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4801 RESEARCH ARTICLE, l" G [7 m( w
INTRODUCTION. ]/ u0 n* O# E, w/ X
Transcriptional networks, signaling cascades, microRNAs and' M2 M/ \9 l2 G- k! `8 \
other factors coordinately regulate stem and progenitor cell
6 M c" W7 K+ a6 l2 m( Kpopulations to give rise to the mesodermal lineages (Davis and Zur
) ^! M Y9 _ ]/ w, FNieden, 2008; Ivey et al., 2008; Omelyanchuk et al., 2009; Park et; b: N$ ? B1 o4 f0 Q+ u" d( C
al., 1998). During embryogenesis, a set of anterior progenitors( ] f5 Z5 Z$ }& ]) r! i# }3 ]
coalesce to form the heart. These progenitors, along with the blood, }3 s R9 {& f
and vasculature, comprise the circulatory system, which is the first
2 S% s0 O$ ~3 ]4 _% [, Zorgan system to develop in mouse and human. Disruption or) U0 U' c. E- J* ~. k
elimination of any one of these lineages results in early embryonic
& h0 Q# U5 F" s( Wlethality. However, the factors that regulate the co-emergence of q9 I H. b6 o" @* K: j5 v6 d" S
these lineages from the mesodermal progenitor pools are- N' t- j5 o2 }; [ |
incompletely defined.& {0 G7 V# Y& e$ r
Recent studies have identified a multipotent progenitor cell" W; a9 ^4 T, t
population that is capable of giving rise to the cardiomyocyte and
4 @% j" s9 h$ ^2 zendothelial lineages during embryogenesis (Kattman et al., 2006;
$ C0 `# w- ~. W M; |# v) C% A( OMoretti et al., 2006; Wu et al., 2006). Although these multipotent( J% a; @( D9 o- Q) V6 S
progenitors have been identified based on the expression of Flk1
( y3 r! ^& M8 c" i; ~% N(also known as Kdr), Nkx2-5 and Isl1, the transcriptional networks
. ]& }) x$ W# G% S! J8 {2 sthat govern the fate of these progenitors are unknown. These and
/ u, T1 M/ g' R) i+ d/ n* hother studies emphasized the plasticity and context dependence of$ d( |! n1 n2 O: s
regulatory cascades in the progenitor cell populations. For example,
* @- Q3 ]. L; Uour previous studies demonstrated that Nkx2-5 has a dual- V6 Z- A; u. b. f' ?8 ]
transcriptional regulatory role in the promotion of the cardiac3 @% [% U0 P" ^) V
lineage and suppression of the hematopoietic lineage (Caprioli et
( E7 s( |! g' `) d5 `/ |2 @" Oal., 2011). In this fashion, we proposed that the stoichiometry of
. P7 N( h2 A) G+ X. l. A) Tregulatory transcription factors and interacting factors could
) m4 F- }8 |7 ^! ]dynamically regulate the fate of progenitors. Definition of4 R2 b' V, u# F2 ]! G
additional networks and signaling pathways that promote and
8 [7 u; Y$ S/ {+ O, j* L h" Gsuppress the fate of distinct lineages that arise from multipotent
: Z k, E/ k- }: V: {. m8 z0 ]progenitors will enhance our mechanistic understanding of their3 b% K: b, d! V! Z1 ?
developmental potential.
1 p! e, z9 v# Q6 v& t, v/ D REts-related protein 71 (ER71; Etv2) is a member of the Ets. Z( t0 T3 z% h, B0 o7 x
transcription factor family that we and others have shown to be0 j# P: X1 q4 I# ~% M
essential for formation of the endothelial and hematopoietic
0 j2 o5 Y! U2 w1 q+ g* ]lineages (De Val et al., 2008; Ferdous et al., 2009; Lee et al., 2008).
5 W! {! t$ B" r* i: pEr71 can be induced by BMP, Wnt and Notch signals to promote p6 t L& J. j
hematopoiesis (Lee et al., 2008) and synergizes with FoxC2 to" Q0 R: D0 N$ \( w
regulate the endothelial program by directly targeting Scl (also
* J) G4 S! O9 {" }# H# Mknown as Tal1), Notch4, Cdh5 and Tie2 (also known as Tek) (De
R x2 D1 v5 e, u' {Val et al., 2008; Ferdous et al., 2009; Lee et al., 2008). In Er712 F V1 r7 C0 V |
mutant mouse embryos, there is a complete lack of hematopoietic
. g, Y: i) h' Tand endothelial lineages and the embryos are nonviable by# d3 t# R/ C: _1 z) k, G
embryonic day (E) 9.5 (Ferdous et al., 2009; Lee et al., 2008).
# l; w$ U; x, ]+ H, q% j2 RAnalysis of the Er71 mutant embryos revealed no differences in
. @! P" U$ M/ R) F$ @* |- [cellular proliferation or cellular apoptosis compared with age-* f& e/ X2 h# d0 r& r+ c* V$ C( v
matched wild-type littermates (Ferdous et al., 2009). These results5 Z; y0 z- D! e ^
raised the question of whether the hematopoietic and endothelial0 h; c& h: p' a9 `7 r5 x
precursors are still present but unable to differentiate to hemato-
! q) L1 i; K% }$ l, vendothelial lineages, whether they had been redirected towards, y, U# k; n% w" u. s9 {* u) H
other lineages or whether these cells never arose during
- w5 N! C* B- R/ ddevelopment.; Q) R' S8 L- a4 `6 Z/ q! e
Development 138, 4801-4812 (2011) doi:10.1242/dev.0709124 V8 S# c& R4 K G3 V' P
© 2011. Published by The Company of Biologists Ltd) `" J% {$ s, U7 p: H: i2 Q
1Lillehei Heart Institute, University of Minnesota, Minneapolis, MN 55455, USA.
: n# J- H/ Q" p# B% F( ]( q2Department of Integrative Biology & Physiology, Medical School, University of: ?! X7 y/ P- k9 o) |& e" L1 h
Minnesota, Minneapolis, MN 55455, USA.
" G% _( E3 Y8 T8 V9 o8 ^3Biolead.org Research Group, LC
5 |$ k+ B, I' L5 p M; w4 FSciences, Houston, TX 77054, USA. 3 l" I. i( C0 R5 }
4University of Texas Southwestern Medical6 A- x3 `, v1 y: ]3 g
Center, Dallas, TX 75244, USA.( {7 E* O. q$ B! \6 Z1 t8 p* ?
*Author for correspondence (garry@umn.edu)
$ N! s; p- i5 |$ |Accepted 5 September 2011: _2 m( O+ h* U" ?- C: x9 g H
SUMMARY2 W1 j7 n4 f, o2 i
Er71 mutant embryos are nonviable and lack hematopoietic and endothelial lineages. To further define the functional role for
. U$ s& c' x* o: t' ~9 ?8 @& nER71 in cell lineage decisions, we generated genetically modified mouse models. We engineered an Er71-EYFP transgenic mouse
3 F% B7 _' r7 g7 kmodel by fusing the 3.9 kb Er71 promoter to the EYFP reporter gene. Using FACS and transcriptional profiling, we examined the
1 h8 F: ^- U8 m9 m: GEYFP+ population of cells in Er71 mutant and wild-type littermates. In the absence of ER71, we observed an increase in the
4 p K6 E$ h4 Tnumber of EYFP-expressing cells, increased expression of the cardiac molecular program and decreased expression of the hemato-+ a, w) u# f: p0 J6 e, P: h9 ?# c
endothelial program, as compared with wild-type littermate controls. We also generated a novel Er71-Cre transgenic mouse
! t9 ]4 h0 g. E, F8 ]model using the same 3.9 kb Er71 promoter. Genetic fate-mapping studies revealed that the ER71-expressing cells give rise to the5 k, [. Q/ F" h: N
hematopoietic and endothelial lineages in the wild-type background. In the absence of ER71, these cell populations contributed
* l+ l# t+ l, j. l4 d. ^) b& }' Lto alternative mesodermal lineages, including the cardiac lineage. To extend these analyses, we used an inducible embryonic
# F+ a6 X9 ]" z: S" q# B nstem/embryoid body system and observed that ER71 overexpression repressed cardiogenesis. Together, these studies identify ER712 V: j; Y: O# ^& H
as a critical regulator of mesodermal fate decisions that acts to specify the hematopoietic and endothelial lineages at the expense) H8 J7 v# m/ b1 Z% f; k& s) Y1 O
of cardiac lineages. This enhances our understanding of the mechanisms that govern mesodermal fate decisions early during
2 D j& P; [$ Oembryogenesis.
3 d) b# h* h* U0 D0 OKEY WORDS: Transgenesis, Er71 (Etv2) knockout, Mouse
% ~, i- R* K) p1 c! q0 \ER71 directs mesodermal fate decisions during
1 M6 g% M; H) h3 V+ iembryogenesis
- k3 X3 \( d% u# ~: V' fTara L. Rasmussen1, Junghun Kweon1, Mackenzie A. Diekmann1, Fikru Belema-Bedada1,2, Qingfeng Song3, 8 x: s# _% K8 h7 n" A% r: J
Kathy Bowlin1, Xiaozhong Shi
" M+ w4 f! J9 l* S' j' R1, Anwarul Ferdous4, Tongbin Li4 I+ H5 _- `3 P2 e% v O z+ }
3, Michael Kyba1, Joseph M. Metzger1,2,
5 A8 Z' `6 s; DNaoko Koyano-Nakagawa1 and Daniel J. Garry1,
* _7 M& m( h% Y2 ^, Q& V*- X& A- J" ]2 B1 L
DEVELOPMENT4802
" K" K" l, g6 }7 q# O0 O, s q) M4 GIn the present study, we have dissected ER71-mediated% ]5 C* ]6 V: c) V
mechanisms that govern fate determination during murine, s$ n5 l- o$ E( \* I
embryogenesis. We have generated a novel transgenic mouse! l$ G7 F3 }1 o- K1 `2 {
model in which the 3.9 kb Er71 promoter drives the EYFP reporter& H+ i, ]0 c# o: c* Y
(Er71-EYFP), and crossed it into the Er71 wild-type and mutant ]9 A0 j4 a9 [! E" J7 }4 U% w
backgrounds. This strategy facilitates the isolation and
/ m0 L# A F: K: Qcharacterization of cells that would normally express ER71 from
2 ^! {) i9 |6 [9 |! P O5 GEr71 mutant embryos. In the absence of ER71, we observed the
& p" O. z7 ?* h8 q7 S7 e, gconversion of cells that would normally give rise to lateral plate
* x, O& ?1 H# _" Zmesoderm into cells that produce paraxial and cardiac mesoderm.
* N0 h! s5 c& l, MFurthermore, ER71 overexpression in vitro using an inducible. g2 L+ @& l8 X/ h7 w( K, h& w
embryonic stem cell/embryoid body system demonstrated5 e% d0 K+ {8 o! t# g( F o5 ]
decreased cardiogenic potential. Collectively, these data
5 H( G2 |* s* V7 Pcomplement and extend our understanding of the functional role of
$ K1 t- y [% O0 ~# LER71 to differentially promote mesodermal fate decisions during: n' y* j, A$ ]. o
embryogenesis.
