Comparative Analysis of mRNA and Protein Expression of Popdc1 (Bves) During Early Development in the
RESEARCH ARTICLE
Comparative Analysis of mRNA and Protein Expression of Popdc1(Bves)During Early Development in the Chick Embryo?
Angela Torlopp,?Stephanie S.Breher,?Jan Schlu¨ter,?and Thomas Brand*?
The isolation of the Popeye gene family was based on its preferential expression in striated muscle tissue. Recently,a monoclonal antibody against chick Popdc1(also known as Bves)became available and was used in this study to comparatively analyze the expression pattern of Popdc1at both the protein and mRNA level during early chick https://www.sodocs.net/doc/4617557509.html,ing whole-mount immunohistochemistry,expression in the heart was ?rst observed at Hamburger and Hamilton(HH)stage10in the presumptive left ventricular segment. Cardiac expression was con?ned to differentiated cardiac myocytes,and undifferentiated myocytes at the anterior and posterior pole showed little expression.After looping,the outer curvature myocardium showed prominent Popdc1staining,whereas the inner curvature was unlabeled.Despite previous reports, Popdc1protein was not detectable at any time point in the proepicardium,epicardium,or the smooth muscle layer of the coronary vessels.Whole-mount in situ hybridization using a full-length Popdc1probe detected novel expression domains,which have not been described previously.Popdc1mRNA was found in Hensen’s node at HH stage4,and by HH stage5?,expression became asymmetric.In addition,Popdc1 mRNA was found in pharyngeal endoderm and in the notochordal plate.Subsequently,beginning at HH stage9,Popdc1mRNA expression was found in the cardiac mesoderm and expression was maintained in the heart in a pattern very similar to the one observed by antibody staining.Developmental Dynamics235: 691–700,2006.?2006Wiley-Liss,Inc.
Key words:Popdc1;Bves;Popeye genes;chick embryo;proepicardium;epicardium;cardiac myocytes
Accepted13December2005
INTRODUCTION
Members of the Popeye domain con-taining gene family were identi?ed in-dependently by two groups using a subtractive hybridization approach aiming at the isolation of novel heart-restricted cDNAs in the chick embryo (Reese et al.,1999;Andre′e et al., 2000).Orthologues were identi?ed in other vertebrates(Hitz et al.,2002; Ripley,2004),lower chordates(David-son and Levine,2003;Davidson et al.,
2003),as well as insects(Lin et al.,
2002).In invertebrates(Drosophila),a
single gene seems to be present;in
lower chordates(Ciona intestinalis),
two genes have been identi?ed;and
three genes,popdc1(also designated
as Pop1,Bves),popdc2,and popdc3,
constitute the vertebrate Popeye gene
family(Brand,2005).Popdc1and
popdc3genes are present on the same
chromosome(chromosome6q21in
man,10in mouse,and chromosome3
in chick)and are organized as a tan-
dem;both genes are separated by ap-
proximately17kB in mouse and man
and9kB in the chick(Andre′e et al.,
2000).The popdc2gene is on a sepa-
rate locus on chromosome3in man,
chromosome16in mouse,and chro-
mosome1in the chick genome(An-
dre′e et al.,2000).
?This article was accepted for inclusion in Developmental Dynamics235#1–Cardiovascular Special Issue
Cell and Molecular Biology,Technical University of Braunschweig,Germany
Grant sponsor:Deutsche Forschungsgemeinschaft(DFG);Grant numbers:BR1218/9-4,GRK1048(Organogenesis).
?Present address for all authors is Cell and Developmental Biology,University of Wu¨rzburg,Germany.
*Correspondence to:Dr.Thomas Brand,Cell and Developmental Biology,Theodor-Boveri-Institute,University of Wu¨rzburg, Am Hubland,D-97074Wu¨rzburg,Germany.E-mail:thomas.brand@biozentrum.uni-wuerzburg.de
DOI10.1002/dvdy.20687
Published online27January2006in Wiley InterScience(https://www.sodocs.net/doc/4617557509.html,).
DEVELOPMENTAL DYNAMICS235:691–700,2006?2006Wiley-Liss,Inc.
The Popdc proteins have a novel structure lacking any known protein domain;however,one typical signa-ture of this family is a 70amino acid long hydrophobic domain close to the N-terminus,which by computer algo-rithms is predicted to form three transmembrane helices.Recently,it was shown that these putative trans-membrane domains are indeed func-tional (Knight et al.,2003).In addi-tion to this transmembrane domains,a conserved 150amino acid long se-quence is present in each Popdc pro-tein and was termed the Popeye do-main (pfam04831).Analysis of the membrane topology of the Popdc1pro-tein established that the amino termi-nus of Popdc1protein is extracellular and the carboxyl terminus is cytoplas-mic (Knight et al.,2003).Recent evi-dence suggests that Popdc1protein can form homodimers and the dimer-ization motif is part of the Popeye do-main (Knight et al.,2003;Vasavada et al.,2004).It has been proposed that Popdc proteins might act as a modu-lator of cell adhesion;however,its bio-chemical interaction partner or its ex-act function presently is unknown (Reese et al.,1999;Wada et al.,2001,2003).A null mutation for Popdc1in mice has been reported (Andre ′e et al.,2002).In these mice,there was no em-bryonic lethality and normal viability during postnatal life.However,null mutants displayed an impaired ability to regenerate skeletal muscle (Andre ′e et al.,2002).
