eTranscriptome analysis) z( a4 i# I( ?) G
Total RNA was isolated from 1000-5000 FACS sorted cells in TRIzol/ `& B) L: ~+ g/ L$ [9 T& E
(Invitrogen) using the PureLink Mini RNA system (Ambion). RNA was  f2 ~+ u+ J+ M1 Q
subjected to two rounds of amplification using the MessageAmp II aRNA
- T( h8 x6 c  J# n- hAmplification Kit (Ambion) and then labeled using the MessageAmp II-2 Z( b0 v0 F1 B' f
Biotin Enhanced Kit (Ambion) according to manufacturer’s protocols.
0 a3 }- f1 I. s) L/ `/ W$ F8 G: _: AAmplified RNAs were hybridized to Affymetrix mouse 430 2.0 full genome
2 B3 ?1 n, |' l# Q$ P2 }. {& xarray chips at the BioMedical Genomics Center of the University of
( G+ u$ B" x/ l; E5 P* _8 pMinnesota. The CEL data files produced from Affymetrix array experiments8 w8 q' O7 s$ C" g) v( e& E4 f* l$ s/ H
were processed using the affy package included in Bioconductor. The robust. m; p' e+ [& _* W6 m) k2 u# b2 o0 ?
multi-array (RMA) method (Irizarry et al., 2003) was used to perform data
- @6 h1 I6 M; Q# r& w+ gnormalization, background correction and expression quantification. The
; K3 |) k. ?9 n( [# C) Mlimma package (Smyth, 2005) was used to identify differentially expressed
2 f. ]6 c. A2 s' l+ e$ P* M( T2 ggenes by ANOVA analysis, and false discovery rate (FDR) values were
" c3 }6 E8 x" _; q" H1 ~$ [8 Hcalculated using the Benjamini-Hochberg method (Benjamini and Hochberg,
& C4 T3 ^# [( Q3 q9 X1995). Average linkage hierarchical clustering analysis was performed using6 V: o+ m, ?0 g4 c
the cluster program (de Hoon et al., 2004), with uncentered Pearson’s, S6 X* J. K- \  Y. S2 c7 T& l  X
correlation coefficient used to define pairwise similarity in gene expression.( v- t0 q% D3 }' Q9 p5 [9 U
Genes annotated with the gene ontology (GO) term ‘heart development’
) {; Z! x1 P# Z5 W" Y(GO:0007507) were downloaded with AmiGO tool (Carbon et al., 2009) and4 F3 ^2 |+ k" B  ]
used in producing the heatmaps. Microarray data is accessible in Gene+ ?) s. P$ M' ^. g) J4 Y. i
Expression Omnibus (series GSE32223).
& |4 ?6 ~, V* q8 Q- _6 y! iQuantitative RT-PCR0 \) L8 e* }! R- I+ F
Total RNA was isolated from 1000-5000 FACS sorted cells or embryoid' @: B( I& r  o# g# E
body cells in TRIzol (Invitrogen) using the PureLink Mini RNA system.
2 B$ H# D% j+ z$ R! ?RNA from FACS sorted cells was subjected to two rounds of amplification/ H1 h# m+ A2 k( B4 }" n3 V; s' l
as described above, but left unlabeled. cDNA was made using random
; F/ Y: |: h9 ], R; Yhexamers and transcript levels were determined using VIC-labeled (Gapdh,
  d$ B0 _* G2 W, Z4352339E) or FAM-labeled (Er71, mm01176581_g1;  Scl,' u% t) W0 s3 b0 x  ?
mm01187033_m1; Cdh5, mm00486938_m1; Gata4, mm00484689_m1;  S& Z. X8 H0 d8 Z; [$ p
Tbx5, mm00803518_m1; Tnnt2, mm00441922_m1) TaqMan probe sets) G/ H- D' z/ Z" E$ A; t' W
(Applied Biosystems).
/ Y9 F+ w) H* G# p8 R8 AEmbryoid body differentiation and ER71 overexpression/ ~6 A' i; u, @
Doxycycline-dependent ER71-overexpressing mouse embryonic stem
) I9 H5 Z7 @! G( K( t(mES) cells were generated using an inducible cassette exchange strategy
2 ^: e3 q: t5 |) O+ e3 p(Iacovino et al., 2009). In this system, ER71 tagged with a C-terminal HA0 @- p- ]. l5 ~9 \- s( n" _! Y
epitope was overexpressed in response to 0.5 g doxycycline. To mimic
. i! y) w6 Z& s6 o( ]* xearly embryonic development, the mES cells were differentiated into/ I. N# ?3 J. `
embryoid bodies (EBs) using mesodermal differentiation media [1.5% FBS4 b9 M1 M* L8 |2 B& y
(Stem Cell Technology), 1 penicillin/streptomycin, 1 GlutaMAX
) b3 u9 L5 R9 X9 n4 ]' ^7 F(Gibco), 100 g/ml Fe-saturated transferrin, 450 mM monothioglycerol, 50' o6 [% ~$ @- G+ s) p$ s
g/ml ascorbic acid in IMDM (Invitrogen)] (Kennedy et al., 1997). ER71
# m8 o7 q5 |5 i% Zwas induced on day 3 after initiating differentiation and maintained through
  ~7 z/ ^5 V- t6 P3 aday 10 by supplementing the differentiation media with doxycycline. On
7 ]( A$ i, {# z0 bday 10, we fixed the EBs with 4% paraformaldehyde (PFA) and prepared
% i& b+ A/ }; e3 u5 ]cryosections. We stained adjacent sections with Hematoxylin and Eosin
2 t0 S) Q- C% A3 pusing a standard protocol and performed immunostaining with mouse anti-
; i& }# ~9 K) ?/ RTnT serum (1:200, DSHB clone CT3). We assayed the TnT-expressing area: L+ J* P7 h5 a4 h6 c
of each EB using ImageJ software (NIH) and calculated the ratio of the
/ L) U( n4 W( Q7 Fpositive area to the total area from 33 sections of uninduced EBs and 42. N$ g7 e2 e; P
sections of induced EBs. EBs were placed on laminin-coated coverslips2 h" G9 X3 v2 |
and stimulated at 1 Hz. Edge detection (Ionoptix, Milton, MA, USA) was
( W5 @% G7 h+ s8 {3 Tused to measure contractility of intact EBs.6 a# L5 D' A- ~$ [* G4 c
Immunohistochemical analysis2 _" ^; I; ^! s; g% |9 w* \, o; j" S
Stage-specific embryos were harvested from time-mated pregnant females.: S! ]1 ?" m" L; q5 I. E3 z5 z( ]
For paraffin sectioning, embryos were fixed for 4-8 hours at 4°C in 4%' S/ d! q6 _' l& o  @
PFA and embedded. For cryosectioning, embryos were fixed for 1 hour at8 p$ \" R( |7 x+ P1 R
4°C in 4% PFA and embedded in OCT compound (Sakura). Sections were7 v! ?. T8 \) F* c+ H  Q% o
blocked with immunohistochemical diluent (2% normal donkey serum, 1%
! d. H- d' \- B- u) _2 dbovine serum albumin, 0.3% Triton X-100, 0.02% sodium azide in PBS,4 \+ _& R0 z8 v2 n# y+ ~6 P
pH 7.3) at room temperature and incubated overnight at 4°C with primary$ a* q2 W- J/ Q4 j) G9 D$ H+ R
antibodies, including chicken anti-GFP (1:500, Abcam ab13970), rabbit
3 N4 J& B. F* f6 _) `+ P) ganti-desmin (1:200, Novus Biologicals NB120-15200), rat anti-Tie2 (1:100,& F' n* j& s3 v
eBiosciences 13-5987-81), rat anti-Pdgfra (1:200, eBiosciences 12-1401-; j0 \6 T( M4 j7 ~. h8 N7 H) t
81), goat anti-Nkx2-5 (1:500, Santa Cruz SC-8697) and anti-CD41 (1:100,
2 O- f9 Y0 c' S7 F& oBD Pharmingen 550274), anti-Cdh5 (1:100, BD Pharmingen 55289), anti-
, m4 N$ i& V% |' Z" X, _CD31 (1:200, BD Pharmingen 550274), anti-Gata1 (1:100, Santa Cruz SC9 C6 ]+ P9 N' n, t- n# c
265X) and anti-troponin T (1:200, DSHB CT3) sera. Slides were washed* u  M$ C# h2 j  c$ o* _: J1 p: T
and incubated with combinations of secondary antibodies (1:200) including' Y) n9 x( b* G: i' K
anti-chicken Dylight 488, anti-rabbit Cy3, anti-mouse Cy3, anti-rat Cy3+ o7 ^  {. }9 e7 c
and anti-goat Dylight 549 (Jackson ImmunoResearch Laboratories).
0 v( c4 ~4 f% sResults were imaged on a Zeiss Axio Imager M1 upright microscope or a( n: k1 y- f3 v5 O# }' b
Zeiss LSM 510 Meta confocal microscope.
! z; W' N/ A) X  S) MStatistical analysis7 W. o  ^) ?8 P" `) [! V
Data represent the average of at least three replicates and s.e.m.
