nkx genes establish SHF cardiomyocyte progenitors at the arterial pole and pattern the venous

66 Pages·2009·8.24 MB·English

©2018.PublishedbyTheCompanyofBiologistsLtd|Development(2018)145,dev161497.doi:10.1242/dev.161497
RESEARCHARTICLE
Nkx genes establish second heart field cardiomyocyte progenitors
at the arterial pole and pattern the venous pole through
Isl1 repression
SophieColombo1,CarmendeSena-Tomás1,VanessaGeorge1,AndreasA.Werdich2,SunilKapur2,
CalumA.MacRae2andKimaraL.Targoff1,*
ABSTRACT Vincent and Buckingham, 2010; Zaffran et al., 2004). Errors in
specificationanddifferentiationoftheSHFcellsundertheseprecise
NKX2-5isthemostcommonlymutatedgeneassociatedwithhuman
spatialandtemporalconstraintscanleadtoavarietyofcongenital
congenitalheartdefects(CHDs),withapredilectionforcardiacpole
heart defects (CHDs), specifically conotruncal (OFT) and atrial
abnormalities. This homeodomain transcription factor is a central
(IFT) abnormalities.Thesemalformations of thecardiacpoles are
regulatorof cardiac development and is expressed in both the first
foundin10%and30%,respectively,ofindividualswithCHDsand
andsecondheartfields(FHFandSHF).Wehavepreviouslyrevealed
result in significant neonatal morbidity and mortality (Gelb and
essential functions of nkx2.5 and nkx2.7, two Nkx2-5 homologs
Chung, 2014; Hoffman and Kaplan, 2002; Hoffman et al., 2004;
expressed in zebrafish cardiomyocytes, in maintaining ventricular
Loffredo, 2000; Nemer, 2008; Payne et al., 1995; Pierpont et al.,
identity.However,thedifferentialrolesofthesegenesinthespecific
2007; Supino et al., 2006). Despite this clinical impact, the
subpopulations of the anterior (aSHF) and posterior (pSHF) SHFs
mechanismsunderlyingthedevelopmentofthearterialandvenous
haveyettobefullydefined.Here,weshowthatNkxgenesregulate
polesoftheheartareincompletelyunderstood.
aSHFandpSHFprogenitorsthroughindependentmechanisms.We
NKX2-5,ahomeodomaintranscriptionfactorexpressedincells
demonstratethatNkxgenesrestrictproliferationofaSHFprogenitors
oftheFHFandSHF(Stanleyetal.,2002),andaregulatorofcardiac
in the outflow tract, delimit the number of pSHF progenitors at the
development (Bruneau, 2008), is associated with a myriad of
venous pole and pattern the sinoatrial node acting through Isl1
human CHDs. Moreover, individuals carrying NKX2-5 mutations
repression.Moreover,opticalmappinghighlightstherequirementfor
demonstrate a propensity to develop malformations involving the
Nkxgenedoseinestablishingelectrophysiologicalchamberidentity
poles of the heart, such as Tetralogyof Fallot, double outlet right
and in integrating the physiological connectivity of FHF and SHF
ventricleandatrialseptaldefects(AbouHassanetal.,2015;Benson
cardiomyocytes.Ultimately,ourresultsmayshedlightonthediscrete
et al., 1999; Chung and Rajakumar, 2016; Elliott et al., 2003;
errors responsible for NKX2-5-dependent human CHDs of the
Goldmuntzetal.,2001;McElhinneyetal.,2003;Nakajima,2010;
cardiacoutflowandinflowtracts.
Schottetal.,1998;SrivastavaandOlson,2000).Investigationofthe
KEYWORDS:nkx2.5,nkx2.7,Arterialpole,Venouspole,Second genetic basis of CHDs in model systems hasyielded insights into
heartfield,Zebrafish functions of Nkx2-5 in progenitor specification in Drosophila,
Xenopus and mouse (Azpiazu and Frasch, 1993; Bodmer, 1993;
INTRODUCTION Grow and Krieg, 1998; Prall et al., 2007), and in cardiac
Cardiomyocytes(CMs)arederivedfromatleasttwodistinctcardiac morphogenesis in mouse and zebrafish (Barth et al., 2010; Lyons
progenitor populations located in the splanchnic mesoderm. First et al., 1995; Prall et al., 2007; Tanaka et al., 1999; Targoff et al.,
heart field (FHF) cells differentiate early in the lateral plate 2013,2008;Tuetal.,2009).Furthermore,Nkx2-5mutants,whether
mesodermtogiverisetothelinearhearttube(Buckinghametal., inside oroutside the DNA-binding domains (Benson et al., 1999;
2005;VincentandBuckingham,2010).Subsequently,secondheart Gutierrez-Roelens et al., 2002; Ikeda et al., 2002), regulate
field (SHF) cells migrate to the pharyngeal mesoderm, proliferate wild-type targets and also ‘off-targets’, destabilizing the cardiac
and differentiate into CMs at the arterial and venous poles. The transcriptional hierarchy in this context (Bouveret et al., 2015).