The cloning of chick popdc1and
popdc2and recent analysis in the case of the mouse popdc3gene revealed the presence of several different isoforms that are generated by alternative splicing (Andre ′e et al.,2000;Breher et al.,2004).These splice isoforms en-code proteins that differ at the car-boxyl terminus.In case of the mouse popdc3gene,alternative splicing also occurs in exons that encode the trans-membrane domains and the Popeye domain (Wiegand et al.,unpublished).Thus,each popdc gene generates mul-tiple splice isoforms.
The Popdc1and Popdc2transcripts are strongly expressed in myocardium and skeletal muscle as visualized by Northern blot and in situ hybridiza-tion (Andre ′e et al.,2000;Breher et al.,2004).By reverse transcriptase-poly-merase chain reaction (RT-PCR),Popdc1transcripts at low levels are also detectable in stomach,gut,brain,kidney,lung,and spleen (Andre ′e et al.,2000).On the basis of the LacZ staining pattern in tissues of mice with a ?-galactosidase knockin into the popdc1locus,expression in the lung is con?ned to smooth and cardiac muscle cells lining pulmonary veins (Fleige et al.,unpublished observa-tions).Expression in stomach and gut is con?ned to the smooth muscle cell layer of the digestive tract.Searching of the expressed sequence tag (EST)database as well as results of SAGE data consistently revealed expression in cell types other than striated and smooth muscle cell types,including for example the embryonic pancreas or melanocytes.A polyclonal anti-serum (D033)directed against a con-served peptide of Popdc1detected in the chick embryo a protein that is prominently expressed in the proepi-cardial organ as well as smooth mus-cle cells of the coronary arteries (Re-ese et al.,1999).The expression of Popdc1in proepicardial cells was cor-roborated further by the demonstra-tion of expression of Popdc1both at mRNA and protein level in a rat epi-cardial cell line (Wada et al.,2003).However,until today expression in the proepicardium and later in the epicardium remains controversial.No expression of popdc genes was found in the proepicardium of the chick em-bryo by RT-PCR analysis (Breher et al.,2004).Moreover,only in the case of bone morphogenetic protein
(BMP)
Fig.1.Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR)analysis of Popdc1expression during early chick embryogenesis.A:The Popdc1antibody recognizes the cytoplasmic domain of recombinant Popdc1protein.Western blot of bacterial lysate after 2hr induction (?)of Popdc1-MBP fusion protein production.As a control,lysate of a noninduced bacterial culture (?)was used.B:Western blot analysis of Popdc1expression using homogenates of embryonic day (E)7chick embryonic organs.Li,liver;Lu,lung;St,stomach;In,intestine;Bm,breast muscle;Lm,leg muscle;Ht,heart.Only in heart tissue was Popdc1protein detected.C:Detection of Popdc1protein in chick embryos between Hamburger and Hamilton (HH)stages 4and 12.Homogenates from whole embryos of the indicated stages were subjected to Western blot analysis using the Popdc1antibody.Popdc1protein was not detectable in total embryo homog-enates before HH stage 11.D:RT-PCR analysis of Popdc1expression.RNA was isolated from whole embryos at HH stages 4–12.Popdc1mRNA was ampli?ed using primers detecting all known Popdc1splice isoforms.In addition,primers for glyceraldehyde-3-phosphhate dehydrogenase (GAPDH)were used to verify the integrity of RNA samples.
692TORLOPP ET AL.
2–induced trans-differentiation of proepicardial cells to the myocardial cell lineage,popdc gene expression be-came detectable in this cell type (Schlu ¨ter et al.,manuscript in prepa-ration).A monoclonal antiserum di-rected against the carboxyl-terminus
of Popdc1yielded no evidence for ex-pression of Popdc1in the
epicardium
Fig.2.Developmental series showing Popdc1mRNA localization in chick embryos from Hamburger and Hamilton (HH)stages 4to 11.All views of embryos are ventral;anterior is at the top;posterior is at the bottom.A:Popdc1is expressed in Hensen’s node at HH stage 4.B:At HH stage 5?,Popdc1expression in Hensen’s node becomes asymmetric being stronger on the right than on the left side.In addition,expression is seen in the anterior pharyngeal endoderm.C:At HH stage 7,left–right (L-R)asymmetric expression in the notochord is seen.D:At HH stage 8,symmetric expression in the notochord is seen.I:At HH stage 9(7somites),Popdc1expression is con?ned to the anterior segment of the right heart ?eld (arrow).At this stage of development,no expression is seen on the contralateral side (arrowhead).J–L:Arrowheads demarcate the extension of the expression domain along the anteroposterior (A/P)axis.J:At HH stage 9(9somites),expression is con?ned to the presumptive left ventricular segment.Note the different length of the expression domain on the left and right side.K:At HH stage 10(11somites),expression extends further rostrad and caudad.Intensity of expression as well as the extension along the A/P axis differs between the left and right side.L:At HH stage 11(13somites),Popdc1expression has further extended along the A/P axis.E–H,M–P:Transverse sections through the embryos shown in (A–D and I–L).Plane of sectioning is indicated in the individual panel.Arrows in F,G,M demarcate the right-sided expression domain,whereas arrowheads demarcate the absence of expression on the left side.Ec,endocardium.L-R indicates left–right orientation of the embryo.[Color ?gure can be viewed in the online issue,which is available at https://www.sodocs.net/doc/4617557509.html,.]