7 R2 K/ c3 b2 G q% RMATERIALS AND METHODS
0 S3 u2 _3 U. b4 ?4 ?6 ^. FGeneration of transgenic mice3 o7 j4 K* V8 K- A2 G: N
The 3.9 kb Er71 promoter (Ferdous et al., 2009), which harbors the% f2 J! V" F; Z# j) t* W0 V
modules necessary to direct the temporal and spatial expression of ER71,4 X1 y5 O2 M' K# w: y' x
was cloned into the pEYFP-1 vector (BD Biosciences) and into a5 W h1 B7 X& V6 W7 H
promoterless pBS-Cre-pA vector to generate the ER71-EYFP and ER71-
, w/ ]- {/ ?, P' t3 G1 h/ L* VCre constructs, respectively. Transgenic animals were generated at the4 m/ K y3 |; Z/ ~
University of Minnesota Mouse Genetics Laboratory using standard* c/ j' i3 K( E& {5 A) l5 C
methods. Transgenic mice were screened for DNA integration by PCR.
4 E. Y8 B7 k. t% b" a. fExpression analysis of ER71-EYFP lines was performed by examining2 [3 M$ ~: \0 _1 O3 M- W
E7.0-9.5 embryos resulting from timed matings to wild-type CD1 mice
( P9 Q6 ]2 s0 F" u% j(Charles River). We also analyzed E11.0 and postnatal day (P) 3 offspring& I' I# V5 u# \0 g4 `6 s# Z
of timed matings of the ER71-Cre lines and the Rosa-EYFP line (Jackson
- _. f0 y% S; k3 t9 W( K- ELabs 006148). Founder mouse lines were crossed to the Er71* y% Y$ O1 R; e: l- B. l* S
heterozygotes previously described (Ferdous et al., 2009). In all cases, at; L" f" P, Z7 A( M# X5 P
least three transgenic lines were examined to confirm similar temporal and
& v3 b; e8 l7 G& Sspatial expression patterns. All mice were maintained at the University of1 P. ]6 `* ?( b, _- p8 m8 k: p
Minnesota under protocols approved by the Institutional Animal Care and# n- L) P: O( t- L
Use Committee and Research Animal Resources.* m, P' i3 i9 |! k
FACS analysis8 Y F$ |( U+ n, ?
Embryos from timed pregnant females were harvested at specified stages.# @) X; V% J& ^; _7 r
Embryos were separated from yolk sacs, which were used for genotyping,: _7 T6 y5 E# K4 q* J8 V& n
and imaged on a Zeiss Axio Observer Z1 inverted microscope prior to L1 h; q# ]& S1 o- J5 N; l
dissociation. Embryos between E7.5 and E9.5 were digested in 30-50 l# r G4 H4 x: J& x; u0 b
0.25% trypsin plus EDTA at 37°C. Digestion was arrested with 500 l
- _: n/ s+ X$ A) MDMEM containing 10% fetal bovine serum (FBS). Cells were pelleted at
7 {! ?& y' B1 n3 T7 X4°C (1600 g), resuspended in PBS and passed through a 70 m cell8 Z `( L& {- P" |4 p
strainer. Cells were incubated with antibody cocktails for 30 minutes,0 y5 h8 ~* @8 @7 t/ B0 n7 C
washed, and resuspended in PBS. Cells were analyzed or sorted using a
- {0 z2 @* U v6 \5 _: `0 iFACSAria (BD Biosciences). Various combinations of antibodies (1:1000); P1 g. x3 N; z1 o7 G! W/ P
were used, including Flk1-APC (eBiosciences 17-5821), Pdgfra-PE
( x" U0 J1 b$ n& Y(eBiosciences 12-1401), CD31-PECy7 (eBiosciences 25-0311), CD34-PE
r. H% f0 [% C# l(BD Pharmingen 551387), cKit-APC (eBiosciences 17-1171), CD41-" C5 y) ]6 ~! V: o& n& }2 g7 _
PECy7 (eBiosciences 25-0411), CD45-PE (BD Pharmingen 553081)," A7 [( F" e8 D9 G2 _
CD45-PECy7 (eBiosciences 25-0451-81), and Ter119-APC (BD
0 I/ K4 W6 s: E, m( aPharmingen 561033) antibodies.$ E1 h. Z9 {8 Q
Transcriptome analysis9 V+ F$ a9 {$ f3 y% S; ~* ^2 R9 H
Total RNA was isolated from 1000-5000 FACS sorted cells in TRIzol( `4 q0 c5 W1 A4 f% [3 T
(Invitrogen) using the PureLink Mini RNA system (Ambion). RNA was
. b) G" M4 K* K4 E4 I! isubjected to two rounds of amplification using the MessageAmp II aRNA& F u% L* P/ p0 g! g; R: _
Amplification Kit (Ambion) and then labeled using the MessageAmp II-
0 F; g6 e$ k8 D& {% X: `/ [Biotin Enhanced Kit (Ambion) according to manufacturer’s protocols.5 K- c! R: E! a1 `4 K: y! M- j
Amplified RNAs were hybridized to Affymetrix mouse 430 2.0 full genome" r/ p6 O1 ?( J0 v3 D
array chips at the BioMedical Genomics Center of the University of4 i+ s5 I& R: s# k
Minnesota. The CEL data files produced from Affymetrix array experiments
7 F) {' M5 Y7 w7 E0 `6 Iwere processed using the affy package included in Bioconductor. The robust1 p) ]: H: ?. H' x& Y' K0 n
multi-array (RMA) method (Irizarry et al., 2003) was used to perform data( n2 ]7 \ h- T. `" C* `
normalization, background correction and expression quantification. The3 Y% @2 F# s. M
limma package (Smyth, 2005) was used to identify differentially expressed
1 z$ }0 y; H7 H9 m. A9 w5 kgenes by ANOVA analysis, and false discovery rate (FDR) values were0 ^. Y! _& h* G$ m6 R( j
calculated using the Benjamini-Hochberg method (Benjamini and Hochberg,1 v0 I8 l4 L2 R$ H9 ^% y
1995). Average linkage hierarchical clustering analysis was performed using7 D3 y I$ i, r' Z* W2 ?
the cluster program (de Hoon et al., 2004), with uncentered Pearson’s1 i- W k1 M7 |$ z; B ?* h
correlation coefficient used to define pairwise similarity in gene expression.
. B. E( I3 { n3 OGenes annotated with the gene ontology (GO) term ‘heart development’
2 Z. K2 A8 c6 E4 c* W: [(GO:0007507) were downloaded with AmiGO tool (Carbon et al., 2009) and
7 u: Z" ~: l6 p, g7 ~used in producing the heatmaps. Microarray data is accessible in Gene9 |) }* B2 I k6 S
Expression Omnibus (series GSE32223).7 q F) S% v5 m5 I3 }6 u2 _
Quantitative RT-PCR$ m$ @) g" }) Z% g
Total RNA was isolated from 1000-5000 FACS sorted cells or embryoid
" d% _; k- I' O3 i% L/ Gbody cells in TRIzol (Invitrogen) using the PureLink Mini RNA system.9 \9 m/ X; Y" V
RNA from FACS sorted cells was subjected to two rounds of amplification3 k; s" U j$ j6 Q0 {+ r
as described above, but left unlabeled. cDNA was made using random
9 v* `. z4 I) E& b- f5 N$ a( mhexamers and transcript levels were determined using VIC-labeled (Gapdh,
: z! u- {0 @* S( Z2 S& N0 ?4352339E) or FAM-labeled (Er71, mm01176581_g1; Scl,
) p% _1 B. W1 C! C5 r0 o3 tmm01187033_m1; Cdh5, mm00486938_m1; Gata4, mm00484689_m1;
3 f8 K3 p% P' R6 ^7 Z' _! Y& [Tbx5, mm00803518_m1; Tnnt2, mm00441922_m1) TaqMan probe sets
9 [( `. B: ^- _(Applied Biosystems).
5 J8 Q: s% `+ Y3 M2 z }6 cEmbryoid body differentiation and ER71 overexpression
1 ?' z2 D5 c1 F& dDoxycycline-dependent ER71-overexpressing mouse embryonic stem& a1 Y. [3 i1 i. J3 I, `
(mES) cells were generated using an inducible cassette exchange strategy3 I! t0 u) X& e3 [" ^% |8 R! ^
(Iacovino et al., 2009). In this system, ER71 tagged with a C-terminal HA! o$ \5 ?: b: s% @
epitope was overexpressed in response to 0.5 g doxycycline. To mimic
8 h: h* D' q% Y7 S Yearly embryonic development, the mES cells were differentiated into
( h$ \4 E3 G( j9 ]8 }) u" uembryoid bodies (EBs) using mesodermal differentiation media [1.5% FBS$ [+ F4 a Y1 ]8 Q p" P6 i% k/ b8 a6 x
(Stem Cell Technology), 1 penicillin/streptomycin, 1 GlutaMAX
5 G* [" ^7 O: v1 @& I(Gibco), 100 g/ml Fe-saturated transferrin, 450 mM monothioglycerol, 50
: p) a% [; h& k, E* L5 X9 Ig/ml ascorbic acid in IMDM (Invitrogen)] (Kennedy et al., 1997). ER71
6 l: E; i# }- H+ Mwas induced on day 3 after initiating differentiation and maintained through+ u+ q; u/ j# Z5 t7 W
day 10 by supplementing the differentiation media with doxycycline. On
) v' B) w. u! p/ c/ _6 E! [day 10, we fixed the EBs with 4% paraformaldehyde (PFA) and prepared/ n Y4 X; ` x) S- ?# d- v
cryosections. We stained adjacent sections with Hematoxylin and Eosin( ?; h' m( L2 j3 X
using a standard protocol and performed immunostaining with mouse anti-
! ~& u4 u9 _6 Z2 YTnT serum (1:200, DSHB clone CT3). We assayed the TnT-expressing area5 T, } B2 n# L, D1 v
of each EB using ImageJ software (NIH) and calculated the ratio of the* {" i8 Q6 A( m0 S7 F
positive area to the total area from 33 sections of uninduced EBs and 42* w' k: e5 E2 x1 a
sections of induced EBs. EBs were placed on laminin-coated coverslips' O5 d5 k7 j1 h6 G" i) W( H
and stimulated at 1 Hz. Edge detection (Ionoptix, Milton, MA, USA) was
. ~5 w4 s: I& |" Z0 M. G" Wused to measure contractility of intact EBs.
' {4 h1 X( b# ^; Y9 vImmunohistochemical analysis
- k. _ [' [ ~Stage-specific embryos were harvested from time-mated pregnant females.4 O! H) J; [. w) R: a# r' ?