anteriorSHF(aSHF)subpopulationgivesrisetotherightventricle Although previous work has revealed specific roles for Nkx2-5 in
and outflow tract (OFT), whereas the posterior SHF (pSHF) theSHF(Cambieretal.,2014;Clarketal.,2013;Dornetal.,2014;
contributes primarily to the atria and inflow tract (IFT) Georgeetal.,2015;Guner-Atamanetal.,2013;Pralletal.,2007;
(Buckingham et al., 2005; Cai et al., 2003; Dominguez et al., Watanabeetal.,2012;Witzeletal.,2012;Zhangetal.,2014),the
2012;DyerandKirby,2009;Gallietal.,2008;Ranaetal.,2014; distinctcellularandmolecular mechanismsoperatingintheaSHF
andpSHFsubpopulationsremainlargelyunknown.
Recent studies have revealed essential functions of Nkx2-5 T
1DivisionofCardiology,DepartmentofPediatrics,CollegeofPhysiciansand N
Surgeons,ColumbiaUniversity,NewYork,NY10032,USA.2BrighamandWomen’s transcriptionalregulationinOFTdevelopment.Inmouse,Nkx2-5 E
Hospital/HarvardMedicalSchool,CardiovascularDivision,75FrancisStreet, hasbeenshowntodrivearterialpoleformationviaaBmp2/Smad1 M
Thorn11,Boston,MA02115,USA.
negative-feedback loop (Prall et al., 2007) and through a direct P
*Authorforcorrespondence([email protected]) repressive effect on a 1.7 kb Fgf10 enhancer element (Watanabe O
etal.,2012).Inzebrafish,nkx2.5playsaroleinaSHFproliferation L
S.C.,0000-0001-8945-5631;V.G.,0000-0003-1990-4651;A.A.W.,0000-0002- E
1222-1180;C.A.M.,0000-0001-5181-2664;K.L.T.,0000-0002-6066-6002 (Guner-Atamanetal.,2013)andstabilizationofNkx2.5byAjuba V
proteinregulatesprogenitorspecificationinthispopulation(Witzel E
D
Received16November2017;Accepted4December2017 etal.,2012).Moreover,theexpressionpatternsofltbp3andmef2cb,
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RESEARCHARTICLE Development(2018)145,dev161497.doi:10.1242/dev.161497
aSHF markers in zebrafish, overlap with the nkx2.5-expressing normallyrestrictisl1expressiontothevenouspoleduringhearttube
region near the arterial pole (Lazic and Scott, 2011; Zhou et al., formation,whereasoverexpressionofnkx2.5issufficienttoinhibit
2011).AlthoughthesedatadocumenttheimportanceofNkxgenes isl1. Notably, electrophysiological chamber identity is dependent
in arterial pole development, it is unclear how Nkx2.5 controls on Nkx gene dose with elimination of all heterogeneity of
differentiationandexpansionoftheaSHFprogenitorpooltosculpt intercellular coupling in the nkx2.5−/−;nkx2.7−/− embryos. These
theprecisedimensionsoftheOFT. findings support the innovative concept that CM differentiation is
Atthesinusvenosus(SV),extensivestudieshaveelucidatedthe highly graded, with functional partitioning of the heart tube
function of Nkx2-5 in cardiac conduction system (CCS) established and maintained through networks of overlapping
development (Briggs et al., 2008; Jay et al., 2004; Moskowitz regulators rather than through binary fate choices. This gradation
etal.,2007;Nakashimaetal.,2009;Pashmforoushetal.,2004;Ye is consistent with the pleiotropy of phenotypes observed in
etal.,2015).YetthemechanismsbywhichNkxgenesdirectpSHF monogenic forms of CHD and the known redundancy and
progenitorstopatternthevenouspolehavenotbeenfullydefined.It resilience of cardiac transcriptional networks. Taken together, our
is evident from murine models that pSHF cells accumulate in the results suggest key functions of Nkx genes in SHF CM
atrialchambers(Caietal.,2003;Gallietal.,2008;Lescroartetal., differentiation in mediating differential roles in anterior and
2012)andpSHFprogenitorsdirectthemolecularandcellularevents posteriorpopulations.Ultimately,ourstudieshavethepotentialto
contributing to atrioventricular (AV) septation (Goddeeris et al., shed light on the mechanisms underlying conotruncal and venous
2008; Hoffmann et al., 2009, 2014; Mommersteeg et al., 2006; pole malformations, and conduction system defects in individuals
Snarr et al., 2007a,b; Xie et al., 2012). Interestingly, Nkx2-5 is carryingNKX2-5mutations.