PROTEIN AND mRNA EXPRESSION OF Popdc1693
with the exception of embryonic day6 heart(DiAngelo et al.,2001;Vasavada et al.,2004).Moreover,no evidence was found for coronary artery expres-sion using this antibody(Vasavada et al.,2004).Likewise,the knockin of LacZ into the?rst coding exon of the popdc1gene in the mouse revealed no evidence for Popdc1expression in the proepicardium and epicardium or any other nonmuscle cell type of the heart at any time point during embryonic development(Andre′e et al.,2002).An-other monoclonal antibody(B0846)di-rected against Popdc1detected sev-eral epithelial structures in the early chick embryo(Wada et al.,2003;Osler and Bader,2004).This result was sur-prising because the mRNA expression of Popdc1has been reported to start at Hamburger Hamilton(HH)stage11 (Andre′e et al.,2000).
To study Popdc1expression during early chick embryogenesis and to?nd out whether expression of Popdc1pro-tein and mRNA do coincide,we ana-lyzed the expression of Popdc1protein using a monoclonal Popdc1antibody (DiAngelo et al.,2001).To analyze and compare protein expression pattern with that of the mRNA,a full-length chick Popdc1A probe was used for whole-mount in situ hybridization analysis.In the heart,Popdc1mRNA was detectable approximately7.5hr earlier than the protein.Thus,mRNA and protein expression differed tran-siently,which suggests a possible posttranslational control of Popdc1 protein.Both,mRNA and protein analysis revealed a consistently weaker expression level in the newly added anterior and posterior heart segments than in segments that were derived from the primary heart?elds (Kelly and Buckingham,2002;Abu-Issa et al.,2004).Consistent with our previous reports,we found no evi-dence for Popdc1expression in the proepicardium,epicardium,or in the coronary vasculature.Surprisingly, analysis of mRNA expression revealed novel early noncardiac expression do-mains of Popdc1;however,no expres-sion was found at the protein level. Thus,Popdc1mRNA and protein ex-pression differ in the early chick em-bryo;however,in the heart,Popdc1 mRNA and protein localization was similar.RESULTS
Recently,the characterization of a
monoclonal antibody directed against
Popdc1has been reported(DiAngelo
et al.,2001;Vasavada et al.,2004).
The initial description of Popc1pro-
tein expression using this antibody,
however,failed to analyze Popdc1pro-
tein expression during early chick em-
bryonic development.We?rst charac-
terized the antigen speci?city of this
antibody.Western blot analysis with
protein extracts of bacteria expressing
the cytoplasmic part of chick Popdc1
as a fusion protein with maltose-bind-
ing protein revealed that this mono-
clonal antibody recognized the cyto-
plasmic domain of Popdc1protein
(Fig.1A).In addition,detergent ex-
tracts of various organs of embryonic
day7chick embryos were subjected to
Western blot analysis(Fig.1B).We
found exclusive Popdc1protein ex-
pression in the heart sample.Two im-
munoreactive protein bands were vis-
ible,a major protein band of58kDa
and minor protein band of approxi-
mately55kDa.After substituting de-
tergent extraction by an extraction
buffer containing urea,immunoreac-
tive bands were also seen in case of
the skeletal muscle samples(data not
shown),suggesting that popdc1pro-
tein in skeletal muscle is crosslinked
to some other protein complex that
cannot be dissolved by detergents
only,as has been previously reported
(Vasavada et al.,2004).To determine
the developmental time of?rst Popdc1
protein expression,chick embryos of
HH stage4to12were subjected to
Western blot analysis using the
Popdc1antibody(Fig.1C).Popdc1
protein expression was found to start
at HH stage11.We next compared the
timing of protein expression with
Popdc1mRNA expression by RT-PCR
analysis using a set of primer that will
amplify all known Popdc1splice iso-
forms.In contrast to the Popdc1pro-
tein expression,Popdc1mRNA ex-
pression was detectable already at
HH stage4(Fig.1D).The expression
level increased at HH stage5and sub-
sequently decreased until HH stage7.
At HH stage8,the expression level
rose again and reached its maximum
at HH stage12(Fig.1D).The mono-
clonal Popdc1antibody detects pro-
tein expression at HH stage11,
whereas the Popdc1mRNA is already
detectable by HH stage4.
Since we have reported previously
that Popdc1is?rst expressed at HH
stage11in the chick embryo,we made
use of a full-length Popdc1A(Popdc1
splice isoform A,previously named
Pop1A)cRNA probe to perform whole-
mount in situ hybridization of chick
embryos between HH stage4and HH
stage11(Fig.2).Consistent with the
PCR data,we saw expression in Hens-
en’s node already at HH stage4(Fig.