. L) X# n, c- C9 DSignificance was tested by the Kruskal-Wallis test with the Dunn multiple
3 F, L- R: ~7 k; scomparison test for more than two groups; for example, when comparing
, s4 S. }# Q4 x; m7 gRESEARCH ARTICLE Development 138 (21)& A' t4 h2 H3 z2 f0 A* X
DEVELOPMENTwild type, heterozygous and homozygous mutants. Significance was tested  T" W6 [$ ]- d) K' C
by the Mann-Whitney U test for two groups. Analyses were performed
4 g& R. r, r- S6 ?$ `using Prism5 software (GraphPad).% l0 U: |5 z" }! u( h9 P; j
RESULTS
- X: C1 g% e! }4 c& zExpression analysis of the ER71-EYFP reporter+ {. _5 O) G: M( t
Initial analysis of ER71 expression in the developing mouse
) z5 L$ A% g  L. wembryo was reported previously (Ferdous et al., 2009; Lee et al.,
) k1 O5 g5 J! w' v5 F2008). To further examine its potential role in cardiovascular2 r8 o( q1 @' }' F1 t
development, we performed a detailed expression analysis of ER71
7 t" R, k! s& m6 X& Ffrom E7.0 to E9.5, when Er71 mRNA is expressed (Ferdous et al.,$ H3 ]  _0 v( `9 K
2009). None of the commercially available ER71 antibodies3 D9 I  q0 }+ J, ?2 K
detected the endogenous protein, so we utilized an Er71 transgenic
  Y* g5 Q& s5 l2 p% k, U3 ]( O+ Xmouse driving an enhanced yellow fluorescent protein (EYFP)
1 ^$ r" y; m' x$ v7 \reporter. The 3.9 kb region upstream of the Er71 transcription start1 F9 E  w4 O, }" H
site was fused to the  EYFP reporter (Fig. 1A). This promoter
( ^( g; @; h( V- |) V0 v: h$ Afragment was previously shown to drive reporter expression that
! R+ o' c% r) `' M% mrecapitulated endogenous ER71 expression (Ferdous et al., 2009).
4 o/ u; T* j7 {; NFour founder lines were generated with offspring embryos that
8 g- O2 V% e$ H1 T; E2 O. Mshowed the same expression pattern.
0 E5 u3 A' x9 m6 NTo examine the expression pattern of the EYFP reporter, we: t0 w. c. G7 o
compared its expression to that of the angiopoietin 1 receptor Tie2,
7 a% ?( }) A" A0 D" bwhich marks endothelial cells in mice and humans (Dumont et al.,
7 _. {0 o; p% Q% L0 n* m5 G1992; Sarb et al., 2010; Schlaeger et al., 1997; Schnurch and Risau,
# `, L+ @! H' X2 Z0 U5 G: R1993). The EYFP reporter (representing ER71 expression) was6 ]$ w& b5 s; E, _4 d& d
expressed as early as the early to mid gastrulation stage (E7.0; Fig.; |6 V- U9 z. K) Y
1B, bracket). At this stage, reporter expression was limited to the
7 V  Y% T$ s" O. Kextra-embryonic mesoderm (Fig. 1C) and showed co-expression
2 \/ F3 l' I. c; B2 D; M4803 RESEARCH ARTICLE ER71 governs progenitor fates
0 n+ I0 ~  T0 I# a/ DFig. 1. The transgenic ER71-
; p+ v4 A) p( q3 p- R# TEYFP reporter is expressed
* e- ^1 y2 M& M# \& r6 `! Q# g. ], uduring embryogenesis.
; m( V) ~+ q) Y) J0 E  Z(A)The transgenic Er71-EYFP
" h7 f5 t/ X9 pconstruct used in these analysis." T8 {& q7 o" g6 L+ `; P7 O
(B-Q)Expression analysis of mouse
6 q' ^$ |) H0 |embryos between E7.0 and E9.5.: g! O4 \" A1 u8 _) [5 o! F2 o
EYFP protein fluorescence (green)/ k2 y. l6 q5 j. F5 o6 p" m& v
is shown in whole-mount images
& w, P: Q2 Q, M% O3 m' X% }/ h+ Q4 _(B,D,F,J,K,N). Sections were
% J# Q6 i2 Y8 P3 Fstained with anti-GFP (green),
. v+ J6 t! Z( ?4 L8 M0 uanti-Tie2 (red), anti-Nkx2-5 (red)
) r1 j& c) F' l) w) z2 U* @or anti-Pdgfra (red) antibody as0 _, y  d  W, J! t5 g* X
indicated. DAPI nuclear staining is
4 s  W+ G- j- \0 l$ I, vshown in blue. Yellow indicates3 h6 R- O' |5 b. Z& g! j
overlap of green and red
3 T8 B$ T6 _& d1 G8 I& P6 A& e0 M2 tchannels. (B-C) Early to mid
0 Z6 `' z! g* ^0 a! jstreak stage, (D-E) early bud/ V' u+ q, h% Y. B1 i0 C4 R3 Z
stage, (F-I) early head-fold stage,6 N2 Q( O) |$ T" i
(J-M) E8.5, (N-Q) E9.5; embryos
# V9 e' Z! A5 D, w* [6 F0 J* Zwere staged according to Downs
9 Q2 h# s$ _$ ~# ]1 z' g4 b  Dand Davies (Downs and Davies,+ n; M1 O0 K7 D/ K- ?
1993). Boxed regions are shown
% D' p+ Y" b5 h6 xat higher magnification; planes of
6 W# [/ h' k: `) L, l- h2 p5 ssections are indicated. See first
+ B" S& r3 @  dsection of the Results for a
* a4 l7 t. V8 D9 W6 Sdescription of arrows, arrowheads
% M5 |/ q" c/ Z' A; ~and other labels. al, allantois; ba,! P2 f" \. y6 W* V, `8 O
branchial arch; bi, blood island;2 i" [9 l8 G7 h; j
cc, cardiac crescent; cv, cardinal4 u' ~- x! N' F* Q# @
vein; da, dorsal artery; ec,
! `& H% O8 g# l5 vendocardium; h, heart; hf, head  J2 I1 K- b* r
fold; np, neural plate; nt, neural8 `2 b# r. @/ u% a' F
tube; ph, pharynx; pm, paraxial
' F) x, J; P( i. }( j4 s9 emesoderm; s, somite; ys, yolk sac.
8 V& A; c5 l, q  o: d7 zScale bars: 200m; 50m in" d- `8 E$ _7 [
enlargements.+ t) q9 Q4 |' s# J
DEVELOPMENT4804  l2 a; b5 J5 B
with Tie2 in the primitive blood islands (Fig. 1C,C, arrows) (Ema
# E, A6 }0 ], W- A) w$ l3 aet al., 2006b). EYFP was also weakly expressed in the mesothelial
2 g- i$ i+ r6 V% H/ U; S% ]; H! player of the yolk sac, but Tie2 was absent in this non-endothelial
# d, E; b- s; pcell population (Fig. 1C,C, arrowheads). At E7.5, continued
3 S7 k+ d/ ^7 ]! {" v8 B0 cexpression of the EYFP reporter was observed in the extra-
6 r+ q, u5 R% w* uembryonic mesoderm (Fig. 1D, bracket). In addition, scattered
) S1 Z9 |& D2 e* Z# OEYFP+ cells were observed in the lateral plate mesoderm (Fig. 1D,
2 Q, N3 q& X9 I& S. harrows). Transverse sections showed that these scattered cells co-6 o6 N3 {" P' A; A8 p* l, H# o
expressed Tie2 and most likely represent endothelial progenitors% J8 |# \& N) \+ V( G* d
(Fig. 1E,E,E), although Tie2 expression has also been identified
# W& L+ `0 @1 b" `- b5 ]; `: cin a subset of cells committed to the hematopoietic lineage: A  L+ M1 I# Z+ J, _
(Kisanuki et al., 2001; Li et al., 2005).
$ I4 u+ M, e( y/ R' I$ u  ]  pAt E7.75, the EYFP signal localized to discrete structures1 l4 a' P' e# g( q
including the cardiac crescent and the progenitors of the dorsal aortae
$ {4 Q  q' f. K(Fig. 1F, cc, arrows). We examined the expression in the cardiac
1 M8 ~% L: F( A7 x2 a- dcrescent and paraxial mesoderm in detail using Nkx2-5 and Pdgfra4 W9 a. Z! ~; I) t( e
antibodies (Fig. 1G-I). We observed that the EYFP signal overlapped
5 [6 ]* e. |2 y! _2 g3 l% jextensively with Tie2 within the cardiac crescent (Fig. 1G,G).# g! C  ]' K9 A! h7 A# s
These EYFP+ cells are presumably endocardial progenitors (Misfeldt; ]- x5 J! d4 ]3 D
et al., 2009). By contrast, EYFP+ cells either weakly expressed or did$ k& |1 P9 @4 f* p$ {7 }
not express Nkx2-5 (Fig. 1H,H, arrows and arrowheads,
1 E2 p! X- ~/ J0 I6 ?) H5 qrespectively). We did not observe strong co-expression of Nkx2-55 X. t3 Y, A2 }& K! `' M: ?
and EYFP. Pdgfra is strongly expressed in paraxial mesoderm and! n" }- [. Q3 q2 L
cardiac mesoderm (Fig. 1I, pm and asterisk). Double
5 Y* g5 O% h1 n) f& x5 B  ~immunohistochemical labeling of EYFP and Pdgfra demonstrated
+ q9 E( X# E0 p8 B  n+ Ethat most cells either did not co-express these markers or weakly
0 E, w( l1 x0 v/ i" L. sexpressed Pdgfra (Fig. 1I,I,I, arrowheads and arrows, respectively),
5 l0 o& S- z0 [  zwhich is consistent with previous reports that the hemogenic lineage
3 V- W: B  {1 Z! L: Y4 o8 Gis segregated from the paraxial mesoderm early during gastrulation9 d( u, p; \. q
(Kataoka et al., 1997; Takakura et al., 1997).