importantinestablishingabalancebetweensinoatrialnode(SAN)
and atrial myocardial identities (Espinoza-Lewis et al., 2011; RESULTS
Mommersteeg et al., 2007; Nakashima et al., 2014). Another OFTsizeisrestrictedinnkxmutants
homeodomaintranscriptionfactor,Shox2,isalsoessentialforSAN Although our previous studies revealed synergistic functions of
development,inpartthroughrepressionofNkx2-5(Blaschkeetal., nkx2.5 and nkx2.7 in OFT development (George et al., 2015;
2007; Espinoza-Lewis et al., 2009). This Shox2-Nkx2-5 Targoffetal.,2013),thediscretecellularandtemporalmechanisms
antagonistic mechanism inhibits pacemaker properties that define operating downstream of Nkx genes in the aSHF remain unclear.
a ‘default’ primitive cellular phenotype (Ye et al., 2015). Previous studies have identified SHF progenitors in the anterior
Furthermore, studies have shown that Shox2 directly regulates lateralplatemesoderm(ALPM)thatexpressnkx2.5andcontribute
Isl1(Hoffmannetal.,2013)andNkx2-5inhibitsIsl1(Dornetal., to the OFT (Guner-Ataman et al., 2013; Paffett-Lugassy et al.,
2014; Prall et al., 2007; Watanabe et al., 2012), a key cardiac 2017). Thus, we examined the roles of nkx2.5 and nkx2.7 in
transcriptionfactorexpressedinthepSHF(Caietal.,2003;Snarr regulating the spatiotemporal contribution of the aSHF to arterial
etal.,2007a;Sunetal.,2007;vandenBergetal.,2009).Variation poleformation.Employingadevelopmentaltimingassaytoassess
in ISL1 confers genetic susceptibility for CHDs (Stevens et al., the differences in protein maturation time in embryos expressing
2010),thushighlightingacrucialroleinhumandisease.Moreover, Tg(myl7:EGFP)andTg(-5.1myl7:nDsRed2)(dePateretal.,2009;
data have illuminated a pivotal role for Isl1 in SAN development Huangetal.,2003;Mablyetal.,2003)(Fig.1A-I), wevisualized
andfunction(Liangetal.,2015;Tessadorietal.,2012;Vedantham theaccretionoftheaSHF-derivedCMsintheOFTofwild-typeand
et al., 2015), and these findings offer explanations for CCS nkx2.5−/−;nkx2.7+/+,nkx2.5−/−;nkx2.7+/−andnkx2.5−/−;nkx2.7−/−
abnormalities in individuals harboring NKX2-5 mutations. Given (hereafter referred to as nkx mutant) embryos. Progressive loss of
embryonicstemcellworksuggestingthatdirectIsl1repressionby Nkxgenefunctionhighlightssubstantialreductioninthevolumeof
Nkx2-5isnecessarytoestablishtheventricularlineage(Dornetal., OFTmyocardium(Fig.1D-I).Moreover,wedetectedastatistically
2014) and our findings revealing essential roles of Nkx genes in significant reduction in aSHF-derived CMs, or EGFP+ DsRed−
ventricularidentitymaintenance(Georgeetal.,2015;Targoffetal., cells, when comparing wild-type (24 cells±3 cells; n=12) with
2013), we sought to uncover novel Nkx-Isl1 interactions that are nkx2.5−/−;nkx2.7−/− (16 cells±4 cells; n=3) embryos (P<0.005).
requiredtopatterntheIFT. Taken together, these findings underscore that nkx2.5 and nkx2.7
Our previous studies demonstrate that the Nkx2-5 homologs positively regulate the contribution of aSHF progenitors at the
expressedinzebrafishCMs,nkx2.5andnkx2.7(Leeetal.,1996),act zebrafisharterialpole.