2A,E).The expression domain within
Hensen’s node became asymmetric at
HH stage5,being consistently stron-
ger on the right side of the node(Fig.
2B,F).In addition,expression was
found in the anterior pharyngeal
endoderm in the head process.At HH
stage7(2somites),expression in
Hensen’s node still persisted;how-
ever,it was symmetrical at this time
of development(Fig.2C).Of interest,
Popdc1was also expressed in the
forming notochord.Expression was
found to be asymmetric being consis-
tently stronger on the right side(Fig.
2C,G).At this time of development,
expression was also present in the
ventral foregut.At HH stage8,ex-
pression persisted in the notochord
(Fig.2D,H).At HH stage9(7somites),
an asymmetric expression domain in
right heart?eld was observed(Fig.
2I,M).At HH stage9(9somites),
when the tubular heart has formed,
Popdc1expression was con?ned to the
presumptive left ventricular segment
(Fig.2J,N).The expression domain on
the right side of tubular hearts at HH
stage9/10(Fig.2J,K)was consistently
stronger and extended over a longer
distance along the anteroposterior
(A/P)axis.At HH stage10(11
somites),expression expanded ros-
trally and caudally(Fig.2J,O).At HH
stage11(13somites),the Popdc1ex-
pression domain included the right
ventricular and atrial segments,how-
ever,myocardium of the conus as well
as the sinus venosus did not show
Popdc1expression(Fig.2L,P).
To compare the pattern of expres-
sion of the mRNA with the protein,we
subjected chick embryos between HH
stage4and20to whole-mount immu-
nohistochemistry.No expression was
seen in embryos younger than HH
stage10(Fig.3A and data not shown).
At HH stage10,faint expression in
694TORLOPP ET AL.
the embryonic heart was visible(Fig. 3B–D).At HH stage11,robust Popdc1 expression within the cardiac myo-cytes of the atrial and ventricular seg-ment of the tubular heart was seen (Fig.3E,K).Between HH stage12and 15,a rostrad extension of the expres-sion domain was observed and in-cluded?rst the proximal and subse-quently also the distal part of the out?ow tract(Fig.3F–H,O).However, Popdc1was not expressed in the sinus venosus at this time of development (Fig.3L).At HH stage18,myocar-dium of the outer curvature showed strong expression,while the inner cur-vature myocardium had little to none expression(Fig.3I,M).At HH stage 20,the entire myocardium was strongly labeled by the Popdc1anti-body;however,upon sectioning through the ventricular myocardium, the compact layer showed slightly higher expression than the trabecular layer(Fig.3J,N).By this time of de-velopment,the distal out?ow tract myocardium also showed Popdc1ex-pression(Fig.3P).
The presence of Popdc1in the pro-epicardium and in the epicardium is a controversial issue(Andre′e et al., 2000,2003).We have shown previ-ously that microdissected proepicar-dium does not express any of the three popdc genes,when analyzed by RT-PCR(Breher et al.,2004).We used the Popdc1antibody to address this con-troversy.Staining of frozen sections of a HH stage19embryo revealed the absence of Popdc1protein in the pro-epicardium as well as in mesothelial cells that have made contact to the ventricular myocardium(Fig.4A,B). Also at the mRNA level,we found no evidence for proepicardial expression of Popdc1(Fig.4C,D).
We also analyzed expression of Popdc1in later stages of heart devel-opment.At day7,Popdc1expression was present in atrial and ventricular cardiac myocytes but was not found in the epicardium,endocardium,or the atrioventricular cushions(Fig.5A).Of interest,at this time of development, atrial myocardium expressed higher levels of Popdc1protein than ventric-ular myocardium.Costaining of Popdc1and cytokeratin revealed ex-clusive detection of Popdc1in cardiac myocytes and absence of expression in the epicardium at day6,7,and9of development(Fig.5B–D).Previous re-
ports claimed that Popdc1is ex-
pressed in the coronary vasculature.
We therefore analyzed Popdc1expres-
sion in a day18heart and found no
evidence for expression in the coro-
nary vessels by costaining of Popdc1
and smooth muscle?-actin(Fig.5E).
These data demonstrate that Popdc1
protein as recognized by this monoclo-
nal antibody was exclusively present
in cardiac myocytes.
DISCUSSION
This study analyzed Popdc1expres-
sion during early chick embryogene-
sis.In the heart,Popdc1mRNA and
protein expression were temporally
separated by approximately7.5hr.
Both,mRNA and protein analysis re-
vealed a consistent weaker expression
level in the newly added anterior and
posterior heart segments,suggesting
that the level of Popdc1expression is
correlated with the extent of myocar-
dial differentiation.Consistent with
our previous reports,Popdc1expres-
sion was not found in the proepicar-
dium or later in the epicardium.
Surprisingly,analysis of mRNA ex-
pression revealed novel expression do-
mains of Popdc1at HH stage4in
Hensen’s node and,subsequently,in
the notochord.At the protein level,no
expression was found in these struc-
tures.Thus,Popdc1mRNA and pro-
tein expression differ in the early
chick embryo.