For paraffin sectioning, embryos were fixed for 4-8 hours at 4°C in 4%0 t3 I9 z! `% {: J2 z7 |6 }! i+ f
PFA and embedded. For cryosectioning, embryos were fixed for 1 hour at- i# ]6 O$ t# W
4°C in 4% PFA and embedded in OCT compound (Sakura). Sections were- j3 R3 D0 ]+ Y, i0 ~- @& u6 v
blocked with immunohistochemical diluent (2% normal donkey serum, 1%- s* q1 V1 u; ]" U0 N# f
bovine serum albumin, 0.3% Triton X-100, 0.02% sodium azide in PBS,$ a( a% b7 L: J8 t5 l7 e) _
pH 7.3) at room temperature and incubated overnight at 4°C with primary5 y' l: g2 v, O1 B1 `) ]* H6 y9 Z
antibodies, including chicken anti-GFP (1:500, Abcam ab13970), rabbit& o: y7 o+ J' c8 g% w
anti-desmin (1:200, Novus Biologicals NB120-15200), rat anti-Tie2 (1:100,5 d8 T! X0 F( i2 P- u4 Z. L
eBiosciences 13-5987-81), rat anti-Pdgfra (1:200, eBiosciences 12-1401-- Y$ \2 c9 C6 z
81), goat anti-Nkx2-5 (1:500, Santa Cruz SC-8697) and anti-CD41 (1:100,/ u; ]: \% O0 @2 ]8 P( x) j
BD Pharmingen 550274), anti-Cdh5 (1:100, BD Pharmingen 55289), anti-
^$ V9 c. y B# U7 y+ q/ ?CD31 (1:200, BD Pharmingen 550274), anti-Gata1 (1:100, Santa Cruz SC6 ], O% k( s/ z0 ]; R( E0 B! t
265X) and anti-troponin T (1:200, DSHB CT3) sera. Slides were washed3 r/ _$ z3 q, e
and incubated with combinations of secondary antibodies (1:200) including
0 j* J4 g3 U6 j" w+ y) Eanti-chicken Dylight 488, anti-rabbit Cy3, anti-mouse Cy3, anti-rat Cy3
2 f8 H ~& c6 }; d" @and anti-goat Dylight 549 (Jackson ImmunoResearch Laboratories).
) ?+ d x6 M% j! ?2 jResults were imaged on a Zeiss Axio Imager M1 upright microscope or a
6 {' l9 G2 R7 S& q7 c2 DZeiss LSM 510 Meta confocal microscope.: B/ N( Q7 j3 I, ? d! x
Statistical analysis1 J7 P9 `: l( E, _
Data represent the average of at least three replicates and s.e.m.
5 D% X2 s( b1 V+ l4 \$ vSignificance was tested by the Kruskal-Wallis test with the Dunn multiple
6 ?$ B4 b, I+ P) vcomparison test for more than two groups; for example, when comparing0 {) q4 o/ U4 U O; e% p, i3 |8 K2 Z
RESEARCH ARTICLE Development 138 (21)- |9 I. M. R( n3 W. ]4 |4 g
DEVELOPMENTwild type, heterozygous and homozygous mutants. Significance was tested5 ]4 [( R x1 d1 z* k) c- T
by the Mann-Whitney U test for two groups. Analyses were performed
/ h+ i6 r5 ^3 z/ ]using Prism5 software (GraphPad).
y7 s$ a& L _& KRESULTS
4 I' R, c6 W7 V. k. u! JExpression analysis of the ER71-EYFP reporter. Q& b$ U& ~* ^" z7 H; K# {# s
Initial analysis of ER71 expression in the developing mouse
: i: y& }( {9 G: zembryo was reported previously (Ferdous et al., 2009; Lee et al.,$ v6 R9 B# e9 \/ n* |" G8 Q
2008). To further examine its potential role in cardiovascular- Q! Y$ V3 z$ |( b& U# H4 T
development, we performed a detailed expression analysis of ER71
* s3 N% N- g+ J! Kfrom E7.0 to E9.5, when Er71 mRNA is expressed (Ferdous et al.,2 m2 @) M' r+ q0 B4 O6 X& v* w
2009). None of the commercially available ER71 antibodies% I' v, d- ^ S" F7 w
detected the endogenous protein, so we utilized an Er71 transgenic
+ y, v" |' m! M+ k' ~mouse driving an enhanced yellow fluorescent protein (EYFP)
6 l) |5 V. \3 t8 U: Greporter. The 3.9 kb region upstream of the Er71 transcription start9 o: Y' c1 A7 I) [
site was fused to the EYFP reporter (Fig. 1A). This promoter3 [ Z) C2 P+ s, v5 _
fragment was previously shown to drive reporter expression that0 W- W4 |% p9 X
recapitulated endogenous ER71 expression (Ferdous et al., 2009).
: s7 ^9 K( g0 L" e3 w7 L8 uFour founder lines were generated with offspring embryos that
' V. c% v: b1 mshowed the same expression pattern.
8 F+ i ]& E* o' n# s4 ~2 OTo examine the expression pattern of the EYFP reporter, we( R+ `* m7 O6 ]7 K. j
compared its expression to that of the angiopoietin 1 receptor Tie2,, M6 P9 Z0 W* I3 _3 Q9 v
which marks endothelial cells in mice and humans (Dumont et al.,
% S* t6 m. ] t' v; e! p% i1992; Sarb et al., 2010; Schlaeger et al., 1997; Schnurch and Risau,! t( \+ S% r/ a9 P9 i- r
1993). The EYFP reporter (representing ER71 expression) was
5 A; L6 U4 r* k+ Jexpressed as early as the early to mid gastrulation stage (E7.0; Fig.0 r1 G( e! z# e4 {" | h) `
1B, bracket). At this stage, reporter expression was limited to the
8 v# b1 k1 y& j1 Y* b# l: e3 Lextra-embryonic mesoderm (Fig. 1C) and showed co-expression
9 {8 x" f/ ]# N. U* C4803 RESEARCH ARTICLE ER71 governs progenitor fates
$ `; D" F' J" l7 e+ wFig. 1. The transgenic ER71-3 X8 O* \ b# ]2 @& e9 k
EYFP reporter is expressed
; Q1 [; r. q/ s1 Y- Fduring embryogenesis.1 ? g; i+ u1 `' z. q/ N7 s. g
(A)The transgenic Er71-EYFP; ^: f4 N1 x6 |* B) j( H" P
construct used in these analysis.. k) q% g, K- Y
(B-Q)Expression analysis of mouse
2 j' F2 v9 N! Oembryos between E7.0 and E9.5.
* `; q6 I5 Y6 ]EYFP protein fluorescence (green)0 S5 T7 i C* d- m' u8 R) Z$ _
is shown in whole-mount images- X5 R4 s" W/ P2 { j: g. J
(B,D,F,J,K,N). Sections were0 K- N) L# w6 v0 W& {
stained with anti-GFP (green),) L4 f, j5 {2 C$ X* z
anti-Tie2 (red), anti-Nkx2-5 (red)" M. R. i' Z$ i# m- G3 y, Z0 U9 J, r1 H
or anti-Pdgfra (red) antibody as
: t5 x# o! |7 c7 k( uindicated. DAPI nuclear staining is1 ~6 q; p! v& p4 t' C1 Q! `
shown in blue. Yellow indicates
/ ^8 K) X: v T* a0 M. X# @overlap of green and red1 w$ ]( A6 q( x# U9 o8 B
channels. (B-C) Early to mid% B6 f; I- v, C! h4 s X& k
streak stage, (D-E) early bud) E0 b, P$ A) ]" X: S4 r, ^
stage, (F-I) early head-fold stage,+ V, G g- N1 q8 Y/ w, G* Z
(J-M) E8.5, (N-Q) E9.5; embryos% ~. O) j% ^3 e: \) R# l
were staged according to Downs
6 G/ O) I$ v k3 b$ Tand Davies (Downs and Davies,
2 d' N- A* G" m" s1 k! k1993). Boxed regions are shown0 ~( |7 z; n3 T8 z3 i3 f: d, q" C
at higher magnification; planes of& q1 a; l; c" ^& }' ]
sections are indicated. See first
5 ]/ t# a2 \ d" p* Y) P7 Vsection of the Results for a
, S' h" W# R! s; G& s0 L. m cdescription of arrows, arrowheads/ k! F2 }* O. t
and other labels. al, allantois; ba,& q7 H% ]) A4 U
branchial arch; bi, blood island;8 I9 U: m2 h/ `; M% {
cc, cardiac crescent; cv, cardinal
. z' ]: I- C* q4 v; l1 {vein; da, dorsal artery; ec,9 {* h- N F- m$ r2 r& H
endocardium; h, heart; hf, head& D( K2 H# e3 _) ~
fold; np, neural plate; nt, neural
9 C6 w8 ~7 n2 stube; ph, pharynx; pm, paraxial
3 Y) v1 i9 u! o! l0 {mesoderm; s, somite; ys, yolk sac.
S4 \' x+ O/ CScale bars: 200m; 50m in
% R4 N9 e1 Q) W; l- xenlargements.
* ~$ H2 [- V4 O# b6 h* A( tDEVELOPMENT4804! c3 C8 Z5 j0 e" i
with Tie2 in the primitive blood islands (Fig. 1C,C, arrows) (Ema
& w# t; M" j) v7 A' J# E. v- J7 i/ t: Set al., 2006b). EYFP was also weakly expressed in the mesothelial
+ g8 l2 P2 K+ @$ ]( Z4 p2 Qlayer of the yolk sac, but Tie2 was absent in this non-endothelial( |) G! z0 v9 q( U1 d
cell population (Fig. 1C,C, arrowheads). At E7.5, continued
! F) J5 q9 L" p8 d, K" H! }" Hexpression of the EYFP reporter was observed in the extra-, J9 z W* k/ K6 [, @
embryonic mesoderm (Fig. 1D, bracket). In addition, scattered
9 K8 U6 N" b/ n/ s$ a% @: h: cEYFP+ cells were observed in the lateral plate mesoderm (Fig. 1D," V! W; ]( g, y |6 Y
arrows). Transverse sections showed that these scattered cells co-+ x2 W, b; M. |7 e
expressed Tie2 and most likely represent endothelial progenitors
* I0 K0 X/ _/ I" x) n1 W: r(Fig. 1E,E,E), although Tie2 expression has also been identified
5 \. L z( V- \) Z7 kin a subset of cells committed to the hematopoietic lineage
' v( ?0 T: \) p! ]2 f/ w(Kisanuki et al., 2001; Li et al., 2005).# @$ J% u+ S% M, c r$ F
At E7.75, the EYFP signal localized to discrete structures6 r$ a7 U4 i+ J
including the cardiac crescent and the progenitors of the dorsal aortae+ d' X; G I9 `1 ^ @% ]; _
(Fig. 1F, cc, arrows). We examined the expression in the cardiac
& }( D; X, o- d* j1 Z+ Xcrescent and paraxial mesoderm in detail using Nkx2-5 and Pdgfra2 U6 t3 E3 O( S) _7 k* a1 I
antibodies (Fig. 1G-I). We observed that the EYFP signal overlapped3 a, h0 R, N- T7 `: L+ |
extensively with Tie2 within the cardiac crescent (Fig. 1G,G).