redundantly in cardiac morphogenesis (Targoff et al., 2008; Tu
et al., 2009) and are crucial in maintaining ventricular identity by NkxgenesaredispensableforOFTcardiomyocyte
repressingatrialfate(Targoffetal.,2013).Furthermore,temporally proliferation
controlled expression of nkx2.5 is essential to ensure chamber- Prior work has elucidated an important role for SHF CM
specific identity maintenance in both the FHF and SHF during proliferation in zebrafish arterial pole development (Guner-
explicitdevelopmentaltimewindows(Georgeetal.,2015).Despite Ataman et al., 2013; Lazic and Scott, 2011; Nevis et al., 2013;
theseinsights,littleisknownaboutthemechanismsbywhichNkx Schindleret al., 2014; Zeng and Yelon, 2014; Zhou et al., 2011).
genes control the specification and differentiation of SHF Given the decreased number of aSHF-derived CMs in the nkx
progenitors at the cardiac poles. In this study, we find that Nkx mutant OFT, we tested whether diminished proliferation is T
N
genespositivelyregulateaSHFprogenitorsatthearterialpoleand responsible for this observation. We pulsed embryos carrying
E
restrictpSHF-derivedCMdifferentiationatthevenouspole.Atthe Tg(-5.1myl7:nDsRed2) at 24hpf, when aSHF-derived CMs M
arterial pole, Nkx genes are required to promote the aSHF proliferate, with the thymidine analog 5-ethynyl-2′-deoxyuridine P
progenitors, within the proliferating pharyngeal region, to (EdU)(Guner-Atamanetal.,2013;Schindleretal.,2014;Zengand O
differentiate and assemble the OFT. In nkx2.5−/−;nkx2.7−/− Yelon,2014).At48hpf,wecountedEdU+DsRed+MF20+cellsat L
E
embryos, unrestricted expression patterns of SAN genes, bmp4, thearterialpoletoquantifyproliferatingnucleiinthemyocardium V
tbx2b, hcn4 and shox2, highlight severe abnormalities in venous (Fig.S1).Wedetectednostatisticallysignificantdifferencesinthe E
D
poledevelopment.Furthermore,ourfindingsrevealthatNkxgenes proliferation indices between wild-type (1.0%±1.3%; n=15) and
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RESEARCHARTICLE Development(2018)145,dev161497.doi:10.1242/dev.161497
myocardium using Tg(nkx2.5:kaede) (Guner-Ataman et al., 2013;
Zeng and Yelon, 2014). Employing Tg(nkx2.5:ZsYellow) and
Tg(myl7:EGFP), we examined aSHF progenitor cells in the
pharyngeal region distal to the arterial polewhere they proliferate
prior to differentiation and addition to heart (Hutson et al., 2010;
Tirosh-Finkel et al., 2010; van den Berg et al., 2009; Zeng and
Yelon, 2014). We observed dispersion and lower levels of
expression of Tg(nkx2.5:ZsYellow) in the distal nkx2.5+ OFT
progenitors in nkx mutant compared with wild-type embryos at
36hpf (Fig. 2F,G). We quantified this alteration in expression
patterns by counting ZsYellow+ EGFP− cells distal to the arterial
pole and between the branchial arches (Fig. 2H). Our results
demonstrate a dramatic decrease in the number of distal nkx2.5+
progenitors contributing to the OFT in nkx mutant embryos.
Together, these datasuggestthatloss of Nkxgene functionlimits
thenumberofundifferentiatedaSHFcellspriortotheirdeployment
tothearterialpole.
NkxgenesregulateOFTprogenitorproliferationtimingand
differentiation
From our findings demonstrating that Nkx genes are required for
OFTformationbyearlyregulationoftheaSHFprogenitorpoolsize,
as opposed to later influence on SHF-derived CM production, we
Fig.1.OFTsizeandCMnumbersarereducedinnkxmutants.(A-I)Lateral postulated that the decreased number of OFT progenitors in Nkx
view,anteriortowardstheright,at52hpf.Representativereconstructionsof mutants could be due to diminished progenitor proliferation. To
fourz-stacksfromadevelopmentaltimingassayshowdiminishedcell investigate this hypothesis, we performed an EdU incorporation
numbersintheOFTofwild-type(A-C),nkx2.5−/−;nkx2.7+/+(D-F)and assayinembryoscarryingTg(nkx2.5:ZsYellow)at18hpftoidentify
nkx2.5−/−;nkx2.7−/−(G-I)embryoscarryingTg(myl7:EGFP)andTg(-5.1myl7:
dividingprogenitorcellspriortotheirmigrationtothearterialpole.
nDsRed2).