Popdc1Displays Left–Right
Asymmetry in Hensen’s Node
We found asymmetric expression in
Hensen’s node at HH stage5.At this
time of development,many genes such
as?broblast growth factor(FGF)8
(Boettger et al.,1999),Sonic hedgehog
(Shh;Levin et al.,1995),ActRIIa
(Levin et al.,1995),N-Cadherin(Gar-
cia-Castro et al.,2000),BMP4(Mon-
soro-Burq and Le Douarin,2001),Pcl2
(Wang et al.,2004),and NCX-1(Li-
nask et al.,2001)display left–right
(L-R)asymmetric expression domains
in Hensen’s node in the chick embryo.
These asymmetries,although subtle,
are of functional signi?cance,because
any alteration in their expression pat-
tern results in aberrant L-R axis for-
mation(reviewed in Brand,2003).Ac-
tRIIa,BMP4,FGF8,Pcl-2,and
N-Cadherin are all expressed on the
right side of the Hensen’s node;how-
ever,each of these genes also display
expression in the primitive streak.In
contrast,Popdc1and NCX-1expres-
sion is con?ned to Hensen’s node(Li-
nask et al.,2001).At the moment,it is
unknown whether any of the signaling
or regulatory factors asymmetrically
expressed on the right side up-regu-
late Popdc1expression,or whether
Popdc1asymmetry is generated by a
down-regulation of Popdc1expression
on the left side.The null mutant for
Popdc1did not provide any evidence
for an involvement of this gene in set-
ting up the L-R axis(Andre′e et al.,
2000,2003).Of signi?cance in this re-
gard is our recent?nding that Popdc2
in the mouse is expressed in the node
(Froese et al.,unpublished observa-
tions).Nonetheless,coexpression of
Popdc1with N-Cadherin in Hensen’s
node is an interesting?nding,because
Popdc1has been implicated in modu-
lating cell–cell interaction(Wada et
al.,2001;Andre′e et al.,2003).Re-
cently,it has been described that,not
only molecular asymmetries are
present during HH stages4–7,but
that Hensen’s node and its descendent
structures such as the forming noto-
chord also display morphological
asymmetries(Dathe et al.,2002).
Whether Popdc1might be involved in
modulating epithelial polarity in this
context is an interesting question and
should be analyzed further.We also
found expression of Popdc1in the
forming notochord,which is a struc-
ture that is generated by a convergent
extension mechanism(Domingo and
Keller,1995).In this regard,it is note-
worthy that treatment with a morpho-
lino against Popdc1interferes with
convergent extension in Xenopus em-
bryos(Ripley,2004).
Tubular Hearts Diplay L-R
Asymmetric Expression of
Popdc1
L-R asymmetry was also observed
within the heart?eld.Between HH
stage9and stage10,the right heart
?eld displayed stronger Popdc1ex-
pression than the contralateral side.
Of interest,Popdc2in the chick also
showed asymmetric expression,but it
was the left side that showed stronger
expression(Breher et al.,2004).Many
PROTEIN AND mRNA EXPRESSION OF Popdc1695
Fig.
3.
Fig.
4.
Fig.5.Absence of popdc1protein expression in the epicardium or coronary arteries of midges-
tation and late stage hearts.A:Transverse section through a chick embryonic heart at embryonic
day7.Expression is con?ned to cardiac myocytes.No staining is observed in the atrioventricular
septum(AVS),valvular tissue(arrowheads),or in the epicardium(E,arrows).B–D:Immuno?uores-
cent detection of Popdc1(red label)in cardiac myocytes and absence of staining in the epicardial
cells,which were identi?ed by an antibody directed against cytokeratin(green label)in sections of
day6heart(Hamburger and Hamilton[HH]stage28;B),day7heart(HH stage31;C),and day9
heart(HH stage35;D).E:Immuno?uorescent detection of Popdc1(red label)in cardiac myocytes
and absence of staining in smooth muscle cells,which were identi?ed by staining with an antibody
directed against smooth muscle?-actin(green label)in a section of a day18embryonic heart(HH
stage44).B–E:Sections were counterstained with4?,6-diamidine-2-phenylidole-dihydrochloride
(DAPI;blue label).A,atrium;C,cardiac cushion tissue;CV,coronary vessel;Ec,endocardium;LA,
left atrium;LV,left ventricle;N,neural tube;RA,right atrium;RV right ventricle;V,ventricle.
696TORLOPP ET AL.
genes display L-R asymmetric expres-sion during early heart formation.The transcription factor Pitx2is expressed in the left portion of the cardiac cres-cent and in the left side of the heart tube(Campione et al.,2001).In the precardiac mesoderm,the extracellu-lar matrix molecules hLAMP and?ec-tin on the left and JB3on the right side are asymmetrically distributed (Smith et al.,1997).In frogs and ze-bra?sh,BMP4is predominantly ex-pressed on the left side(Breckenridge et al.,2001).Consistent with our ob-servation of asymmetric expression in the presumptive left ventricular seg-ment is the observation that the left and right heart?eld have unequal contributions to the tubular heart along the A/P axis.The right heart ?eld has a greater contribution to the rostral end(presumptive left ventri-cle)of the tubular heart and less to the caudal end(Stalsberg,1969).In addi-tion,there are some differences in temporal progression of cardiac differ-entiation between the left and the right side(Satin et al.,1988).Signi?-cantly,the?rst contractions in the tu-bular heart are observed in the right margin of the ventricular portion at the late9-to10-somite stage(Sakai et al.,1996).Whether and how this re-lates to asymmetric Popdc1expres-sion in the heart requires further analysis.