7 O! G9 _+ }+ w8 MAt E8.5 and E9.5, we observed localization of the EYFP signal to
/ W1 u$ \0 M# f) u. F$ U' cvascular structures throughout the embryo proper (Fig. 1J,N; arrows
' Q7 x. S: I6 I$ [' K! Z& H7 i% jpoint to the dorsal aorta and arrowheads indicate intersomitic vessels)' p- |! b8 e7 ^8 V. J! o
and yolk sac (Fig. 1K). Transverse sections of the embryos (Fig.
1 S8 G6 b  `! ^: }1L,M,O,P) showed localized expression in the vessel structures
5 y% }9 P, [6 Vincluding dorsal aorta, cardinal vein, intersomitic vessels/ g$ n8 l+ G' x9 @
(arrowheads in Fig. 1M) and the endocardium (arrowheads in Fig.
2 x  w0 A# R# I/ d9 c1L). The expression of EYFP largely overlapped with that of Tie2,' t5 A8 t  B* P% I6 V
indicating that the  Er71 promoter is active in early embryonic0 V( M( B( E( e* G; G' B
endothelial and hematopoietic cells (Fig. 1O-P). We also observed
, k- k' u9 Z! O3 ]that EYFP was co-expressed with the hematopoietic transcription
! e# @0 [7 J5 ^( [factor Gata1, the cell surface marker of hematopoietic progenitors# n; a$ c% F7 G3 F
CD41 (also known as Itga2b), the marker of hematopoietic and
$ d1 T& P7 ~: w/ R+ `; dendothelial lineages Cdh5, and with the primarily endothelium-! [; `6 F, {+ I
specific cell surface marker CD31 (also known as Pecam1)& i0 A( R" ?6 C4 U! z/ r! |* l9 y
(supplementary material Fig. S1). By E9.5, Nkx2-5 was localized to
: h1 [, C$ p+ B, othe myocardial layer of the heart and did not overlap with EYFP: }: S2 A  U) T) |8 n  d+ C8 _
expression (Fig. 1Q, arrowheads; compare with 1O).
5 m4 _4 O' j. }Reporter expression is mislocalized in the Er713 y  J; l1 O% U2 P. C
mutant
) u% t( q+ z# D% J* r# QIn the Er71 mutant, the hematopoietic and endothelial lineages are) A+ l1 l% S) X: p6 N( n0 R2 a; G" ~8 O
absent. However, no changes were observed in cellular
: n$ k* t% |0 J( O0 _7 ^proliferation or apoptosis in the Er71 mutant embryo (Ferdous et
( \, ~' w0 J# o' \4 x$ xal., 2009). Therefore, we crossed the ER71-EYFP reporter line into
3 v) Z! R1 P. k% lthe Er71 mutant background to define and characterize the cells
" ~- C6 H  m! |2 B2 v/ ethat should give rise to these lineages (Fig. 2). In the E7.75 wild-
4 W8 k/ s  x+ p$ @! W" \; etype embryo, EYFP expression was observed throughout the
+ I: y* O0 t" k! m- a3 n7 j5 }; Hcardiac crescent, the dorsal aortae and the extra-embryonic- C3 E) t' B0 w9 J3 k9 s) ^
mesoderm (Fig. 2A). By contrast, in the E7.75 mutant embryo the2 k5 D7 B4 O, S4 y
expression pattern was less definitive. EYFP-positive cells were5 O/ D& V  f6 _4 c) C( r7 N
found in the extra-embryonic tissue; however, expression of EYFP
8 ?' }# O! c4 ^- v* K% jwithin the embryo proper was mislocalized (Fig. 2B, arrow). EYFP5 D5 Q4 ]! i- b* M$ y( T0 d9 l
expression appeared diffuse throughout the cardiac crescent (Fig.
$ ~. `# c0 C" }2B, asterisk). In the wild-type embryo at E8.0, EYFP expression$ T7 l5 c' {- r, J& }! d
was observed specifically in the dorsal aortae, but not on the
3 o/ V8 o) x! @" ^periphery of the embryo (Fig. 2C, arrowheads). EYFP expression
7 I5 X& t  Q9 s% o1 vwas also observed in the presumptive endocardium within the6 f! W$ O3 |- z! Y1 M- n3 S: ?) ^& n
linear heart tube (Fig. 2C). By contrast, the Er71 mutant embryos
& r1 H1 v, p) _- u% D9 g( ?$ Wexpressed EYFP only at the periphery of the embryo, which is) V. y, y0 u1 w# ^
likely to represent paraxial mesoderm (Fig. 2D).( t. E" P2 W8 i3 ^4 @3 S) ~; t
Immunohistochemical analysis of ER71-EYFP and Pdgfra, a
+ e9 u% j8 \6 A% Q0 D, @" w. Qmarker of paraxial mesoderm, expression in E8.5 wild-type and
! }3 W6 g- r6 U4 u, e2 H1 f- e8 Q: umutant embryos demonstrates that EYFP-positive cells are8 U3 c$ q4 b" y2 m0 f! I
interspersed within the Pdgfra-positive mesoderm (supplementary3 w2 [/ y/ q- P' i% k; `9 D
material Fig. S2). FACS profiling showed that the frequency of; i2 b$ Y2 A& ]* H. R
EYFP+ cells was increased in the Er71 mutant compared with the
; s  h( `9 `) Y8 q2 }0 F1 c- Z) f( Uwild-type control at E8.0 (Fig. 2G-I), but not at E7.75 (Fig. 2E,F,I).& O, U& E% I2 F
The apparent increase in the percentage of EYFP+ cells could be8 Z- |4 T- G7 s* w
due to a reduction in the total number of cells without a change in
! I& k# p3 U) y5 P1 zthe number of EYFP+ cells, as cell viability can be reduced by a
& R8 J! J1 A% U1 Q. hlack of vascularization. However, we ruled out this possibility as