( n8 j. z4 H" O" iThese EYFP+ cells are presumably endocardial progenitors (Misfeldt3 m5 ~% W; C! i4 r; P+ J
et al., 2009). By contrast, EYFP+ cells either weakly expressed or did
$ d3 S: s5 D4 u1 e; d) B* |not express Nkx2-5 (Fig. 1H,H, arrows and arrowheads,9 T- j' X6 {. B
respectively). We did not observe strong co-expression of Nkx2-5: [1 D( t. W; u2 e5 D
and EYFP. Pdgfra is strongly expressed in paraxial mesoderm and
* H- y" v) m: x. d+ r6 i: Ecardiac mesoderm (Fig. 1I, pm and asterisk). Double. w0 o2 G4 y! L% z) C& m6 [) N$ C
immunohistochemical labeling of EYFP and Pdgfra demonstrated
! y6 Y& R; K: X0 t$ ?- o0 ^( i2 a2 lthat most cells either did not co-express these markers or weakly
4 Z& ]4 A0 u" lexpressed Pdgfra (Fig. 1I,I,I, arrowheads and arrows, respectively),
' S; D+ a9 w8 X0 U! Zwhich is consistent with previous reports that the hemogenic lineage
# }- c8 _6 I" _9 F6 f; O/ `# xis segregated from the paraxial mesoderm early during gastrulation) u" H9 ~' F6 C0 `) f7 {4 S) s
(Kataoka et al., 1997; Takakura et al., 1997).
2 I' J, f' Y* Y/ s) \2 e1 e" cAt E8.5 and E9.5, we observed localization of the EYFP signal to
; {8 ~. X- Y* o" ^ ~vascular structures throughout the embryo proper (Fig. 1J,N; arrows0 N8 |% J6 ^) y' b( @1 {0 `: r+ @( J
point to the dorsal aorta and arrowheads indicate intersomitic vessels)
2 t& W" C. D. {# X) @and yolk sac (Fig. 1K). Transverse sections of the embryos (Fig.
8 `2 N3 e" Q+ o6 e6 }9 F1L,M,O,P) showed localized expression in the vessel structures# U$ \8 @) O5 e6 O
including dorsal aorta, cardinal vein, intersomitic vessels7 }* [6 D- P$ D0 W$ i
(arrowheads in Fig. 1M) and the endocardium (arrowheads in Fig.- f: B1 D: P7 t+ \! p4 I4 o" p. z
1L). The expression of EYFP largely overlapped with that of Tie2,
4 x7 q5 y% W" D M6 n8 E Aindicating that the Er71 promoter is active in early embryonic
: V0 n; `( q. @# h, s/ L) d$ Wendothelial and hematopoietic cells (Fig. 1O-P). We also observed% v5 b! l% ]$ M9 ` Q+ L1 K
that EYFP was co-expressed with the hematopoietic transcription* {8 ~& K+ C* V2 i6 ~* S+ |; M
factor Gata1, the cell surface marker of hematopoietic progenitors
' J X' t4 C) _$ t. hCD41 (also known as Itga2b), the marker of hematopoietic and; C/ l6 \/ \5 Y
endothelial lineages Cdh5, and with the primarily endothelium-
; X- N, u1 c7 T0 L+ x6 k6 k$ w8 Tspecific cell surface marker CD31 (also known as Pecam1)# ^3 ~- t; V( \0 K; I8 J* d
(supplementary material Fig. S1). By E9.5, Nkx2-5 was localized to6 ]1 w0 D3 l2 S9 K9 n9 n$ N# `" x
the myocardial layer of the heart and did not overlap with EYFP' x$ d C. g$ b/ y
expression (Fig. 1Q, arrowheads; compare with 1O).2 S- b* f% J1 |# e& v6 Z
Reporter expression is mislocalized in the Er71
# Z7 w$ H$ H, Z7 Emutant
1 d1 V5 F" ?2 Y7 z( t+ ~' yIn the Er71 mutant, the hematopoietic and endothelial lineages are
3 v: Y+ Z* i8 B! F' S$ j7 G# }7 zabsent. However, no changes were observed in cellular
& H3 {0 c3 I5 q. R% o5 k8 ?' M% {proliferation or apoptosis in the Er71 mutant embryo (Ferdous et
1 F) k: ^3 `) m' L& y$ @al., 2009). Therefore, we crossed the ER71-EYFP reporter line into# g) r& u6 a- s2 w6 L
the Er71 mutant background to define and characterize the cells2 q' a3 N4 \! U$ ?* n$ v- s
that should give rise to these lineages (Fig. 2). In the E7.75 wild-
c5 a7 A4 q% X) X$ g# {type embryo, EYFP expression was observed throughout the
+ i, q" m; P* U/ o/ x! t' rcardiac crescent, the dorsal aortae and the extra-embryonic Y. [9 y2 S! y2 v* X6 H4 Y( n
mesoderm (Fig. 2A). By contrast, in the E7.75 mutant embryo the2 }( D; x C6 p# j9 o4 s- I; R
expression pattern was less definitive. EYFP-positive cells were
2 U* V; }) `2 r! ]found in the extra-embryonic tissue; however, expression of EYFP, b0 h: s( R7 l* C3 }1 Y, d; u
within the embryo proper was mislocalized (Fig. 2B, arrow). EYFP
' F5 j' A) H8 Iexpression appeared diffuse throughout the cardiac crescent (Fig.4 e. ], O* p) g" s5 \- s
2B, asterisk). In the wild-type embryo at E8.0, EYFP expression. Y5 a( v: g; w! J1 M
was observed specifically in the dorsal aortae, but not on the
! _# R% w2 E/ u9 E0 [9 dperiphery of the embryo (Fig. 2C, arrowheads). EYFP expression
) `1 |* ^8 G; rwas also observed in the presumptive endocardium within the
6 R' J7 n' ?% x8 X$ k) [& p _% olinear heart tube (Fig. 2C). By contrast, the Er71 mutant embryos
$ x. X. f7 r/ h1 dexpressed EYFP only at the periphery of the embryo, which is
- h( y6 m) t" e: b. x! |/ s+ hlikely to represent paraxial mesoderm (Fig. 2D).+ L* m. K, m2 [2 M
Immunohistochemical analysis of ER71-EYFP and Pdgfra, a
3 t5 X" ~; W1 q! c; d5 _8 Y$ Nmarker of paraxial mesoderm, expression in E8.5 wild-type and
" ^/ [$ M0 v6 @/ Q6 b; S- A5 mmutant embryos demonstrates that EYFP-positive cells are+ `$ g) {$ X6 G7 u$ ^( `& ^- I
interspersed within the Pdgfra-positive mesoderm (supplementary% z8 X# Q( L* u0 M( K
material Fig. S2). FACS profiling showed that the frequency of
8 ?4 P- R6 n* [! s+ d' t; ]- gEYFP+ cells was increased in the Er71 mutant compared with the
7 Y+ C3 W8 I9 b9 M* p% V8 f- Zwild-type control at E8.0 (Fig. 2G-I), but not at E7.75 (Fig. 2E,F,I).
( e c! z. Z4 oThe apparent increase in the percentage of EYFP+ cells could be" n$ I! X" x7 f2 J; U
due to a reduction in the total number of cells without a change in2 b& l+ W1 J$ n' o" D8 O
the number of EYFP+ cells, as cell viability can be reduced by a' V& k0 H' _3 o
lack of vascularization. However, we ruled out this possibility as6 o, G% e3 t- @# q+ R! Z: P
we also observed a doubling in the absolute number of EYFP+ cells
& l+ a) A0 k( P( U7 v Oin E8.0 Er71 mutant embryos as compared with wild-type+ a7 N% q9 e+ Y. j0 ~
littermates (Fig. 2J).1 v+ i; A( i _4 U3 K3 P4 |$ J. }. J2 f
These analyses demonstrated that cells that expressed the ER71! N$ j Y8 S, o4 v5 C4 r5 t
reporter were not only present in the Er71 mutant embryo, but were
. i+ Z2 o( f5 ]0 {/ S: @also expanded in number. Since the Er71 mutant embryo lacks6 i: m& p, o9 T7 k# ^3 d8 C+ F
differentiated endothelial and hematopoietic lineages, we examined
3 \- D$ n& S$ ywhether these EYFP+ cells were arrested progenitors or cells& Z5 y4 k1 x$ v
redirected to other lineages. We used FACS analysis to examine the
3 K, Q8 L) v( |/ ?cell surface markers of the EYFP+ cells in the Er71 mutant and: ~+ C, `, h) M0 J; i/ b; @* Z7 H. s
wild-type backgrounds. Previous studies have established that& C t: ^. |! d
Pdgfra is expressed in the primitive streak at E7.0, but is restricted
L4 M2 F8 F$ `+ t6 mto the paraxial mesoderm at the early head-fold stages (Takakura
. U6 }- y' r' Z6 o5 w' c$ \, Bet al., 1997). Flk1 is expressed more broadly in mesodermal
- ] q: f" |" }. R+ Z! K* Dprecursors (Ema et al., 2006a; Motoike et al., 2003), including the* Y- b6 r, M. Q! N$ [, s, v
posterior portion of the primitive streak. Flk1+/Pdgfra–9 O% S- m& r6 j
cells mark7 E+ p: _$ _0 S& a3 a9 T. `' t! B! E
lateral plate mesoderm that will give rise to the endothelial and h0 h: _2 b& l# N$ ~* r3 h
hematopoietic lineages and Flk1–
& b& \4 N7 f: t+ v& Q; B/Pdgfra+ cells mark the paraxial6 E' e# f3 T+ q3 l
mesoderm. Flk1 and Pdgfra are known to be co-expressed in the5 j7 V" R: j% F3 s( d% q
cardiac crescent (Kataoka et al., 1997) and in cardiac progenitors4 P4 B: a. v. R5 ^; g( w
of embryoid bodies (EBs) (Bondue et al., 2011; Kattman et al.,6 d* v+ u0 P/ z! k& q! H; J" l
2006). In the present study, the frequency of Flk1+ cells within the s% H" T. r# G# K8 Q
EYFP+ population was significantly reduced at E7.75 and E8.0
5 }: \( x% t7 H; v(Fig. 3A,B,D,F). CD41 (Fig. 3D,E) and CD34 (Fig. 3E,F)
* s) _/ _ _7 y7 O, h" ]5 eexpression was essentially absent at E8.0 in the EYFP+ cells of the9 F' x% ?' B& P& n7 Y8 p
Er71 mutant. These results show that the lateral plate mesoderm% A/ u3 e' _3 a( \& c
lineage and its derivatives, i.e. the hematopoietic and endothelial
9 ` F5 ~' [7 M8 Y) S5 t: g7 m3 Q# qlineages, are reduced in the Er71 mutant.1 n. X) M- t7 P& G
In order to assess which lineages are expressing ER71-EYFP, we
7 X6 [+ G4 E, hanalyzed the EYFP+ cells using Flk1 and Pdgfra antibodies. ]/ S; e9 c$ O3 _$ l3 }0 |3 ^
Concurrent with the decrease of Flk1 expression at E7.75 and E8.0,1 \; ?" x" j! g! v
we observed an increase in the frequency of EYFP+ cells that
. D# A, C9 x: T* d5 F; O" R8 o9 zexpress Pdgfra (Fig. 3A,B). This correlated with a significant. G: \0 ], v) U7 N9 J
increase of Pdgfra single-positive cells at E7.75 and a significant
% D: h6 q* Q9 ], {# G! \8 nincrease of Flk1+/Pdgfra+ cells at E8.0 (Fig. 3A,C). There was also
1 } y3 \+ P# M9 Z+ ~* aa trend toward expansion of Pdgfra single-positive cells within the
! t3 D. v1 r7 g F( I6 j( UEYFP+ population at E8.0 (Fig. 3A,C). To support these& F# i/ j: y. w! n) c% J; `/ k
observations, we used immunohistochemistry to analyze the EYFP, T/ L" ?) \* L q. H2 |
expression patterns. In wild-type embryos, EYFP expression is/ U& n: X, k7 N( e1 a) s
distinct from Pdgfra+ paraxial mesoderm. A select number of! g- r) ?4 }) I! K' p
EYFP-positive cells are found within this region; however, these
9 ~4 E9 p# r% t( m1 ?2 i5 ^9 acells are Pdgfra–
+ K. M) l, b L" r2 E4 Y(supplementary material Fig. S2A,B). In mutant3 f7 M h5 o+ a* j/ L5 f
RESEARCH ARTICLE Development 138 (21)) k" W6 o$ _, }1 V" `2 e" ?