Popdc1Expression in the Tubular Heart Seems to Be Associated With
Differentiated Cardiac Myocytes
Popdc1also displayed a restricted ex-pression pattern along the A/P axis.Initially,expression at the mRNA
level was con?ned to the left ventric-
ular segment at HH stage9.Subse-
quently,expression gradually ex-
tended both caudally to include the
atrium and rostrally to include?rst
the future right ventricle,the conus,
and?nally the truncal region of the
out?ow tract.The stepwise addition of
positively stained cardiac segments is
also seen at the protein level.When
heart looping progressed,there was a
higher level of Popdc1expression in
the myocardium of the outer curva-
ture and less in the inner curvature,
consistent with our previous descrip-
tion at the mRNA level(Andre′e et al.,
2000,2003).It has been proposed that
the myocardium of the outer curva-
ture is the future chamber myocar-
dium,whereas the inner curvature
myocardium represents remnants of
primary myocardium(Christoffels et
al.,2004).Genes such as Nppa,
Chisel,and Cx40in the mouse heart
are representatives of a chamber-spe-
ci?c gene expression program,which
is under the control of various tran-
scription factors of the Tbx gene fam-
ily(Habets et al.,2002;Singh et al.,
2005).To what extent Popdc1expres-
sion at the post-looping stage is under
the control of this chamber-speci?c
gene expression program is an unre-
solved question.At embryonic day7,
myocytes in the compact layer appar-
ently had a higher level of expression
than trabecular cardiac myocytes and
the atrium displayed higher levels of
expression than the ventricular myo-
cardium.It is well known that gene
expression in the compact layer is
modulated by signaling factors de-
rived from the epicardium.
Popdc1Is Not Expressed in
the Proepicardium or in the
Epicardium
Previously,Popdc1has also been
named bves(blood vessel/epicardial
substance),based on its expression in
the proepicardium,the epicardium,
and its derivatives the coronary arter-
ies(Reese et al.,1999).We have re-
ported previously that none of the
three Popdc cDNAs were detected by
PCR ampli?cation of mRNA isolated
from microdissected proepicardia
(Breher et al.,2004).We now show
that that there is no evidence for
Popdc1expression in the proepicar-
dium,or subsequently in the epicar-
dium,both at the mRNA and protein
level.Moreover,we show here that
Popdc1is not found to be expressed in
the smooth muscle layer of the coro-
nary vessels.Expression of Popdc1in
the proepicardium has been observed
previously by immunohistochemical
staining using antibodies directed
against conserved peptide sequences
within the cytoplasmic domain of the
Popdc1protein(Reese et al.,1999;
Wada et al.,2001,2003;Osler and
Bader,2004).At present,we do not
know whether these monoclonal anti-
bodies detect only Popdc1or whether
these antibodies crossreact with some
other antigen that is expressed in the
proepicardium.In addition,it was
proposed recently that Popdc1is ex-
pressed in the early gastrula in the
epiblast and later in various epithelial
structures such as neural tube and
somites(Osler and Bader,2004).Our
RNA localization does not provide any
evidence for expression in the epiblast
at HH stage4but rather very speci?c
expression in Hensen’s node and sub-
Fig.3.Whole-mount immunostaining for Popdc1protein in chick embryonic hearts of Hamburger and Hamilton(HH)stage9to20.All views of embryonic hearts are ventral;anterior is at the top;posterior is at the bottom.A:HH stage9(9somites).B:HH stage10(10somites).C:HH stage 10(11somites).D:HH stage10(12somites).E:HH stage11(13somites).F:HH stage12(15somites).G:HH stage14.H:HH stage15.I:HH stage 18.J:HH stage20.Arrowheads in E–H demarcate the anterior border of Popdc1expression.K–P:Transverse sections through the embryos shown in E,G,I,J.Plane of sectioning is indicated in the individual panel.A,atrium;CL,compact layer;EC,endocardium;FG,foregut;G,Gut;NT,neural tube; TL,trabecular layer;V,ventricle.
Fig.4.Popdc1at mRNA and protein level is not expressed in the proepicardium or epicardium.A:Transversal cryostat section through a Hamburger and Hamilton(HH)stage19embryo immunostained for Popdc1and counterstained with Mayer’s hematoxylin.Strong staining is observed in cardiac myocytes(M)of the ventricle,whereas the sinus venosus myocardium(SV)and the proepicardium(P)are unstained.Boxed area is shown enlarged in A?.B:Section through a HH stage18heart showing immunoreactivity in cardiac myocytes but not in proepicardial mesothelial cells.C:Whole-mount in situ hybridization of a HH stage18embryo heart showing an unlabeled proepicardium(arrow).In contrast,the myocardium is labeled by the Popdc1 probe.Plane of sectioning is indicated by a bar.D:Transverse section of the embryo shown in C.A–atrium,C,cardiac cushion tissue;Ec, endocardium;M,myocardium;N,neural tube;P,proepicardium;V,ventricle;SV,sinus venosus.