4 U$ J4 v$ C% l" G" j- Pwe also observed a doubling in the absolute number of EYFP+ cells9 _9 D! Q* ?8 O4 q, D! ]
in E8.0  Er71 mutant embryos as compared with wild-type
1 C( Q# F& M6 j4 Y! D) e! Ulittermates (Fig. 2J).
1 M& g/ D. |6 TThese analyses demonstrated that cells that expressed the ER71
/ G% K6 h; Z9 ~2 q- n& e# d1 Hreporter were not only present in the Er71 mutant embryo, but were
6 k1 s% s" Q" A: balso expanded in number. Since the Er71 mutant embryo lacks# ?# T! x( F0 \& j
differentiated endothelial and hematopoietic lineages, we examined
& z( r" _3 q% d9 l, J3 `5 ^whether these EYFP+ cells were arrested progenitors or cells  Z+ x7 j" l3 S8 m9 C# r& I
redirected to other lineages. We used FACS analysis to examine the6 Z4 ]  ]2 K5 q9 n4 d
cell surface markers of the EYFP+ cells in the Er71 mutant and' Z; T/ r' B$ x8 b; |- D& j. t
wild-type backgrounds. Previous studies have established that
5 _8 u# H& u, m# p* v6 R8 C8 GPdgfra is expressed in the primitive streak at E7.0, but is restricted
5 E6 L1 s, [' c8 R7 T, G) T( k' M  Zto the paraxial mesoderm at the early head-fold stages (Takakura
" t: K; |0 A: w8 [' x; H) t2 |3 set al., 1997). Flk1 is expressed more broadly in mesodermal
! Q! _2 h- S1 a9 j% Y3 bprecursors (Ema et al., 2006a; Motoike et al., 2003), including the& v  L" z/ a/ L9 I
posterior portion of the primitive streak. Flk1+/Pdgfra–
' S, o& M; K+ l, u) G1 _cells mark, y) A2 y2 h# Q6 ]( Q
lateral plate mesoderm that will give rise to the endothelial and
- y# S; ?2 ]5 {hematopoietic lineages and Flk1–
& {$ b. T5 ]0 l% C' e3 I/Pdgfra+ cells mark the paraxial4 _) A7 i& _$ K8 c
mesoderm. Flk1 and Pdgfra are known to be co-expressed in the2 Z& E) D* T3 C6 e5 ^0 h
cardiac crescent (Kataoka et al., 1997) and in cardiac progenitors
1 m' i9 g% j# r/ s, zof embryoid bodies (EBs) (Bondue et al., 2011; Kattman et al.,
3 ]$ \2 w6 z/ D  T  {( F( X; ^2006). In the present study, the frequency of Flk1+ cells within the' W: g! Y$ w: V' X7 c8 x' V
EYFP+ population was significantly reduced at E7.75 and E8.0
( A3 p& w- n0 F! \' r+ |(Fig. 3A,B,D,F). CD41 (Fig. 3D,E) and CD34 (Fig. 3E,F)
. d8 y# r9 P- k' mexpression was essentially absent at E8.0 in the EYFP+ cells of the
) Q. ]- T8 d% jEr71 mutant. These results show that the lateral plate mesoderm! M3 b1 n! H# g+ s
lineage and its derivatives, i.e. the hematopoietic and endothelial
! P4 ^( q" w/ Xlineages, are reduced in the Er71 mutant.3 E# c: R. `  p
In order to assess which lineages are expressing ER71-EYFP, we7 v  T, r7 E( D' l/ o
analyzed the EYFP+ cells using Flk1 and Pdgfra antibodies.! x- W" A/ Y+ o5 |3 F
Concurrent with the decrease of Flk1 expression at E7.75 and E8.0,
" U/ V' j" _! I% F  ewe observed an increase in the frequency of EYFP+ cells that
- i+ d! I+ P% Dexpress Pdgfra (Fig. 3A,B). This correlated with a significant1 J0 o6 x! G# @7 A  g3 ]1 v1 O
increase of Pdgfra single-positive cells at E7.75 and a significant3 B6 y; r  P* a
increase of Flk1+/Pdgfra+ cells at E8.0 (Fig. 3A,C). There was also
& {9 w1 i/ C8 B: H, q  Sa trend toward expansion of Pdgfra single-positive cells within the$ F) W% C% M  ~' t
EYFP+ population at E8.0 (Fig. 3A,C). To support these/ ]2 Z, m% t1 L- r$ ^5 N  _/ c
observations, we used immunohistochemistry to analyze the EYFP/ q3 j* b, j/ z) \
expression patterns. In wild-type embryos, EYFP expression is
) M" ?( }, O2 E& g9 a, a7 g" z+ L+ xdistinct from Pdgfra+ paraxial mesoderm. A select number of
0 P: a& Q7 y* `* h7 wEYFP-positive cells are found within this region; however, these
+ O3 D3 L/ d; X5 Y9 {! t  Pcells are Pdgfra–
/ T9 A6 L: i% z- F/ _* w7 A9 Q% L(supplementary material Fig. S2A,B). In mutant
9 S! Q3 P) x; c+ n( a  s& Q. gRESEARCH ARTICLE Development 138 (21)
3 d. Z) }  u7 @& I  @+ ]. PDEVELOPMENTembryos, many EYFP+ cells are found within the Pdgfra+ paraxial& k/ V# j2 D3 K& u6 J% Y
mesoderm. Some of these cells are double positive for EYFP and) D8 Q2 }6 C& Z6 N2 Y/ y
Pdgfra (supplementary material Fig. S2C,D). Likewise, in wild-) K; j  `4 Y: k
type embryos, EYFP expression is distinct from troponin T (TnT)
9 j% M; y. X9 e! L$ s+
2 Y  d4 \$ n% K# ]cardiomyocytes (supplementary material Fig. S2E). However, in
# W) V' g/ ]( y3 W8 `mutant embryos double-positive cells are found (supplementary5 @6 W5 |" q$ l5 J9 Y
material Fig. S2F,G, arrows). One interpretation of these data is/ F' U( u3 m; e- B- Z( J6 C2 D
that, in the absence of ER71, the EYFP+ progenitor cells' e4 D! e) o2 t# h! d1 \; G1 a
differentiate towards the paraxial (Flk1–
( ]9 z( D/ n) I  E0 w5 m/Pdgfra+) and cardiac
0 Q9 b8 J8 D$ h1 a* `* i/ H/ Gmesodermal (Flk1+/Pdgfra+) lineages at the expense of the lateral! B& N7 i- h: _1 z
plate/hemangiogenic mesodermal (Flk1+/Pdgfra–
. X9 y7 N+ y& A7 l9 x) lineage.. C) x. p; }: W- M; r& m1 z2 J, @
An alternative explanation of these results is that EYFP is! r' z3 j/ G9 U4 J% i2 w4 q
expressed ectopically in cardiac (Flk1+/Pdgfra+) and paraxial' x( U# {; d! ]7 j
(Flk1–0 R, ]. _5 D& {' x4 Y, Y, N
/Pdgfra+) mesodermal populations in the absence of ER71.
/ \. _3 v  k( D3 f! V6 X' gIf this were the case, we would expect the cardiac and paraxial& h2 v, s  r( ~7 Q5 f9 u3 a
mesodermal populations within the EYFP–
* A- \/ {& |( j3 Jgate to decrease in
$ j1 l& y0 E9 y$ b# [) Rthe mutant embryos because these cells expressed EYFP and
9 V3 e' D, E$ p' S* fwould be included in the EYFP+ gate. Furthermore, we would; Y/ a6 ?( s; {, N
expect no change in the overall representation of these markers
! f: z+ ]/ q$ ^6 {9 Jin the unfractionated (the sum of EYFP+ and EYFP–5 L6 }* d* `# f" N* s  M4 N! u8 |
) gate. To; s* M1 e8 V* Q* A  }
examine this possibility, we analyzed unfractionated cells from* Z4 G- t7 S. g' C$ y9 [
the whole embryo as well as EYFP–
  t- g$ V; g, k: T! Sfractionated cells. In the
* n$ g. j1 |7 E6 @  U/ Z0 n1 SEYFP–# e& V. k, P" C
fraction no changes were observed in the mesodermal# x0 P+ Y& N: X! P4 M
populations at E7.75 (supplementary material Fig. S3C,D) and  l% G7 T5 q& j" e
E8.0 (supplementary material Fig. S3G,H). Analysis of the: @1 f' u) D# R( T. Q+ t* h
whole population (the sum of EYFP–
% L5 H" \' x! o* ?, |7 ^and EYFP+ gates) showed5 k$ @1 e1 {6 M0 R# h  ~$ Q
no change in cardiac or paraxial populations at E7.75
; F" D# P) x; O7 ~(supplementary material Fig. S3A,B); but at E8.0, we observed' c) O- s# p# H3 f+ l7 T
a small, but reproducible, increase in the cardiac mesoderm
- I, v2 F' ?4 F, W# s(Flk1+/Pdgfra+) (supplementary material Fig. S3E,F). We predict( E8 a+ A! ]; g; N" H
that the increase in cardiac mesoderm in the whole embryo is
0 b3 G* J6 M- w) K8 n$ _small because the EYFP+ population is a minor fraction of the
& K0 F* S6 ~) C- u8 M3 s2 Y7 Uentire embryo.2 F5 u& M1 [0 j0 d" f4 T: d  M
Collectively, our results support the model that, in the Er71
, N7 d1 `( E" E/ i: Smutant background, EYFP+ cells differentiate to the cardiac- E* a: s3 D7 G0 ]! H; c
mesoderm lineage and contribute to the increase in cardiac' J6 t: t3 s6 ^( U# i% r0 X! T
mesoderm (Flk1+/Pdgfra+) markers, rather than cardiomyocytes or
6 P6 T( D' u/ w4 `2 \, L8 V! Ncardiac progenitors ectopically expressing EYFP.
' t% P; z: d8 ^7 f4805 RESEARCH ARTICLE ER71 governs progenitor fates! O; D" x+ S/ u- {
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); v: E5 ^  ~( I" t9 ^
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). Arrowheads7 q; o8 Y. h% a! _2 A4 _8 J( \7 y
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).; I: [: X5 ]6 c2 z
Representative FACS profiles of individual embryos (E-H) show the percentage of EYFP positive cells per embryo. Scale bars: 200m. (I)The
0 K3 V) d/ ?) ^percentage of EYFP+ cells for all experiments. (J)The average number of EYFP+ cells sorted per embryo. WT, wild type; HET, Er71 heterozygote; MT,# O/ G8 L! T/ D. B
homozygous Er71 mutant. ***, P<0.001. Error bars indicate s.e.m.1 T  w' Q$ |! B3 [1 }" g
DEVELOPMENT4806
- C, p8 L# ?( kCardiac and skeletal muscle genes are
, M" i1 H1 i3 z& W" E' H- X, Supregulated in ER71-EYFP+ cells in Er71 mutant
: T; A; A& _5 A) m- M) r7 D5 \+ pembryos5 x& L. ^+ Z" w8 P" X  n6 ]  h
To further examine whether the EYFP+ cells in the absence of0 Z, P8 W8 }8 C
ER71 were redirected to other mesodermal lineages, we performed, J' [, [1 J, [
a transcriptome analysis. We used an Affymetrix microarray: F6 F! L$ c3 g; ^
platform and examined the EYFP-positive and EYFP-negative
# @3 F  A. z% G' i6 v- }/ `% Dcells from wild-type and Er71 mutant littermate embryos. Genes
  c' Z6 ?% c: d# {  G/ Sthat were significantly dysregulated were filtered based on the false
4 @9 O0 h! N( Cdiscovery rate (FDR<0.01 for EYFP+ versus EYFP–: ~2 E. L% G* ]5 Y0 ?. X
; FDR<0.35 for
1 {6 ~: X- J+ Z6 m  [) ~wild type versus mutant; FDR<0.01 for the interaction between
1 c: q$ R# o0 u! e" |3 n# Sthese two variables). Clustering analysis was carried out
) j% p  w0 l, V: U  m$ Vsubsequently (Fig. 4A). Interestingly, there were no transcripts that" {; V) b! w. _" j9 m" _% m
showed differential expression between EYFP–
0 l& T# a$ w$ Y5 y$ }populations in the) m% V( g: M7 ^' O5 {( M- B2 p
Er71 wild-type and mutant backgrounds. Also, the cluster analysis# M- J# j* `$ U
was unable to resolve differences between the EYFP–9 E4 h9 m% \0 v2 L3 n1 b
populations0 D5 t4 h5 x+ y. X. O/ \0 C
RESEARCH ARTICLE Development 138 (21)1 t3 X% E" f$ _0 o/ p! R2 |6 Y8 R
Fig. 3. ER71-EYFP+ cells in Er71 mutant mouse embryos lack markers of hematopoietic progenitors in exchange for those of other
* q" ?  v% O1 _4 {( Q0 e: H5 z1 x. S# zmesodermal lineages. (A)Using fluorophore-conjugated antibodies and FACS analysis, we observed an enrichment of Pdgfra+ cells in ER71-EYFP+: p5 L: [5 y" S1 {+ z' z
mutant cells at E7.75 and E8.0. (B,C)Summary of multiple experiments comparing individual fluorophores Flk1 and Pdgfra (B), and combinations of, D/ K# t: J% }$ {6 D: e* [7 D% R* Y+ o
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
+ q, _, [/ Q7 i/ ?reduction in CD41 (D) and CD34 (F) at E8.0. (E)Summary of multiple experiments comparing hemogenic and endothelial markers CD41 and CD34.