DEVELOPMENTembryos, many EYFP+ cells are found within the Pdgfra+ paraxial3 r0 d6 o) g/ f' F
mesoderm. Some of these cells are double positive for EYFP and) Y! ^& A4 m/ Q0 G' k
Pdgfra (supplementary material Fig. S2C,D). Likewise, in wild-
0 T, H: c# |" E" H' A* ]$ x5 g. ~type embryos, EYFP expression is distinct from troponin T (TnT)$ u/ G- ]' A3 P7 b4 _6 X
+
7 L' z( y, h+ V9 p: \8 Ucardiomyocytes (supplementary material Fig. S2E). However, in
+ t- q7 ?, x A( a% [, {mutant embryos double-positive cells are found (supplementary+ d, s d8 p' [0 l* o ]
material Fig. S2F,G, arrows). One interpretation of these data is
, \* c3 B5 }3 a% o1 E1 H7 mthat, in the absence of ER71, the EYFP+ progenitor cells
$ t- G* l6 N1 Z3 O* @6 odifferentiate towards the paraxial (Flk1–
. ~+ [9 _/ E, F( e+ ]/Pdgfra+) and cardiac
/ ?" T/ w+ Q$ Y& |; |0 _& g' imesodermal (Flk1+/Pdgfra+) lineages at the expense of the lateral, ^& G- Z' x! h+ t+ Z. _
plate/hemangiogenic mesodermal (Flk1+/Pdgfra–" ?9 t" {7 [- D/ M* m
) lineage.! A* v5 Y! _& f8 L9 @
An alternative explanation of these results is that EYFP is- c1 N1 Q1 V$ ]0 X# T7 U
expressed ectopically in cardiac (Flk1+/Pdgfra+) and paraxial
' S4 E6 p4 K; f" y- ~8 f5 |# P(Flk1–6 t$ ]+ |: }! j( v
/Pdgfra+) mesodermal populations in the absence of ER71. p$ X3 T J1 l2 i: K, j
If this were the case, we would expect the cardiac and paraxial' J/ }/ s j0 f" w" Y# }
mesodermal populations within the EYFP–# E3 R$ b& Y% z" o& U- s* m
gate to decrease in
: \" x* ]$ x1 qthe mutant embryos because these cells expressed EYFP and
! h& \$ [! J$ e5 {3 `: _, t& t! C9 _would be included in the EYFP+ gate. Furthermore, we would
2 {4 D( @, U- R0 I1 d+ h. w7 _expect no change in the overall representation of these markers, l# \9 I( m! q
in the unfractionated (the sum of EYFP+ and EYFP–
& K4 x5 f* [' O* ^/ a0 q" j) gate. To
) s9 }9 q) v7 a4 Kexamine this possibility, we analyzed unfractionated cells from/ n) [; P8 i g! x8 }
the whole embryo as well as EYFP–
: V3 m! `9 V$ y, Vfractionated cells. In the D, B/ {2 r: T& M9 Y1 e
EYFP–
& ^ S1 H1 Z% C: Ufraction no changes were observed in the mesodermal0 D: d+ C3 |6 D# n) p; f
populations at E7.75 (supplementary material Fig. S3C,D) and5 g6 W7 @$ R7 l9 w5 x( z) f
E8.0 (supplementary material Fig. S3G,H). Analysis of the* Y* K8 @7 }. s, }
whole population (the sum of EYFP–8 O# P5 k) ?1 m
and EYFP+ gates) showed8 y0 N6 Q- T* d& c
no change in cardiac or paraxial populations at E7.75
$ y/ H; D5 H& S8 k" m(supplementary material Fig. S3A,B); but at E8.0, we observed& _7 S, w% r5 ]* l' x
a small, but reproducible, increase in the cardiac mesoderm" t2 }, p* k+ T7 P% f
(Flk1+/Pdgfra+) (supplementary material Fig. S3E,F). We predict) ~3 [- Z4 r& p- |7 }) `2 g
that the increase in cardiac mesoderm in the whole embryo is
& h+ \5 X, I3 H. m6 S6 Ysmall because the EYFP+ population is a minor fraction of the
5 Z" b0 z9 j+ Jentire embryo.+ d2 i6 \. \( t2 c& L1 K
Collectively, our results support the model that, in the Er71! i" b7 r* N! W- ?
mutant background, EYFP+ cells differentiate to the cardiac( k% Z2 u/ B6 J' z: t5 x
mesoderm lineage and contribute to the increase in cardiac w1 h( N# I5 Y1 w
mesoderm (Flk1+/Pdgfra+) markers, rather than cardiomyocytes or6 J5 i' Y# |$ s& u$ _% t
cardiac progenitors ectopically expressing EYFP.5 ?* F% f, W9 u, m6 n" f
4805 RESEARCH ARTICLE ER71 governs progenitor fates2 `& w: w/ Q& S
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)
: S, T- c8 O* a" Cmouse 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
. g: b0 z9 v6 b i7 M. m8 j. Aindicate 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).* z) I3 ]; p/ j& k+ {
Representative FACS profiles of individual embryos (E-H) show the percentage of EYFP positive cells per embryo. Scale bars: 200m. (I)The) [9 m( P2 h! b, r$ G# H
percentage of EYFP+ cells for all experiments. (J)The average number of EYFP+ cells sorted per embryo. WT, wild type; HET, Er71 heterozygote; MT,
6 ]- w- A# Q( Z: ~, K) k) Vhomozygous Er71 mutant. ***, P<0.001. Error bars indicate s.e.m.5 \, m1 K" O" D" T; @, O
DEVELOPMENT48061 V, T6 _, @) } F( [, T. f
Cardiac and skeletal muscle genes are' z: ` Y2 i# b. X
upregulated in ER71-EYFP+ cells in Er71 mutant7 N1 [/ L7 p7 {' P6 D9 K$ \# y2 o: s
embryos
" t5 Z) J. Q% g8 {9 nTo further examine whether the EYFP+ cells in the absence of
( O! m0 m! U7 X4 j2 }( c: ^ER71 were redirected to other mesodermal lineages, we performed
; L s# y- B* M6 n {+ Qa transcriptome analysis. We used an Affymetrix microarray. M& Q. h4 r5 h2 R2 s7 C% C
platform and examined the EYFP-positive and EYFP-negative& j8 z2 Q% `2 e0 e4 c j& d7 O
cells from wild-type and Er71 mutant littermate embryos. Genes* W' W+ {& {* Q" A# u; E- K
that were significantly dysregulated were filtered based on the false
- L* C. L5 C% _) [1 K" Zdiscovery rate (FDR<0.01 for EYFP+ versus EYFP–
1 a7 }3 v/ N* a; l: s; FDR<0.35 for
8 E5 }% m: v# s# H! C( z1 [; Cwild type versus mutant; FDR<0.01 for the interaction between
& N" O4 N+ W6 Z6 V6 A$ |2 Dthese two variables). Clustering analysis was carried out3 r8 M& a, D9 c2 q
subsequently (Fig. 4A). Interestingly, there were no transcripts that" I8 D7 T! ?2 _8 o/ J- o
showed differential expression between EYFP–
- \, x) K2 ^: {" W" w( y8 R: ~populations in the
* c7 M# U+ s' Z/ `9 PEr71 wild-type and mutant backgrounds. Also, the cluster analysis; x# [+ e& X) h* q$ K
was unable to resolve differences between the EYFP–
z) g% g5 x8 E) ~- ]( Qpopulations
5 Y# @0 H5 e# W' W; T& WRESEARCH ARTICLE Development 138 (21)" l' a# j' s5 X' j
Fig. 3. ER71-EYFP+ cells in Er71 mutant mouse embryos lack markers of hematopoietic progenitors in exchange for those of other$ {3 m8 r7 _( e8 I, n$ U0 r( G
mesodermal lineages. (A)Using fluorophore-conjugated antibodies and FACS analysis, we observed an enrichment of Pdgfra+ cells in ER71-EYFP+' y: M, {' s' q+ \, P& f4 A
mutant cells at E7.75 and E8.0. (B,C)Summary of multiple experiments comparing individual fluorophores Flk1 and Pdgfra (B), and combinations of
& B8 q. J; ?! F, @% w6 \4 PFlk1 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
) S G9 g0 U9 ?0 lreduction in CD41 (D) and CD34 (F) at E8.0. (E)Summary of multiple experiments comparing hemogenic and endothelial markers CD41 and CD34.
u" F5 b; i9 k' `+ C* @**, P<0.01; *, P<0.05. Error bars indicate s.e.m. LPM, lateral plate mesoderm; CM, cardiac mesoderm; PM, paraxial mesoderm.