PROTEIN AND mRNA EXPRESSION OF Popdc1697
sequently expression in the forming notochord and in the pharyngeal endoderm.Thus,the easiest explana-tion for these divergent results ob-tained with the D033and B0846anti-bodies,generated and characterized by the Bader laboratory(Reese et al., 1999;Wada et al.,2001,2003;Osler and Bader,2004),is that these anti-bodies do crossreact with another pep-tide unrelated to Popdc1.Alterna-tively,there might exist Popdc1 isoforms generated by alternative splicing that are not recognized with the antibody used in this study.
In a previous study of Popdc1pro-tein expression using the same mono-clonal antibody,it was reported that Popdc1was transiently expressed at day6in the epicardium(Vasavada et al.,2004).We were unable to con?rm this?nding and,rather,show here that Popdc1is exclusively expressed in the cardiac myocyte and is not ex-pressed in the epicardium,particu-larly not at day6of development. These differing results are probably due to some technical reason. Popdc1Antibody Does Not Recognize the Early Noncardiac Expression Domains of popdc1mRNA There are some differences between mRNA localization and protein detec-tion in our data.The early expression of Popdc1at the mRNA level in Hens-en’s node and in the notochord as well as in pharyngeal endoderm is not seen by antibody staining.One possibility is that the Popdc1mRNA is not trans-lated before the heart reaches a func-tional state.There are some prece-dents for this:both smooth muscle ?-actin and Troponin T mRNA are de-tected already in the early heart?eld, whereas translation only occurs shortly before contraction starts(Co-las et al.,2000;Antin et al.,2002).An alternative explanation would be that the protein produced by the mRNA that is present in early gastrulating embryos is not detected by the anti-body used here.The isoforms for Popdc1that are known presently are generated through alternative splic-ing and differ at the carboxyl termi-nus(Andre′e et al.,2000,2003).In case of the related gene popdc3in the mouse,we have obtained evidence re-cently for the presence of an alterna-
tive splicing event within the Popeye
domain.These splice products may
not be recognized by the monoclonal
antibody used here.To distinguish be-
tween these alternatives,we will
characterize the splice isoform pro-
duction in case of the Popdc1gene
during chick development.
EXPERIMENTAL
PROCEDURES
Chick Embryos
Fertilized chicken eggs(White Leg-
horn,Gallus gallus)were obtained
from Lohmann,Cuxhafen.Eggs were
incubated at38°C and75%relative
humidity.Staging of the embryos was
performed according to Hamburger
and Hamilton(Hamburger and Ham-
ilton,1951).
Immunohistochemistry
For immunohistochemical detection of
Popdc1,a monoclonal antibody(3F11-
D9-E8)developed by Melinda Duncan
(DiAngelo et al.,2001)was obtained
from the Developmental Studies Hy-
bridoma Bank developed under the
auspices of the NICHD and main-
tained by The University of Iowa,De-
partment of Biological Sciences(Iowa
City,IA52242).Chick embryos were
?xed for2hr with4%paraformalde-
hyde,sucrose in?ltrated,and embed-
ded in OCT.Ten-micrometer sections
were taken on Superfrost slides.The
sections were?rst pretreated with
0.3%hydrogen peroxide in MeOH/
Aceton(1:1)for30min at room tem-
perature to block endogenous peroxi-
dase,washed for2–3min with water,
and incubated with blocking solution
(5%horse serum in1?phosphate
buffered saline[PBS]).This procedure
was followed by an incubation with
the3F11-D9-E8monoclonal antibody
at a1:10dilution.After several
washes with PBS,the sections were
incubated with a1:200dilution of a
horseradish peroxidase conjugated
horse anti-mouse antibody(Vector
Laboratories).After several washes
with PBS and0.05M Tris(pH7.6),
the immunoreaction was color devel-
oped using diaminobenzidine.Some
sections were counterstained with
Mayer’s hematoxylin.
For immuno?uorescent detection of
Popdc1and costaining of Popdc1with
smooth muscle?-actin(clone1A4,
mouse monoclonal smooth muscle
?-actin antibody SIGMA)or cytokera-
tin(clone LU-5,mouse monoclonal
pan-cytokeratin antibody Acris anti-
bodies),the frozen sections were incu-
bated with blocking solution and sub-
sequently with antibodies directed
against Popdc1(1:10dilution),cyto-
keratin(1:200),or smooth muscle
?-actin(1:200).Popdc1was detected
with a donkey anti-mouse antibody
conjugated with Alexa Fluor555(Mo-
lecular Probes)and binding of the cy-
tokeratin and smooth muscle?-actin
antibodies were detected with a don-
key anti-mouse Fab fragment conju-
gated to?uorescein isothiocyanate
(Dianova).After the?nal wash,nuclei
were counterstained with4?,6-diami-
dine-2-phenylidole-dihydrochloride.