& r  `9 T% v0 l* \: d9 ]( \' W**, P<0.01; *, P<0.05. Error bars indicate s.e.m. LPM, lateral plate mesoderm; CM, cardiac mesoderm; PM, paraxial mesoderm.
  e9 y; z6 ?; l4 t- n' l0 F) aDEVELOPMENT(Fig. 4A). These transcriptome results for the EYFP–/ p+ _, G5 J/ K, i
cells support% [7 l$ A7 r. Y
the notion that EYFP is not being expressed ectopically.
: K% q( b) D) fFurthermore, this indicated that there were no significant non-cell-
* ^: C) n, t" Kautonomous effects on gene expression resulting from the lack of
' R; J* b5 i" D" QER71 at this time point." B$ ?# n- M' {; J4 y
Within the EYFP+ populations, the Er71 mutant and wild-type
' n0 M. o; _) D+ O& }cells were significantly different (Fig. 4A). A large number of; ~+ g4 w7 R! {* Y8 A
hematopoietic and endothelial transcripts were decreased in7 k# Q9 m% i6 h; c6 J
expression between  Er71 wild-type EYFP+ and  Er71 mutant$ }- u. i) i, n5 S  f
EYFP+ cells (Fig. 4C). Importantly, ER71 expression was restricted$ X4 P; c' N0 n0 d/ H4 J: O. {
to the EYFP+ cells in the wild-type background (Fig. 4C, arrow),* M! C; C+ m1 n/ {: ?
confirming that the  Er71-EYFP transgenic model reflects1 `' c# m) g5 D& `
endogenous ER71 expression. Other transcripts that were6 q8 P+ P9 L) u: B6 S( O& j: j
downregulated in the mutant EYFP+ cells compared with the Er719 f$ A8 o! c+ ~7 i" _! O  z
wild-type EYFP+ cells included endothelial and hematopoietic6 k6 }; R: F. R+ D4 `
transcripts such as Hbb-y, Fli1, Erg, Tal1, Cldn5, Cd93, Sox18,
9 ~$ R* W1 X# S3 L( pCdh5, Sox7, Mmrn1, Eng and Nos3 (Fig. 4C). These data indicated3 D: D) C: ]6 D, J: m! J
that the EYFP reporter-positive cells contributed to the endothelial( I" G9 [3 Z  U6 o5 H
and hematopoietic lineages and did not express markers of those) t  T1 A4 Q+ I# r
lineages in the absence of ER71, consistent with the previously# H, a/ |: T! f: q
described phenotypes (Ferdous et al., 2009; Lee et al., 2008).0 Q+ e7 G9 q4 e: o1 S2 X
Transcripts that were significantly upregulated in the Er71 mutant
% w  W, j: j2 e5 L* o6 W$ e$ h. U% uEYFP+ cells compared with the Er71 wild-type EYFP+ cells were* h$ N1 a3 Y9 [1 s6 x
associated with muscle lineages (Fig. 4B). These transcripts
' J  N  Y' e( S8 Z# W8 Vincluded Smarcd3, Myl3,  Irx4, Tnni1, Myl2 and Tbx5 (cardiac-; Z$ Y/ t( d) C7 Q% f
restricted transcripts); Acta1, Myl1, Svep1, Dpf3 and Cdo1 (skeletal( c7 ]; C( T% ]" ~0 E  {0 I
muscle-expressed transcripts); and  Acta2 and  Myl9 (vascular" ~8 p, v  [: i3 d' S9 Y
smooth muscle-specific transcripts). These results suggested that0 i% C, Q5 c' ^  X' b
the EYFP+ cells gave rise to other mesodermal lineages in the' L; r7 [; |, r4 F
absence of ER71.
' u5 \6 e' y- n. H% R, P5 X$ S( x/ BIn order to determine whether the cardiac lineage was broadly
% T3 _; n) o0 ~' Q# v/ L' |affected, we analyzed transcripts from the cardiac development+ P3 T/ c* |" K& a$ Y$ h2 {
gene list in gene ontology (GO) terms and performed a hierarchical
5 {/ ]% Z) S5 s( m9 k* Cclustering analysis. Similar to the results obtained from whole-
6 M) G2 N8 y/ _genome analysis, wild-type EYFP+ cells segregated from the
# [0 E# R  [% }: V+ tmutant EYFP+ cells (and all EYFP–
# l+ n) f$ c* x; }cells) (supplementary material
' G4 h) V" G# g& `Fig. S4A). This result demonstrated that the difference between; ^  p8 b; m! f) i
these two populations can also be detected based only on cardiac+ k1 H  ~, q( d" D3 J& h/ G/ J* u
development criteria. Some transcripts were decreased in EYFP+
4 y9 \1 A, l! H, TEr71 mutant populations compared with EYFP+ wild-type cells,6 \$ Z+ b) g$ ~. z$ q9 Q4 h
including a number of transcripts that are important for endocardial. `/ C' i& A( E! ]. v) m/ S0 p  j
development (including  Eng,  Hhex,  Sox18,  Nfatc1,  Sox17)' _3 I4 C: ]& [$ p* }# {1 Y
(supplementary material Fig. S4B). A number of transcripts were
2 D  y3 C0 |# d& J, o$ w. j$ Pincreased in expression in the EYFP+ Er71 mutant population
8 d, d, G& \0 a# H; e* gcompared with the other three groups (EYFP+ wild-type and both$ j" _* `$ P5 P# z! B" Y  ?
EYFP–
# K: s7 c2 ~  Y/ f' Zpopulations), including a number of cardiac transcription3 ~4 R2 _/ G: j. m/ x
factors (Myocd, Nkx2-5, Irx4, Isl1, Gata6, Tbx20, Tbx5, Gata4,; K3 n5 C) s/ `) l% B/ C
Smyd1 and Srf) and structure-function transcripts (Myl2, Myh7,: t8 b! {7 n& \+ C6 s
Myl3, Tnnc1, Tnnt2, Tnni3) (supplementary material Fig. S4C). We
0 c1 Z; o! K( p/ N; d4 Tvalidated changes in selected transcripts by qPCR on independent
# i# ]% c* g/ {* X3 v  R; lsamples and confirmed decreased expression of Er71, Tal1 and
! u& e/ Z0 v! U  FCdh5 and the overexpression of cardiac transcripts including Nkx2-
! d# T9 G* _) p6 `3 ~+ ]# g  p5, Gata4 and Tbx5 in Er71 mutant EYFP+ cells (Fig. 5).
; d# O' ~1 l1 v4 GIn summary, our transcriptome analysis indicated that cardiac
- ^( I" w" O- i. o4 p+ ?genes are over-represented in Er71 mutant EYFP+ cells, whereas
5 n7 h. K! ^* g: T7 h  @# n9 pendothelial, endocardial and hematopoietic genes are under-! D# h2 G5 E, [1 v. ~( i9 k
represented.
; p: {  f7 }* P% d" N( e; TA novel Er71-Cre transgene marks endothelial and
8 L5 |* `- S* B1 X1 l9 g! thematopoietic lineages/ c; M; ^2 y/ U$ ~# ?
In order to determine whether ER71-expressing cells give rise to
) s% ]2 @6 r5 T" n+ borgans or programs other than the endothelial and hematopoietic4 d9 S- G! ~) z
lineages, we generated an Er71-Cre transgenic mouse model (Fig.