4 d* n4 P J+ z$ o8 eDEVELOPMENT(Fig. 4A). These transcriptome results for the EYFP–6 S, S3 {. r, D ]8 @4 d q1 H
cells support( E+ Z4 U; e; K$ X" Y* j# q/ @
the notion that EYFP is not being expressed ectopically.0 P0 l: h# } j {2 m
Furthermore, this indicated that there were no significant non-cell-8 ]% U* k( q3 E8 I* y$ ]: R
autonomous effects on gene expression resulting from the lack of, v% f& w$ M' _* I \5 @
ER71 at this time point.- [# o" ]. z' J( K9 m
Within the EYFP+ populations, the Er71 mutant and wild-type
1 a! [: h/ X7 ~6 R, Icells were significantly different (Fig. 4A). A large number of2 G% L8 X, F( a3 U9 s& M/ k- k; Z' f* _
hematopoietic and endothelial transcripts were decreased in
/ t, l2 Z- B- Y& ]expression between Er71 wild-type EYFP+ and Er71 mutant# x+ P8 }4 W: _9 N9 }
EYFP+ cells (Fig. 4C). Importantly, ER71 expression was restricted
3 y6 v. B5 ?! H; p K P6 m7 c" kto the EYFP+ cells in the wild-type background (Fig. 4C, arrow),3 p7 u/ U# g: V/ V/ p
confirming that the Er71-EYFP transgenic model reflects
[/ L$ V4 j2 X. d2 H% ~endogenous ER71 expression. Other transcripts that were6 ^9 K' @/ V$ C8 X9 |' o
downregulated in the mutant EYFP+ cells compared with the Er71
, {: c+ X4 o; C# L: kwild-type EYFP+ cells included endothelial and hematopoietic! N2 j2 ]# U6 d
transcripts such as Hbb-y, Fli1, Erg, Tal1, Cldn5, Cd93, Sox18,8 k! Z, k9 Y& x) |6 T
Cdh5, Sox7, Mmrn1, Eng and Nos3 (Fig. 4C). These data indicated; T4 t3 b$ i" Q+ J0 {
that the EYFP reporter-positive cells contributed to the endothelial
3 o/ T* P' }8 N6 F% m0 eand hematopoietic lineages and did not express markers of those6 N% d# E, ?2 l; R' ~
lineages in the absence of ER71, consistent with the previously6 S; u1 b5 f) r1 V
described phenotypes (Ferdous et al., 2009; Lee et al., 2008). x' ?+ X5 t+ i# [, n
Transcripts that were significantly upregulated in the Er71 mutant
9 y: p6 \. Z2 OEYFP+ cells compared with the Er71 wild-type EYFP+ cells were
) ^: k3 R9 ^9 Vassociated with muscle lineages (Fig. 4B). These transcripts
( V* i! {* {5 P9 R% u2 u, v4 tincluded Smarcd3, Myl3, Irx4, Tnni1, Myl2 and Tbx5 (cardiac-! ]1 H' z4 N8 U( q8 J
restricted transcripts); Acta1, Myl1, Svep1, Dpf3 and Cdo1 (skeletal; a1 B# m" C. K* B, Z
muscle-expressed transcripts); and Acta2 and Myl9 (vascular" H' M3 r8 g* b& d) @
smooth muscle-specific transcripts). These results suggested that
* D z/ K( ]* ?the EYFP+ cells gave rise to other mesodermal lineages in the
7 h( M9 y% B/ w# Yabsence of ER71.
& ^+ [, T2 N5 `, aIn order to determine whether the cardiac lineage was broadly
: G# C! j0 X( N3 _2 s2 t! aaffected, we analyzed transcripts from the cardiac development' Y8 C! r {, U5 L+ ^2 N
gene list in gene ontology (GO) terms and performed a hierarchical7 l, B1 R7 B; \3 ~3 X" T
clustering analysis. Similar to the results obtained from whole-
* n9 I0 l6 n @0 n( _" ]genome analysis, wild-type EYFP+ cells segregated from the
0 X+ }1 k2 ^; g' ?- R0 umutant EYFP+ cells (and all EYFP–
0 v# s( h( E7 z5 ?3 Q& ^: Ccells) (supplementary material
/ C: H! ^2 Q( |5 O, {* _. W% fFig. S4A). This result demonstrated that the difference between
( S: g% B6 J/ l; l8 g4 o# |these two populations can also be detected based only on cardiac3 J. Y b) k5 v4 V. c; o9 M
development criteria. Some transcripts were decreased in EYFP+
9 N( A( w" B$ t1 G2 u! O# SEr71 mutant populations compared with EYFP+ wild-type cells,; G+ X; i' t# _/ |& v. R) ^
including a number of transcripts that are important for endocardial
; q7 A1 d6 Y& g+ s0 q0 Adevelopment (including Eng, Hhex, Sox18, Nfatc1, Sox17)
" U" p/ B* v' k5 f* L6 S8 R8 }( u) k(supplementary material Fig. S4B). A number of transcripts were
. o& W0 {8 n0 s0 W Pincreased in expression in the EYFP+ Er71 mutant population
( S' ]% v- \6 C9 K4 y: q+ ?' E/ mcompared with the other three groups (EYFP+ wild-type and both
7 n, _9 m! L, H; ?$ DEYFP–
! l3 v. U$ |9 H' Dpopulations), including a number of cardiac transcription4 r* ~: t6 w% Q' G2 i z
factors (Myocd, Nkx2-5, Irx4, Isl1, Gata6, Tbx20, Tbx5, Gata4,
0 @4 Q. w; h7 c p1 ~9 b$ J+ E+ PSmyd1 and Srf) and structure-function transcripts (Myl2, Myh7,
1 m+ c$ j7 z2 I$ z+ p/ M1 uMyl3, Tnnc1, Tnnt2, Tnni3) (supplementary material Fig. S4C). We6 k% ?# b$ ~5 c" M5 O/ J* _
validated changes in selected transcripts by qPCR on independent
6 q" Y9 o+ p9 p9 W4 lsamples and confirmed decreased expression of Er71, Tal1 and
( m( g# `% D( OCdh5 and the overexpression of cardiac transcripts including Nkx2-
- z( N) _( D: r* `' U5, Gata4 and Tbx5 in Er71 mutant EYFP+ cells (Fig. 5).
2 A. v6 z; z7 ~$ X2 K0 ~" ]( KIn summary, our transcriptome analysis indicated that cardiac
+ y3 B/ Z I# t0 y6 _genes are over-represented in Er71 mutant EYFP+ cells, whereas
& `3 V& ]2 S* u, f, g6 B" rendothelial, endocardial and hematopoietic genes are under-
/ V' ]; N+ Y! R8 }represented.
: s& `3 J. A! j/ W$ PA novel Er71-Cre transgene marks endothelial and
! D8 s; @- Z7 X, E6 zhematopoietic lineages% r: f8 I9 Q X) T3 Z( f- P1 @0 M
In order to determine whether ER71-expressing cells give rise to
+ R4 O, y7 A! A. t/ J9 a) qorgans or programs other than the endothelial and hematopoietic
, S/ C2 U2 D4 J' l3 xlineages, we generated an Er71-Cre transgenic mouse model (Fig.
3 d$ `5 |/ x6 P7 \' G6A). We obtained a total of six founder lines, with embryos that% J. O' k9 N$ M9 ?3 C8 H/ ?" |
showed similar expression patterns after breeding with the Rosa-5 m y% B" |0 S/ F. ]3 G% E6 b
4807 RESEARCH ARTICLE ER71 governs progenitor fates
. x( w, l, K5 R$ \Fig. 4. Transcriptome analysis reveals that hemato-endothelial# @# n+ u: T, k2 `; [
and cardiac lineages are dysregulated in the Er71 mutant
; O$ x! b5 @0 m& bembryo. Transcriptome analysis was performed on EYFP+ and EYFP–
4 f s, p5 X# m4 L" `! ?cells sorted from wild-type and Er71 mutant littermate mouse embryos.4 ~4 N8 x6 o: ?- W
(A)Clustering analysis shows that wild-type and mutant EYFP–4 W( L0 }. c7 T
cells are
$ t9 l" s2 {! d! Tindistinguishable, whereas EYFP+ cells from wild-type and mutant
7 y- m7 i# Q1 Y4 N* Q! j( bembryos show distinct gene expression profiles. (B)Transcripts
, Q: b [& } A. E4 Bupregulated in the EYFP+ mutant progenitors include cardiac and8 N" a4 w5 U6 y# g+ t/ H
skeletal muscle genes. (C)Downregulated transcripts in the Er71, p- d9 G- D$ [- n' S) ]& V
mutant background include hemato-endothelial transcripts, including
) Z' f! f w: f# @5 Q; GEr71 (Etv2) (arrow).0 a! y2 x# s# ?" R- j# N6 j5 W
DEVELOPMENT4808; ]3 k4 z) @& H5 Z; ?: o! _! I
lacZ reporter mice (supplementary material Fig. S5). Two' A2 j( E8 ^# k7 ^1 F
transgenic lines were crossed to the Rosa-EYFP reporter mice and
( m! y3 h: @2 i' p" o: o! ^8 T3 Aused for immunohistochemical and FACS analyses. Our results
' C+ N# O: r2 a8 p/ H# q' @- i* Rrevealed that E11.5 embryos were marked by Rosa-EYFP or Rosa-' S' l. o1 f7 h! E3 y& r- D0 |4 w
lacZ in the endocardium, cardiac cushions, vasculature,* t% S; D) p/ T% b% a( x
mesenchyme and the fetal liver, which is the stage-appropriate site1 ]" w! l/ U' `5 Q r2 _3 i
of hematopoiesis (Fig. 6B; supplementary material Fig. S5G,H;. A+ m( E4 @3 j3 p; Z+ v
data not shown). Hearts of P3 neonates were marked by Rosa-
+ M) K2 \0 W5 K7 B* x+ w1 lEYFP in the vascular structures (Fig. 6C). Co-staining for EYFP
1 B& D) [3 T1 Oand desmin, an early muscle-specific structural protein, showed
0 `# _4 c3 o8 p- j4 L1 X t$ g7 i' dthat the Rosa-EYFP reporter did not overlap with desmin staining
% L0 J$ v3 {1 g+ O! |" G1 oin the heart at either time point (Fig. 6B,C). FACS analysis of1 i5 H2 s: n8 i# l( u( k1 \* ^; \5 S
whole E11.5 embryos showed that 7-8% of cells in the E11.5 z! E1 z: ]& l! @
embryo were derived from ER71-expressing cells (Fig. 6D,E). Of
# N' f5 Z0 d# |, ` `these EYFP+ cells, ~12% were endothelial (CD45–1 U2 T* B, V3 A7 [& ]8 s0 C
CD41–6 x l h* R5 }9 n M
Tie2+;
! J$ H; L. K$ dFig. 6F), 23% were non-erythroid hematopoietic cells (CD45+, also6 ?. G5 R* m2 a% Q' H
RESEARCH ARTICLE Development 138 (21)1 h2 ^0 o3 {6 A# ?: m
Fig. 5. Representative transcripts from the cardiac program are overexpressed in the Er71 mutant. (A-F)qPCR analysis was performed on7 a1 J2 `7 b4 `, c( 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),
4 |* a' s" N' n" T: s; NCdh5 (B) and Scl (C) expression and an increase in the expression of representative cardiac program transcripts including Nkx2-5 (D), Tbx5 (E), and
; d% Y7 l) {* N) J4 b- LGata4 (F). Error bars indicate s.e.m.