Whole-Mount
Immunohistochemistry
Embryos were washed three times in
PBS and?xed in a methanol/dimethyl
sulfoxide(DMSO)mixture(4:1)at4°C
overnight.Endogenous peroxidase was
blocked by incubating the embryos with
methanol/DMSO/30%H
2
O
2
mixture(4:
1:1)for2hr at room temperature.Un-
speci?c binding sites were blocked by
incubating the embryos2?1hr in2%
BSA,0.1%Triton X-100in PBS at room
temperature.Embryos were incubated
overnight in a1:200dilution of the
3F11-D9-E8antibody in TBST(0.8g of
NaCl,20mg of KCl,25mM Tris-Cl,
pH7.5,1%Tween-20)containing1%
horse serum.After5washes for1hr
with TBST,the embryos were incu-
bated with a1:200dilution of a horse-
radish peroxidase conjugated horse
anti-mouse antibody(Vector Laborato-
ries).After5washes for1hr with
TBST,the immunoreaction was color
developed using diaminobenzidine.For
cryostat sectioning,the embryos were
in?ltrated overnight at50°C with a
7.5%gelatin/15%sucrose solution and
snap-frozen in dry ice–cooled isopen-
tane.
Whole-Mount In Situ
Hybridization
Whole-mount in situ hybridization
was carried out as described(Andre′e
698TORLOPP ET AL.
et al.,1998).For expression analysis of Popdc1,a1.4-kb full-length cDNA clone(ChEST59k12)was identi?ed in the ChickEST Database(Boardman et al.,2002)and obtained from the MRC geneservice.For cryostat sectioning, the embryos were in?ltrated over-night at50°C with a7.5%gelatin/15% sucrose solution and snap-frozen in dry ice–cooled isopentane.The section shown in Figure4D was counter-stained with Fast Red.
Western Blot
For biochemical analysis,tissues were lysed in RIPA buffer(1?PBS,1%Ige-pal Ca-630[Sigma],0.5%Natrium-desoxycholate,0.1%sodium dodecyl sulfate[SDS],10?l/ml of a protease inhibitor cocktail;Roche).Each sam-ple(40?g of protein)was run on a 10%SDS polyacrylamide gel and elec-troblotted to a nitrocellulose mem-brane(Bio Trace;Pall).The mem-brane was blocked in5%skim milk, 1?TBS,and0.1%Tween-20and then incubated with a1:1,000dilution of the monoclonal3F11-D9-E8Pop1an-tibody.The membrane was washed three time with1?TBS-0.1% Tween-20solution and then incubated with a1:1,000dilution of a horserad-ish peroxidase–conjugated horse anti-mouse antibody(Vector Laboratories). in1?TBS,0.1%Tween-20,5%milk solution.The membrane was again washed three times,immersed in sub-strate solution(ECL,Amersham)for1 min,and then exposed to X-ray?lm. PCR Analysis
For RNA isolation,chick embryos of the indicated stages,from which the ex-traembryonic tissue had been removed were used.cDNA was synthesized from DNase treated total RNA by using AMV reverse transcriptase.PCR was performed by using the primer pairs: Popdc1fwd,5?-TTGCTCACCGTAGGA-TGTGC-3?;and Popdc1rev,5?-CGG-TTCATCTGAGTTGATCG-3?.cDNA in-put was controlled by ampli?cation reactions with glyceraldehydes-3-phos-phate dehydrogenase(GAPDH)primer: GAPDHfwd5?-ACGCCATCACTATC TTCCAG-3?,GAPDHrev5?-CAGGCC-TTCACTACCCTCTTG-3?.The PCR products were size separated on1% agarose gels.Production of a Maltose
Binding Protein–Popdc1
Fusion Protein
The carboxyl-terminus of Popdc1was
ampli?ed by Ex-Taq polymerase
(Cambrex Bioscience)using primers:
Pop1MBPA,5?-GATAATTCAGCCTGT-
ACAAGAGA ATGTTTGAACCACTC-3?;
Pop1MBPB,5?-GAATCTAGATCAAG-
GCAGCCGCT GCAGCTCAAGCTT-
TTC-3?.The resulting fragment was
restricted with Eco RI and Xba I and
subcloned into pMal-2vector(New
England Biolabs).The resulting plas-
mid was transformed into BL21cells.
For the production of Popdc1fusion
protein,the recombinant bacterial
cells at logarithmic growth were in-
duced with IPTG for2hr.For control
purposes,cells that had not been in-
duced by IPTG(0hr)were used.In
each case,500-?l cell suspension was
centrifuged and the resulting cell pel-
let was taken up in SDS sample buffer
and size-separated on a12%SDS-
PAGE.The resulting gel was pro-
cessed for Western blot analysis.
ACKNOWLEDGMENTS
This project was funded by Deutsche
Forschungsgemeinschaft,BR1218/
9-4,“Functional Characterization of
the Popeye gene family”and GRK1048
“Organogenesis.”
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