! `7 t8 u, B* _: j. r6A). We obtained a total of six founder lines, with embryos that; I- j) u# r' o7 o
showed similar expression patterns after breeding with the Rosa-1 I% n* k# d: N) M5 V$ G6 @
4807 RESEARCH ARTICLE ER71 governs progenitor fates8 u) m0 ?3 K# l: t
Fig. 4. Transcriptome analysis reveals that hemato-endothelial7 y5 ]. `8 {. h6 F9 f* r
and cardiac lineages are dysregulated in the Er71 mutant4 l- y/ y* F, h4 A# Q
embryo. Transcriptome analysis was performed on EYFP+ and EYFP–
3 H4 o- Z- T1 C& k; S' `3 ycells sorted from wild-type and Er71 mutant littermate mouse embryos.$ `1 X  O  Z: [8 H- w
(A)Clustering analysis shows that wild-type and mutant EYFP–: _1 q% c( f- \
cells are
  V6 A7 X/ Q# e& {indistinguishable, whereas EYFP+ cells from wild-type and mutant
2 a0 t4 X; i$ E  M5 M8 vembryos show distinct gene expression profiles. (B)Transcripts
. X. R2 q8 l- {- ~upregulated in the EYFP+ mutant progenitors include cardiac and: A" W' K4 ?2 I' ~+ j
skeletal muscle genes. (C)Downregulated transcripts in the Er71
9 U, r9 p. a8 Y* C) C- Tmutant background include hemato-endothelial transcripts, including" r/ F8 F( d$ O: [# Z; Y
Er71 (Etv2) (arrow).
7 `% X. I$ A9 ^7 p/ j+ YDEVELOPMENT4808
1 _' H4 C  `0 b0 Q5 b2 ^! Q; t" P* UlacZ reporter mice (supplementary material Fig. S5). Two
/ I" o7 j' o4 htransgenic lines were crossed to the Rosa-EYFP reporter mice and/ m% i- E% ^5 n0 I/ M5 R6 j1 ~
used for immunohistochemical and FACS analyses. Our results
5 ^0 k+ Y- C$ K6 P/ F8 m: u1 jrevealed that E11.5 embryos were marked by Rosa-EYFP or Rosa-6 B2 N* ?6 I2 J' O) m- k9 Q  d
lacZ in the endocardium, cardiac cushions, vasculature,
( p% ^1 z' g' L, G4 r( V1 Bmesenchyme and the fetal liver, which is the stage-appropriate site
3 R8 _8 l2 z) j8 f1 V9 V* I/ \of hematopoiesis (Fig. 6B; supplementary material Fig. S5G,H;/ J/ d# A0 D. s# F2 i
data not shown). Hearts of P3 neonates were marked by Rosa-
- Q! E; p* L% l5 ^& T7 bEYFP in the vascular structures (Fig. 6C). Co-staining for EYFP, Q* {! Y" d1 C. C1 y2 ]2 {  x
and desmin, an early muscle-specific structural protein, showed0 I3 H/ ]/ }+ |
that the Rosa-EYFP reporter did not overlap with desmin staining
9 D/ D4 ^: d+ t1 R& Ein the heart at either time point (Fig. 6B,C). FACS analysis of* l7 t6 G* K* t" c+ D# i( [
whole E11.5 embryos showed that 7-8% of cells in the E11.50 A5 Z( B  P& t  t
embryo were derived from ER71-expressing cells (Fig. 6D,E). Of) W9 x0 \0 e; E1 {. v+ n9 E9 J6 V
these EYFP+ cells, ~12% were endothelial (CD45–
) {$ a" k5 l/ B, cCD41–/ E; I6 g. A. U
Tie2+;* M* ?9 {7 B  h9 ^. [: ]  P
Fig. 6F), 23% were non-erythroid hematopoietic cells (CD45+, also9 L9 b9 T5 i9 l* I7 i( {* \' y( A
RESEARCH ARTICLE Development 138 (21)+ _# v- a% C1 `2 r" W
Fig. 5. Representative transcripts from the cardiac program are overexpressed in the Er71 mutant. (A-F)qPCR analysis was performed on
* B* B7 e; o0 HRNA 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),% `8 N# N+ a% l  d
Cdh5 (B) and Scl (C) expression and an increase in the expression of representative cardiac program transcripts including Nkx2-5 (D), Tbx5 (E), and
6 J) w) J3 i5 S1 r. w5 B: iGata4 (F). Error bars indicate s.e.m., i, a$ l6 Y$ E" ]1 F4 ]
Fig. 6. Genetic fate-mapping studies indicate that ER71-expressing cells give rise to the endothelial and hematopoietic lineages." e: B* b. ^; y$ N" Q( b; V
(A)The transgenic Er71-Cre mouse model used for these studies. (B,C)Immunohistochemistry for GFP (green) and desmin (red) and DAPI staining
: D) a( H- |% |( ]5 m9 _3 `0 \(blue) in E11.5 (B) and P3 (C) heart. a, atria; bw, body wall; cu, cardiac cushion; fl, fetal liver; v, ventricle. (D)A representative FACS profile showing' M% [6 @7 Y5 I  ?& H& [  |5 P+ r" L
EYFP+ versus side scatter width (SSW). (E)ercentage of Rosa-EYFP+ cells from all experiments (n9). (F-H)EYFP-positive cells were gated and* \" V! C6 v# h) R
analyzed for lineage contribution. Representative FACS profiles are shown for (F) endothelial lineage (CD41–0 f( \" u5 P  F( [/ O$ S
CD45–, X3 s4 _7 g8 Z$ h% ^
Tie2+) cells and (G) CD41+# z6 |. C% X0 N! p* j& \+ [! Q
CD45+, (H) CD45+ Ter119–1 G7 x; Y/ |4 W$ D: ~; I
and Ter119+ CD45–8 O' s' K: K0 L1 m1 i4 F. c# i
hematopoietic lineage cells. (I)ercentage of Rosa-EYFP+ cells of each lineage from all experiments+ S$ m! J1 ?# l% I  H( k
(n9). Error bars indicate s.e.m. DEVELOPMENTknown as Ptprc; Fig. 6H), and 13% were erythroid (Ter119+; also5 k: n5 z7 H, C6 W
known as Ly76; Fig. 6H). Interestingly, the EYFP+ population
( l$ }( ]' {3 ssegregated into two populations of varying EYFP expression (Fig.8 g# T+ p  L5 E/ j
6D). Ter119 primarily marked the EYFP-low population, whereas2 r9 o8 m5 I" ^, e
endothelial and other hematopoietic markers primarily marked the
9 O9 c+ {. O2 a/ B! c# @EYFP-high population (data not shown). This suggests that the' ?7 l' l# ?& E. j
Rosa reporters are expressed weakly in erythroid cells and might4 D& Q* w+ l0 g! m# j2 r, M
therefore inefficiently mark this population.2 _( M& D5 C6 a9 u% M2 b  H- M
ER71-EYFP-expressing cells give rise to0 h! ~- H  G0 z: S
myocardium in the Er71 mutant
1 d: o2 m( o6 [2 uTo determine whether ER71 reporter-positive cells could produce
0 E' `5 q  {+ z9 z2 L$ ~' ?alternative lineages in the Er71 mutant, we crossed the Er71-Cre
# o' B5 H# J  g! _and  Rosa-EYFP alleles into the  Er71 mutant background. As
2 R- _; @0 G8 D4 b/ N7 K& U. U6 C1 q3 Xobserved with the ER71-EYFP reporter, Er71 mutants carrying the
3 \) d+ m$ T% D2 cEr71-Cre and  Rosa-EYFP alleles displayed altered EYFP
: i! |7 ?. p$ i* [expression patterns. Compared with the Er71 wild-type embryos at
- G( p! R3 C  O# K! `, h) vE8.0, in which Rosa-EYFP is expressed in the yolk sac, vessels and
/ h: V" s4 O( G6 |heart tube (Fig. 7A), the Er71 mutant embryos had robust EYFP
3 M1 k+ U; Y! P8 [; `- ~expression localized in the allantois, posterior mesoderm and* j- o. G/ F& E, M% i' Q' C
paraxial mesoderm (Fig. 7F). We speculate that the increased
& o6 w* H% o% E5 I2 j1 aexpression in the allantois and the posterior mesoderm is due to the
3 }2 g2 ]& r8 b4 }( tprogenitor cells differentiating to alternative mesodermal lineages,
5 m/ @4 n% X/ i* v1 t5 u) qperturbed migration patterns, or a combination of the two. Embryos$ M$ ~. S( J% N0 A# h) p
were analyzed using immunohistochemical techniques for co-3 Z' H/ C  _, J4 M, i4 r. i0 }
expression of EYFP and Nkx2-5, a cardiac transcription factor,8 \; q! H7 t! Y' }# L. z
EYFP and desmin, or EYFP and troponin T, a cardiac sarcomeric
2 N. v$ v5 m# {( p- Q* G4 B; gprotein. In wild-type hearts at E8.0, EYFP was expressed in the% ~, _: F8 @% n1 @; w/ X
endocardium, the major vessels such as the dorsal aortae and in
; R. l4 Y7 k1 N& Xhematopoietic cells within the heart (Fig. 7B-E). There was no$ Z4 @4 m, f# ^, X4 U
overlap of EYFP expression with Nkx2-5, desmin or troponin T.* S* n3 {6 I, X
In the Er71 mutant embryo, the vessels and endocardium were& h" X  r: J0 p) l3 _- w# O5 y
absent. However, EYFP expression persisted in the heart. In the
, Y9 V4 E3 z3 x. h4 A3 zheart of the Er71 mutant embryo, all EYFP-expressing cells had an6 Z& m- i* O* ~, A' _2 L) v1 w
Nkx2-5-positive nucleus (Fig. 7G,H). Some of the EYFP-$ _2 |! d" V) ]( o6 C) a# i
expressing cells were also positive for the structural proteins+ ]" Z0 E7 v& ^( x! p1 X2 ]  _
desmin and troponin T (Fig. 7I,J, arrows). These results support the& D( H) n; z7 J$ b
notion that the EYFP+ cells are capable of differentiating to the
8 y" j  X$ M: q; g' O: a" Xmyocardial lineage in the absence of ER71. It is likely that
+ t& k  o. j) j. |progenitor cell populations are also capable of differentiating) Z3 i. P# h8 \+ m6 T
toward other lineages depending on the context of the spatial and+ G+ \1 V% J% }
temporal cues that they are exposed to during embryogenesis.