$ P9 F/ Z3 L1 CFig. 6. Genetic fate-mapping studies indicate that ER71-expressing cells give rise to the endothelial and hematopoietic lineages.0 f h; `9 P+ O* W/ z2 E
(A)The transgenic Er71-Cre mouse model used for these studies. (B,C)Immunohistochemistry for GFP (green) and desmin (red) and DAPI staining3 Z0 J8 ~ k7 u! F, `
(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: L0 m& r7 q' U3 h9 k+ U% O( |
EYFP+ versus side scatter width (SSW). (E) ercentage of Rosa-EYFP+ cells from all experiments (n9). (F-H)EYFP-positive cells were gated and
/ V* ~8 j4 q; Z( z- {, E( C& t% \analyzed for lineage contribution. Representative FACS profiles are shown for (F) endothelial lineage (CD41–. z, Q, E6 }4 |' b3 v
CD45–5 V& }& f. ]) Z6 K3 i
Tie2+) cells and (G) CD41+
# X/ Z8 t2 y7 p" DCD45+, (H) CD45+ Ter119–
6 o# S0 t# x0 M' t% U6 `and Ter119+ CD45–% d9 F/ K9 q. V( h
hematopoietic lineage cells. (I)ercentage of Rosa-EYFP+ cells of each lineage from all experiments
- k3 Z( N4 {( Y: d" S9 r; B(n9). Error bars indicate s.e.m. DEVELOPMENTknown as Ptprc; Fig. 6H), and 13% were erythroid (Ter119+; also
0 L' S4 {0 F. g! yknown as Ly76; Fig. 6H). Interestingly, the EYFP+ population) P, C! X3 }7 X( ?- R2 J
segregated into two populations of varying EYFP expression (Fig.! ]3 W+ v+ S- c1 h
6D). Ter119 primarily marked the EYFP-low population, whereas
# S5 R# Z) J8 s% W2 rendothelial and other hematopoietic markers primarily marked the# x, L& Y! B0 W6 E' J3 e' |) i
EYFP-high population (data not shown). This suggests that the
0 X! ?- y" q) T) lRosa reporters are expressed weakly in erythroid cells and might8 a/ h) R0 f+ U0 x$ x. E5 n) P V
therefore inefficiently mark this population.
. h* [& a. D' c" KER71-EYFP-expressing cells give rise to
" F4 m) D$ `! zmyocardium in the Er71 mutant
3 {+ b# y7 I8 Z$ I, cTo determine whether ER71 reporter-positive cells could produce
0 m! ?* q! N( f2 g# f2 {alternative lineages in the Er71 mutant, we crossed the Er71-Cre! ]& I% I. o# b
and Rosa-EYFP alleles into the Er71 mutant background. As2 N# [8 F7 _- }$ I1 x- `( L
observed with the ER71-EYFP reporter, Er71 mutants carrying the
. Y+ ~! Y7 `! O$ iEr71-Cre and Rosa-EYFP alleles displayed altered EYFP* n: E8 ?6 }* m- {: `7 \
expression patterns. Compared with the Er71 wild-type embryos at' p3 k" J3 Q$ P0 B% _
E8.0, in which Rosa-EYFP is expressed in the yolk sac, vessels and4 O# j" Q, F/ j2 N9 E
heart tube (Fig. 7A), the Er71 mutant embryos had robust EYFP+ q% ]2 \- `4 B" C
expression localized in the allantois, posterior mesoderm and: y, B, b; c" |0 r4 {9 L e
paraxial mesoderm (Fig. 7F). We speculate that the increased8 H* a+ z1 q/ X' d, P: N; c
expression in the allantois and the posterior mesoderm is due to the0 D& g: s! n* P
progenitor cells differentiating to alternative mesodermal lineages,
7 L# h- |& a0 D' x8 n3 Wperturbed migration patterns, or a combination of the two. Embryos
* i3 C* h9 i# G4 V4 ~were analyzed using immunohistochemical techniques for co-
. [; L2 h( ]4 T* Aexpression of EYFP and Nkx2-5, a cardiac transcription factor,
8 Q3 D+ G1 K# A V8 B6 IEYFP and desmin, or EYFP and troponin T, a cardiac sarcomeric8 u4 m7 M, `. }8 K, d" N3 o! w
protein. In wild-type hearts at E8.0, EYFP was expressed in the8 c8 P3 ~" P7 _2 \) y3 ?( }8 @- u
endocardium, the major vessels such as the dorsal aortae and in% i" ~$ n* N: T/ [1 q1 \
hematopoietic cells within the heart (Fig. 7B-E). There was no1 N: O7 w1 F( Z6 d9 I* o
overlap of EYFP expression with Nkx2-5, desmin or troponin T.+ [& `# d; M$ b0 {' ^5 u2 Q2 K
In the Er71 mutant embryo, the vessels and endocardium were
3 L' ~$ a+ W* o. l& u7 d5 Zabsent. However, EYFP expression persisted in the heart. In the7 ~1 l1 X+ t: N) N* H
heart of the Er71 mutant embryo, all EYFP-expressing cells had an! ?4 K( t K0 a. H
Nkx2-5-positive nucleus (Fig. 7G,H). Some of the EYFP-
. B& p4 d6 ~% [expressing cells were also positive for the structural proteins
1 P( V- w5 b0 s, \* @, }2 x8 |( ]) H* Jdesmin and troponin T (Fig. 7I,J, arrows). These results support the
r0 ] ~" o$ E9 T/ |6 s/ Qnotion that the EYFP+ cells are capable of differentiating to the
/ Z) A0 {8 Z7 ]& E$ Xmyocardial lineage in the absence of ER71. It is likely that
( C; I* }5 d' V" O' K0 [) n" K3 Zprogenitor cell populations are also capable of differentiating7 k) u! `9 t- M* O
toward other lineages depending on the context of the spatial and
' Q) h9 d. o4 v& t6 otemporal cues that they are exposed to during embryogenesis.7 a6 c! o6 T& T: ]
However, the heart is one of the few organs present at E8.0 and+ [* J/ C+ t3 n: N
Er71 mutant embryos are nonviable by E9.5. Therefore, the# M$ W; Z# ~ ?& s
lethality of the Er71 mutant limits our ability to evaluate the
% V1 S" L: ] [) \7 Z3 qcontribution of EYFP+ progenitors to other lineages that develop7 V2 ?! y/ }/ Y" K' ~
later during embryogenesis.
5 A0 m! L' j% R! n8 o UER71 overexpression suppresses development of
, n" {( r' H T+ Jthe cardiac lineage U6 V: U2 S0 @% G) O7 q
To complement our analysis of the Er71 mutant embryo, we
: f: ~( E1 Z9 H2 s' Q8 w! ~% Boverexpressed ER71 using a doxycycline-inducible embryonic; w$ G6 Q. W) f% b6 G
stem cell (ES)/EB system. An ES cell line in which expression of
+ \' ?$ j# N6 y8 ^' dER71 can be induced by doxycycline treatment was generated by
4 Y6 g. H7 }( v6 G( O; S8 ma cassette exchange method (Iacovino et al., 2009). ES cells were
+ {+ R% y2 W9 J" Ginduced to form EBs in differentiation media and cultured for 10. @1 _7 O+ Z2 a/ B8 {1 ` F5 N t+ h0 l
days with or without 0.5 mg/ml doxycycline from day 3. In the& `) M* S& t- d; l. M) P+ ?2 w* v
ER71-overexpressing EBs, we observed a decrease in the
3 [& a# a$ O- o0 mexpression of troponin T, which we quantified by comparing the
+ @0 ~- d3 y0 r. `5 g- mpositively stained area with the total area of the EB (Fig. 8A-E).
# p) C& w# t; DWe also observed a decrease in cardiac gene expression (Nkx2-5,! N. t3 o7 F$ M
Tbx5 and Gata4), as quantified by qPCR analyses (Fig. 8F).. t, u; h# g+ u( g3 c+ m
Finally, we quantitated the ability of the EBs to beat with edge
' F4 Q& w, K2 A* ]detection. The doxycycline-induced EBs had a dramatic reduction
2 \( a9 o$ A% u6 l3 q5 s7 C0 ^in contractility (Fig. 8G) and in percent shortening (Fig. 8H).
$ ?$ x. G3 f/ d4 B5 O( F& b' rCollectively, these results further support the notion that ER712 l6 Z1 r, y1 C" E( c
suppresses the myocardial fate of mesodermal progenitors.
- _. ]* ] N+ {4 D+ w% `: U4 }/ T8 v0 _7 dDISCUSSION! ~, {9 Y4 `4 H6 H' }$ Z- x
An array of signaling cascades and networks govern the fate of. o. b4 w) W( L4 J" p) n
progenitor cell populations and their contribution to mesodermal
! x/ V7 k7 f& d" {lineages during embryogenesis. Recent studies have demonstrated
& L5 \( _) a6 A4809 RESEARCH ARTICLE ER71 governs progenitor fates$ h8 |# h4 u7 d# B# z! r( f
Fig. 7. Genetic fate-mapping studies in the Er71 mutant show that the affected lineages give rise to myocardium in vivo. (A,F)Whole-
! Q0 `( Q$ ]8 H$ ~3 e, ]- Vmount 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
1 R( U3 w& R @! `( ~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). E, `, ]6 ]) f0 A. |; p) F
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,% d. B: R( z5 v" R E* N
endocardium; mc, myocardium. Arrowheads indicate EYFP colocalization with desmin or troponin T. Scale bars: 200m in A,F; 20m in B-E,G-J.
- R' l3 x0 z% U7 ]6 n0 y$ ^DEVELOPMENT48109 {9 q& n! m2 C9 w1 z9 d; X
that various progenitors give rise to multiple mesodermal lineages.$ t1 z6 S9 }# f1 Z# a
This broad developmental capacity is evident from the fact that
# K* {- O, ^- w7 ?2 J7 k5 e" Kmultipotent progenitors have been shown to differentiate to diverse! y3 w r% Y3 R
lineages, including hematopoietic, endothelial, cardiomyocyte and
`1 a1 a7 N# h& \ \* l' Msmooth muscle lineages. We have previously demonstrated that
% o4 `( M1 J$ \% d' s/ W) GER71, a member of the Ets transcription factor family, is essential7 s+ F# R" q2 g$ S' s
for the development of mesodermal lineages. Er71 mutant embryos. K) W+ a' [8 D) K# [* W
lack hematopoietic and vascular lineages and are nonviable. Here,
5 Q& w" ^. p) Y$ J2 e0 xwe have further defined the role of ER71 during mesodermal3 {% Y2 V# a, _; k, P
patterning and have made three new discoveries that advance our
4 @! F3 r9 `6 r$ imechanistic understanding of mesodermal fate decisions during
9 z p# v9 x& ^) h; [development.
3 F) E/ X0 m# PFirst, we have defined the expression pattern of ER71 during
; e# \, q% t1 `embryogenesis using a newly generated transgenic reporter
8 E1 j- r+ a5 b' W- R6 omouse model. Using immunohistochemical techniques, we( l0 z) O9 u) G- z' ~
demonstrated that ER71 is largely co-expressed with Tie2 in! P+ S7 l2 Y! M8 Z# K: `
endothelial and hematopoietic progenitors. In the absence of$ c/ z( R3 ^8 X
ER71, we observed that these progenitors are expanded in
4 t3 O; A% j z/ a9 |+ G. \number but do not represent the hematopoietic and endothelial
% y5 j2 w, I0 `6 olineages; rather, they are redirected to other mesodermal
5 \! M+ k! U3 N3 rlineages, including the cardiac lineage. These data also show that$ J. |: E7 _3 G4 _7 e
ER71 is not required for the initiation or maintenance of its own
! ]% M" B6 W' @- B7 aexpression. In the absence of ER71, this increase in the number" d1 D+ J5 N$ L+ z. t3 C4 l
of progenitors (EYFP+ cells) might represent a delay in cell
* U- u/ g q% N9 Q. ~9 acycle withdrawal due to the lack of distinct mesodermal lineages% ^6 O- g9 v* ^4 k& n) O1 A" I
(i.e. hematopoietic and endothelial lineages) or, alternatively,
- v. P2 H$ _$ _6 O9 _- K2 `decreased programmed cell death involving these progenitors.0 J6 ^! a7 n3 N( _+ v5 k v
We did not observe an increase in apoptosis in the Er71 mutant
- m: m# D$ f5 E: v- e# Vembryo compared with its wild-type littermate control (Ferdous( Q0 H7 |9 A: h6 y/ I& V. k
et al., 2009). Thus, we hypothesize that the progenitor cell
* P. U% k( {7 p cexpansion in the absence of ER71 is a result of the lack of) X. S- w$ R: U% ~9 L: P
cellular differentiation to hemato-endothelial lineages.