7 R4 g$ o+ W& H' R# H& X( Y) ^4 ]However, the heart is one of the few organs present at E8.0 and
- w6 n1 E! O$ }0 A! U. j+ K1 c4 d5 HEr71 mutant embryos are nonviable by E9.5. Therefore, the
2 Z  ?8 ^* j$ V' n3 Blethality of the  Er71 mutant limits our ability to evaluate the3 p/ f. H6 A- p# |
contribution of EYFP+ progenitors to other lineages that develop% F8 E; ~0 I! Y
later during embryogenesis.
# U0 D2 V) J$ i" M7 a3 r# _( s) d' L, mER71 overexpression suppresses development of9 g1 Z! N3 C2 h5 V3 @
the cardiac lineage
1 J. r/ Q! D8 X- N' lTo complement our analysis of the  Er71 mutant embryo, we$ D' |# k1 ]7 w/ i) `
overexpressed ER71 using a doxycycline-inducible embryonic
0 L+ g  t4 _7 v  @stem cell (ES)/EB system. An ES cell line in which expression of) g% q. R& \0 I( M4 o  x" ^
ER71 can be induced by doxycycline treatment was generated by+ t  c2 t' G! }0 l8 h2 T
a cassette exchange method (Iacovino et al., 2009). ES cells were. H1 Z0 s8 t+ T' w* [5 P; g
induced to form EBs in differentiation media and cultured for 104 \0 E* U3 o" [' m( U
days with or without 0.5 mg/ml doxycycline from day 3. In the
8 C, G% v3 ]" Z4 q; p# AER71-overexpressing EBs, we observed a decrease in the4 T! T9 V7 P& M# O' t& l  ~
expression of troponin T, which we quantified by comparing the# |. u! ]$ c5 V  z% a3 e2 |
positively stained area with the total area of the EB (Fig. 8A-E).! b; e* J/ K, ~# D6 D- r
We also observed a decrease in cardiac gene expression (Nkx2-5,% a$ V* w" C: A3 L
Tbx5 and  Gata4), as quantified by qPCR analyses (Fig. 8F).
9 v' y2 l, N1 M. p( N7 ]Finally, we quantitated the ability of the EBs to beat with edge
/ F5 o* T4 j4 L8 F. u" m! Ydetection. The doxycycline-induced EBs had a dramatic reduction
/ O9 s1 G8 q. i8 X6 u4 O2 Pin contractility (Fig. 8G) and in percent shortening (Fig. 8H).# V" X" \% E1 g5 g$ I
Collectively, these results further support the notion that ER71& G. n; ^$ r$ p- \4 L3 j% t
suppresses the myocardial fate of mesodermal progenitors.
% Z; K6 ?! a1 d$ [DISCUSSION8 m8 K' u) o4 A; B7 h8 |! k, A% u
An array of signaling cascades and networks govern the fate of
  ^& X8 S* A# X' cprogenitor cell populations and their contribution to mesodermal
$ B& E' I  {( t, W/ S1 Qlineages during embryogenesis. Recent studies have demonstrated. g7 k2 o3 g' ~8 Q
4809 RESEARCH ARTICLE ER71 governs progenitor fates' r2 f( R$ [) G9 M
Fig. 7. Genetic fate-mapping studies in the Er71 mutant show that the affected lineages give rise to myocardium in vivo. (A,F)Whole-
. D' a' i5 F& B( f& l; y# w8 Pmount 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
% X2 y+ F! s5 _% Tcorresponding 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)
4 o, ?0 O" o; n& P( s& N& _7 YImmunohistochemical analysis of EYFP and desmin (D,I) or troponin T (E,J) expression. ys, yolk sac; h, heart; al, allantois; da, dorsal aortae; ec,
0 _/ z5 l! k, h6 ]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.+ G& `! D5 @- M; }7 ?
DEVELOPMENT4810" `' @7 |, B4 ~% Y
that various progenitors give rise to multiple mesodermal lineages.
, @( e! Y4 p, O: I7 ?This broad developmental capacity is evident from the fact that' S  X5 ~. H6 S  `& `
multipotent progenitors have been shown to differentiate to diverse) y5 |. f! S- y. T
lineages, including hematopoietic, endothelial, cardiomyocyte and( M$ G" L" o. s/ i2 [: K6 Z
smooth muscle lineages. We have previously demonstrated that
! `/ S& O% K# e0 Q4 kER71, a member of the Ets transcription factor family, is essential
* }5 {/ N* V4 p7 q0 E, P. ^( h- Tfor the development of mesodermal lineages. Er71 mutant embryos! P" b. f6 u' l* z! `1 S
lack hematopoietic and vascular lineages and are nonviable. Here,$ V5 Y" L* _: D
we have further defined the role of ER71 during mesodermal* A- I. b9 N: Q! Z; s
patterning and have made three new discoveries that advance our
% f! m" z8 ^, \2 b5 Umechanistic understanding of mesodermal fate decisions during
0 T# X8 T8 l/ k' }development.% k% V# {; T6 `% X. b
First, we have defined the expression pattern of ER71 during/ b2 P3 h; D$ `9 d; S3 x
embryogenesis using a newly generated transgenic reporter
/ F* d1 Z; c# v6 w+ kmouse model. Using immunohistochemical techniques, we
1 F5 n$ N$ s$ c* i8 D/ H& M( vdemonstrated that ER71 is largely co-expressed with Tie2 in
2 d  Y: C' _% z* @* \* fendothelial and hematopoietic progenitors. In the absence of
2 t, N; v4 K2 @ER71, we observed that these progenitors are expanded in6 q9 b& ]+ B4 V$ J- M; h! \0 q9 d, Y
number but do not represent the hematopoietic and endothelial) g+ Z# N% ]% N7 \* X$ ^. T2 P4 l1 {
lineages; rather, they are redirected to other mesodermal
8 \' W7 b1 v5 e0 Vlineages, including the cardiac lineage. These data also show that) p( D0 {- x/ L
ER71 is not required for the initiation or maintenance of its own
; w/ i" \. s  w$ P# Nexpression. In the absence of ER71, this increase in the number
4 K! n! V' d! X/ o. C$ A" ^0 B0 qof progenitors (EYFP+ cells) might represent a delay in cell+ G5 _" Z3 N( V5 t5 V% P! f! R9 w
cycle withdrawal due to the lack of distinct mesodermal lineages% r- S& ~/ p0 p, S9 M8 Y7 W4 T8 ]1 j
(i.e. hematopoietic and endothelial lineages) or, alternatively,0 X0 R; F: @9 v  J% J. {8 r6 `
decreased programmed cell death involving these progenitors.& O2 u$ |, R; ?4 q0 {& j* ]
We did not observe an increase in apoptosis in the Er71 mutant3 p" j1 f* y; I+ L3 ?- _" p/ ^
embryo compared with its wild-type littermate control (Ferdous
. `2 Z4 v. i4 ~" U- _/ \6 J: a" Vet al., 2009). Thus, we hypothesize that the progenitor cell
; {/ j5 ?' T1 r. \! iexpansion in the absence of ER71 is a result of the lack of
# c: r% v3 N( y  xcellular differentiation to hemato-endothelial lineages.1 N6 `( V" }$ s$ F5 o( W
Our second discovery defined the fate of the ER71 progenitors
0 ^7 I9 A. m" e0 X5 k) [  pin the presence and absence of ER71. Using genetic fate-mapping" _8 h* ^; t% x+ A& M
strategies, FACS and whole-genome analysis, we demonstrated that+ y6 S6 ~* j2 h5 P8 c
ER71 progenitors give rise to hematopoietic, endothelial and
& a3 H' V! W$ i3 G4 Fmesenchymal lineages. In the  Er71 mutant background, these
9 ]3 E0 ~/ T2 T1 J+ Vprogenitors (EYFP+ cells) produced other mesodermal lineages," [7 M+ o+ M, D. q) u+ M8 ~2 J
including the cardiomyocyte lineage (supplementary material Fig.
3 i! y3 h1 n; W- H4 IS6). Moreover, our FACS and transcriptome data supported the
" }! c6 Q3 f* e+ Rnotion that paraxial mesodermal programs (transcripts enriched in
+ B+ @' W% `; i+ n0 {$ xthe skeletal muscle lineage) were increased in the Er71 mutant
! s3 Y: e8 a0 u  v+ Qprogenitors. Accumulating evidence suggests that the cardiac and5 O+ f8 X) W" @( g$ ^( W9 p& h* ~
hematopoietic lineages develop from a common progenitor and that
' E! ~% L: [$ X3 p5 A: Ethe specification of these lineages is inversely regulated. We7 N8 f" J9 d% q
recently demonstrated that Nkx2-5 has a dual transcriptional; Q3 K& r5 l7 }: W1 \$ n6 R
regulatory role during embryogenesis as it represses Gata1-
4 a4 k& F8 J3 o' g+ F8 F7 Hmediated hematopoiesis and promotes cardiogenesis of the
- H( j/ X* I1 ?multipotent mesodermal progenitors (Caprioli et al., 2011). The
% M( f8 M3 L8 e3 {" `" g2 hresults of the present study further support the plasticity of the: T/ H% D! X8 N" ^' `
ER71 progenitors and their capacity to respond to cues and
( Q; g6 [0 H3 R2 G; x# E8 ?contribute to other mesodermal lineages.6 A& D4 j8 R" L8 b6 x$ b( X) M
The origin of endocardium relative to the myocardium has( j1 w9 j# |+ R( {; f) C" `
been controversial (Harris and Black, 2010). Studies in zebrafish6 \  D7 e, R2 U1 r6 D$ V
and chick suggest that endocardial and myocardial lineages are
' C, M3 H. u# @distinct from the point of gastrulation, and there are no
4 R; p! b0 |3 I( h. t) a% Rmultipotent progenitors (Cohen-Gould and Mikawa, 1996; Wei
2 K! ?# J4 e( C: ]3 _9 ]+ oand Mikawa, 2000). By contrast, lineage-tracing studies in0 w6 W2 W) ]8 L- L1 \
mouse embryos and ES cells suggest that endocardial
5 m: _3 X( j% V9 n: }7 vprogenitors are specified as multipotent progenitors in the/ U) T) E5 j0 n. d1 e
cardiac crescent and cell fate is later specified to either
* k; Q: W" }2 D3 G1 cendocardial, myocardial or vascular smooth muscle fates4 Z0 O0 [+ |$ w4 i+ C
(Kattman et al., 2006; Misfeldt et al., 2009). In our novel ER71-( w; Z' q# }4 n& ^# h
EYFP reporter mice, we observed weak co-expression of Nkx2-
9 p  Z( e# g5 W1 j) K& _& g5 protein and EYFP in the cardiac crescent at E7.75. However,; Q. B3 ~/ T* O
lineage-tracing experiments showed that cells that expressed  F3 d4 r0 q7 ?) [) c3 \
ER71 during any point in their development did not become
. p0 m* B0 f5 k8 Ycardiomyocytes. By contrast, Nkx2-5-Cre lineage-tracing
& j) u+ f: I1 Q; q* c5 o! kexperiments show that at least some of the endocardial lineage
/ d, y5 ?5 M& V! m2 G3 Lderives from cells that expressed Nkx2-5 (Stanley et al., 2002).
4 Q' Z) I+ Q4 fTaken together, these observations favor a model in which
' n% B0 H8 e* w. u' C5 L! Jcommon progenitors of endocardial and myocardial lineages are
7 l9 O" u1 L- a/ g5 s  L& Cspecified as Nkx2-5+ cells in the cardiac crescent, but those that
7 p  F0 _# }: k9 l; m. WRESEARCH ARTICLE Development 138 (21)
) Z! C- k8 ]$ x  ZFig. 8. Overexpression of ER71 in differentiating EBs inhibits
# h5 C9 K, x  A/ u! J9 {; k" W- dcardiac development. (A-D)Sections of representative EBs either
2 J5 e, J7 b1 p; k: tuntreated (A,C) or induced to overexpress ER71 (B,D) stained with) o/ d3 z+ [  h! `$ C. w+ `, k* |" L& S
Hematoxylin and Eosin (C,D) or anti-TnT (A,B). (E)Quantitative analysis of
3 D3 @6 D$ Z' k! i+ p! t; pthe troponin T-positive area in 33 induced (Dox) and 42 untreated
& z6 I( b8 t) w$ U) {sections. (F)qRT-PCR confirms that representative cardiac program7 V+ n7 R+ V/ ?" M% ?0 @" M
transcripts (Nkx2-5, Tbx5, Gata4) are downregulated in ER71-
0 f$ c  S' J$ K" Ooverexpressing EBs. (G,H)Extent of contraction and relaxation after 2
, }4 P4 A8 v' J2 d. q) C- nseconds of stimulation (G) and percent shortening (H) are reduced in" }- U2 U- d4 R
ER71-overexpressing EBs. ****, P<0.001; *, P<0.05. Error bars indicate
: ~7 {/ u# [( X7 |3 Q3 d* j5 }s.e.m.
/ Y' l* c/ w8 Z6 A: Z; V4 lDEVELOPMENTwill become endocardium will downregulate myocardial
( }1 |9 R: q. R/ C1 `. ^! N" Fpotential, including the expression of Nkx2-5. Once ER71 is1 u/ t* n5 d( `
expressed, the fate is restricted to endocardium, and not
) E/ d( r& P- Fmyocardial lineages. Future studies are warranted to decipher the
( M; g4 a* Y+ b- X3 n2 }/ h; X9 imechanism that governs the specification of endocardial cells5 o% e5 y# g( r, G
within the cardiac progenitor pool.: ^2 N5 ?" j, B
Our third finding is that ER71 overexpression repressed cardiac) D4 ]' H% X. s- ?$ Q9 C' l
potential. These results support the notion that ER71, like Nkx2-5,
$ G' E' r) Q/ E' c8 V; Lhas reciprocal and overlapping dual roles in the specification of, g6 @0 k6 G+ O8 e* ?' j6 L0 L
mesodermal lineages. Our previous studies demonstrated that
: t- c7 n0 m; S/ Z& BNkx2-5 overexpression suppressed hematopoiesis, but not the8 {' |1 {# |) }( b- w& S/ J9 a
endothelial program (Caprioli et al., 2011). We further defined that6 E8 h7 j6 Z" Q! M
Nkx2-5 could, in a context-dependent fashion, transcriptionally
. r. u$ i" V1 }% w& e' Xactivate  Er71 and promote endocardial lineage development( s9 k+ _3 z) i8 y9 U/ z
(Ferdous et al., 2009). Collectively, our previous studies and recent
7 h$ z6 h% `8 I& ?1 zresults support the notion that distinct transcriptional networks can,7 P- H; c- \1 N) k
in a context-dependent fashion (and dependent on the local
; N8 c# O# c: r) s2 Q! O9 m, aenvironment), activate a hematopoietic or endothelial program and
! d7 v5 u* u3 wantagonize the cardiac program. These studies further emphasize5 I3 T8 D6 X7 Y$ d" q/ k
the plasticity of progenitors and their contribution to distinct* Z' N+ Z$ T* Z6 u3 k
lineages.
2 d2 T" Q" R+ K. Q7 p- i6 m) G" K5 zIn summary, our data support a dual role for ER71 in the
7 Y9 B5 z+ T/ o2 D% Fspecification of the endothelial and hematopoietic lineages and the# V8 o0 R! x0 w* K  E
suppression of other lineages (i.e. myocardial and paraxial8 W" _. p2 r( R" D0 z- r
mesodermal cell fates) (supplementary material Fig. S6). Our
/ D* j3 [( J0 ~/ Nstudies further support the notion of multipotent mesodermal
/ W8 L( v, r( V+ {! i  cprogenitors that are dependent on transcriptional cascades and other
' C' K! P; A0 P9 x8 T. Mcues to direct them to populate one or more lineages. Moreover,
. O$ g9 P5 U0 h6 d! s" ~6 Y+ uour studies reveal that global deletion of a single gene (i.e. Er71)
5 _: Z/ x# H6 k% r8 [aborts one or more lineages and redirects the cells to other lineages
: l3 a6 `9 }8 @. j  X3 w% [that would not typically arise from those progenitors. These studies5 N% O" w2 ~" J8 P9 g7 g: X7 g
further our understanding of the regulatory mechanisms that govern/ J7 o& Z1 c, c( \
mesodermal cell fate decisions and embryogenesis.
" S) Y/ ~! Z6 ]* n1 WAcknowledgements5 ^5 m, }8 {6 U9 I7 X& S5 z1 V
We thank Jennifer L. Springsteen and Alicia M. Wallis for assistance with' [' B6 H3 z: R+ X. J; x4 ^
histological analyses and animal care.; m" X9 d2 i/ D! ~1 R/ f7 ~# r. G
Funding
' r$ b% @5 ]" @Funding support was obtained from the National Institutes of Health [U010 d7 {) P/ W; T  X, G
HL100407 and R01 HL085729 to D.J.G.]; and the American Heart Association
& k. E+ Z1 y( t7 l  Z& F& x& k[Jon Holden DeHaan Foundation 0970499 to D.J.G.]. Deposited in PMC for7 m" \: w  [, x: u& l5 Y6 l
release after 12 months.
" c5 E0 f& F- I& p7 HCompeting interests statement8 f3 Q* x; F/ A, x8 U1 V+ O
The authors declare no competing financial interests.
; O3 C0 L" `9 |Supplementary material
+ y% }. p. Y5 i' ^Supplementary material available online at
/ G. F, [. `7 Z  U  q" K! ohttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.070912/-/DC1' l+ U9 A) M& ?: D, W, H
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) E; ?0 T5 p, A1 X/ R0 m* uRESEARCH ARTICLE Development 138 (21)6 f- c! G$ K2 e3 f. c$ T
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