4 t, e* |/ w* n! D. o+ d6 pOur second discovery defined the fate of the ER71 progenitors2 L: v& T$ C' c
in the presence and absence of ER71. Using genetic fate-mapping5 G5 m( M. X/ h0 E9 j& R# K
strategies, FACS and whole-genome analysis, we demonstrated that
+ \* w6 M J3 ?$ a' xER71 progenitors give rise to hematopoietic, endothelial and
% c' l% K/ R1 z8 t6 Rmesenchymal lineages. In the Er71 mutant background, these
; ^0 o$ O$ f& X6 C1 d8 N; Cprogenitors (EYFP+ cells) produced other mesodermal lineages,
( Z1 T( P$ K: j+ d% Z* aincluding the cardiomyocyte lineage (supplementary material Fig.4 M4 F8 n7 ~) z% k& T) D
S6). Moreover, our FACS and transcriptome data supported the
7 I) R" \8 M% d, s$ Nnotion that paraxial mesodermal programs (transcripts enriched in1 I6 U6 I' Q: ^/ Q+ A' g' N h
the skeletal muscle lineage) were increased in the Er71 mutant
' W' e) A7 B \ O9 E, }progenitors. Accumulating evidence suggests that the cardiac and
% t0 X5 g* H. v0 s' Q, i" H/ V3 Nhematopoietic lineages develop from a common progenitor and that* v$ }! T' M0 o$ i6 n
the specification of these lineages is inversely regulated. We( R/ j8 ]6 a8 s+ Y5 E
recently demonstrated that Nkx2-5 has a dual transcriptional
: [$ t* B% ?5 O* G% g! |regulatory role during embryogenesis as it represses Gata1-0 F" U8 |& d, R& q: |
mediated hematopoiesis and promotes cardiogenesis of the
) h/ M) e& h& a# }! A9 Cmultipotent mesodermal progenitors (Caprioli et al., 2011). The- o5 ]' ^. `% j- S8 D# K' B
results of the present study further support the plasticity of the
3 J- X" Z/ l. Q ^1 SER71 progenitors and their capacity to respond to cues and- \3 O9 ?+ Q4 C) I' _
contribute to other mesodermal lineages. @& X+ l: P* V) w" u
The origin of endocardium relative to the myocardium has) V+ O9 c% w: t" q8 ?! J
been controversial (Harris and Black, 2010). Studies in zebrafish- `; Z o# m/ a
and chick suggest that endocardial and myocardial lineages are# C& X1 }* V- x- O" M1 V
distinct from the point of gastrulation, and there are no- W$ ~4 P' K' J: ^4 h+ i+ o
multipotent progenitors (Cohen-Gould and Mikawa, 1996; Wei7 F7 q. n6 h* I& z7 n( V" G" C$ R0 S
and Mikawa, 2000). By contrast, lineage-tracing studies in
8 J+ A! \+ q3 i/ gmouse embryos and ES cells suggest that endocardial
% ~6 Y( ~. D% }4 }2 Sprogenitors are specified as multipotent progenitors in the$ J9 |( }! }; _ K m6 l5 i
cardiac crescent and cell fate is later specified to either
/ v1 L( ^6 E# I- q9 Oendocardial, myocardial or vascular smooth muscle fates
: a# s6 L6 g6 l% ?5 M(Kattman et al., 2006; Misfeldt et al., 2009). In our novel ER71-
4 d: X+ b8 \3 g2 q* G) F7 ]1 oEYFP reporter mice, we observed weak co-expression of Nkx2-
6 `7 j# ]+ X) r5 protein and EYFP in the cardiac crescent at E7.75. However,) }! d" t: s7 b, n& Y
lineage-tracing experiments showed that cells that expressed
; W# T4 m" c M0 u, v- ~% _3 VER71 during any point in their development did not become
4 s" o$ v) c% {. Fcardiomyocytes. By contrast, Nkx2-5-Cre lineage-tracing' K" l& A$ j$ v
experiments show that at least some of the endocardial lineage
" i/ ?- p/ c6 b& L- g) D S/ O/ \$ Bderives from cells that expressed Nkx2-5 (Stanley et al., 2002)./ u0 J0 p8 b7 n5 n1 U8 F
Taken together, these observations favor a model in which
0 F1 `+ |& f( T9 Ycommon progenitors of endocardial and myocardial lineages are" E# a' ~" T" H9 r- e7 K7 Q
specified as Nkx2-5+ cells in the cardiac crescent, but those that% w* {& ~! {: a I X/ h
RESEARCH ARTICLE Development 138 (21): z9 C: F# i2 H+ t% ?' R f
Fig. 8. Overexpression of ER71 in differentiating EBs inhibits
W; J# S5 W4 p3 ~2 P( qcardiac development. (A-D)Sections of representative EBs either
& L* l& K2 |" D2 Duntreated (A,C) or induced to overexpress ER71 (B,D) stained with5 u( R/ V5 `8 t( v, t R
Hematoxylin and Eosin (C,D) or anti-TnT (A,B). (E)Quantitative analysis of; g3 }1 C. ]! L# r
the troponin T-positive area in 33 induced (Dox) and 42 untreated
, m6 Y7 x+ `/ P. M) O# z+ Isections. (F)qRT-PCR confirms that representative cardiac program+ P' E: P- q2 O! c
transcripts (Nkx2-5, Tbx5, Gata4) are downregulated in ER71-. T# p$ |2 o; C: D, v/ D
overexpressing EBs. (G,H)Extent of contraction and relaxation after 2
% d6 R. F$ R( f: Hseconds of stimulation (G) and percent shortening (H) are reduced in- F. l; l7 @1 A: d/ G" `" Z+ b0 w
ER71-overexpressing EBs. ****, P<0.001; *, P<0.05. Error bars indicate! y8 L$ f) E( W% P
s.e.m.: l1 }; z& M, U
DEVELOPMENTwill become endocardium will downregulate myocardial
9 I- h% \, R+ A; ~7 l) C' d, Y R2 \potential, including the expression of Nkx2-5. Once ER71 is
) i& p E0 \7 z$ D- N% Vexpressed, the fate is restricted to endocardium, and not5 b# G# |" P; O$ S2 a% u5 _4 I
myocardial lineages. Future studies are warranted to decipher the
+ h m! J- t# _3 {; Hmechanism that governs the specification of endocardial cells
6 O+ o( v) ]; w" }+ s, k* hwithin the cardiac progenitor pool.
9 F3 J* u( }" `% W: fOur third finding is that ER71 overexpression repressed cardiac
+ R( a" \( V6 c Z/ n Cpotential. These results support the notion that ER71, like Nkx2-5,! P6 s; {2 ~9 Q% u! T' V
has reciprocal and overlapping dual roles in the specification of
7 i3 i( q1 ^, u7 smesodermal lineages. Our previous studies demonstrated that
1 A f' u: ]) ]8 z: _7 h8 N: H$ R8 tNkx2-5 overexpression suppressed hematopoiesis, but not the) `1 I' Q) u' b
endothelial program (Caprioli et al., 2011). We further defined that& N$ q1 T# k+ F, z6 S
Nkx2-5 could, in a context-dependent fashion, transcriptionally
6 X1 C# ?( X3 E0 Eactivate Er71 and promote endocardial lineage development
8 d% _) p j8 b(Ferdous et al., 2009). Collectively, our previous studies and recent
7 k, D9 G; N2 Yresults support the notion that distinct transcriptional networks can,8 D1 E- l/ \1 D3 h/ C* _0 C+ Y/ B
in a context-dependent fashion (and dependent on the local
% C8 s; ^0 J$ z( Y, V% _# q$ qenvironment), activate a hematopoietic or endothelial program and( J0 |# ^5 D6 C% |: Q- c" q
antagonize the cardiac program. These studies further emphasize6 Z2 c9 p- E: B) }; F
the plasticity of progenitors and their contribution to distinct
0 r* ]" i' L& s1 [$ |6 s0 n, h! `lineages.8 t* o* i& ^6 p6 f4 H/ Z
In summary, our data support a dual role for ER71 in the
, W: f4 B5 O- A: {% d5 dspecification of the endothelial and hematopoietic lineages and the: J) p& h- x9 T0 B, P8 W
suppression of other lineages (i.e. myocardial and paraxial( d. ]8 b; d/ n3 L$ v
mesodermal cell fates) (supplementary material Fig. S6). Our
/ b( U9 p! D, y( z8 zstudies further support the notion of multipotent mesodermal
; K/ r I9 `, s* k( eprogenitors that are dependent on transcriptional cascades and other
% ]. H0 C! x# [cues to direct them to populate one or more lineages. Moreover, g( Q- Y: s) K$ z/ p9 {
our studies reveal that global deletion of a single gene (i.e. Er71), l# W: }5 j# ~; }( r& \
aborts one or more lineages and redirects the cells to other lineages9 g$ e: [; i9 N0 D: Y& V7 @/ c5 {
that would not typically arise from those progenitors. These studies; x# n7 Q' }9 A# o& h# F
further our understanding of the regulatory mechanisms that govern) V# D& _0 @0 k+ m, T( l( x
mesodermal cell fate decisions and embryogenesis.& X$ N8 K1 F/ t2 w: f% s
Acknowledgements @8 }! y) f- \( _5 w4 K
We thank Jennifer L. Springsteen and Alicia M. Wallis for assistance with, E Q ^- B3 N
histological analyses and animal care.
; Z7 V8 T/ q- {* }$ Q( YFunding
' r1 q2 }, S. P" PFunding support was obtained from the National Institutes of Health [U01
2 t0 e# s9 `# u2 _; B- \HL100407 and R01 HL085729 to D.J.G.]; and the American Heart Association
, q) h, w0 b, |( B8 y0 K8 F[Jon Holden DeHaan Foundation 0970499 to D.J.G.]. Deposited in PMC for' w9 X# x8 x# ^- M& J$ w
release after 12 months.
1 L, `6 P" ^5 K# x% I' r8 wCompeting interests statement1 ]0 V7 D5 t5 ]5 g' o
The authors declare no competing financial interests.
5 @. r& ]: ~, YSupplementary material; c& {* b, _% N) O) S- [, ?
Supplementary material available online at
: E# u" Q4 i5 R( r) O/ S7 t! vhttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.070912/-/DC1
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RESEARCH ARTICLE Development 138 (21)1 ?4 `8 ^) Q; ^! o8 I& z5 b
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