At 40hpf, we performed MF20 immunostaining to delineate
proliferative ZsYellow+ cells that had been incorporated into the
nkxmutant (1.5%±1.2%; n=8) embryos, indicating thata reduced OFTinwild-type(Fig.3A,B)andnkxmutant(Fig.3C,D)embryos.
rateofOFTproliferationisnotresponsibleforthedecreaseinCMs Surprisingly,wefoundnosignificantdifferenceintheproliferation
accruedatthearterialpole. indexofthetotalOFTprogenitorpopulationwhencomparingwild-
type with nkx mutant embryos (Fig. 3E). We next tested whether
NkxgenesdelimittheOFTprogenitorpool theremight be differences in OFT progenitor proliferation indices
WethenpostulatedthatashrunkenarterialpolesizeinNkxmutant betweenwild-typeandnkxmutantembryosintheproximalearly-
embryos could represent an earlier influenceof Nkx genes on the differentiating (ZsYellow+ MF20+) and distal late-differentiating
reservoir of undifferentiated aSHF progenitors. The SHF-derived (ZsYellow+ MF20−) populations. The proximal OFT progenitor
population at the OFT can be divided into two cell types: population is recruited to the arterial pole while the distal OFT
differentiated CMs that strongly express myl7, vmhc and nkx2.5, progenitor population remains in a proliferative state in the
andaprogenitorpoolthatfaintlyexpressesthesesamemarkersand pharyngeal region (van den Berg et al., 2009). We found that our
stronglyexpressesmef2cbandltbp3(LazicandScott,2011;Zeng results from the EdU incorporation assayare comparable between
and Yelon, 2014; Zhou et al., 2011). Indeed, our findings reveal wild-type and nkx mutant embryos in the proximal precursor
diminished mef2cb expression in the arterial pole of wild-type populationcontributingtothearterialpole(Fig.3F).However,there
compared with nkx mutant embryos (Fig. 2A-E). Specifically, is a statistically significant increase in the proliferation index of
mef2cbexpressionisslightlydecreasedinnkx2.5−/−;nkx2.7+/+and those OFT progenitors that reside in the distal pharyngeal region
nkx2.5−/−;nkx2.7+/− embryos (Fig. 2C,D), and further diminished when comparing wild-type and nkx mutant embryos (Fig. 3B
in nkx2.5−/−;nkx2.7−/− embryos (Fig. 2E) when compared with comparedwithD;Fig.3G).Insummary,ourdatasuggestthatNkx
wild-type and nkx2.5+/+;nkx2.7−/− embryos (Fig. 2A,B). These genes regulate OFT size by restricting the proliferation of aSHF
results corroborate our previously published studies illustrating a progenitors in the distal pharyngeal region and promoting
progressivedeclineinltbp3expressionwithsuccessivereductionof differentiationastheymigratetoformthearterialpole.
Nkx gene dose (Targoff et al., 2013). Altogether, our findings
suggest that Nkx gene function is required to establish the proper isl1functionsdownstreamofNkxgenesatthevenouspole
size of the aSHF progenitor pool. However, in assessing this In light of our evidence suggesting that Nkx genes positively T
N
population through gene expression profiles of SHF markers, we regulatetheaSHFOFTprogenitorpool,weexaminedpossibleroles
E
mayfailtoidentifyallcellsdestinedtobecomeaSHF-derivedCMs. in pSHF development given the relatively high incidence of atrial M
Given the importance of the temporal regulation of aSHF septal defects in individuals carrying NKX2-5 mutations. In the P
precursor accretion to the arterial pole (Cambier et al., 2014; absence of clearly defined regulators of the IFT in zebrafish, we O
Jahangirietal.,2016;ZengandYelon,2014),wesoughttoquantify investigated Isl1, a LIM homeodomain transcription factor, as a L
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aSHFprogenitorsbytrackingtheselate-differentiatingcellsduring potential candidate downstream of Nkx genes at the venous pole. V
OFTrecruitment.Recentstudieshaveconfirmedthecontributionof Extensivestudieselucidatedthatisl1isessentialfornormalcardiac E
D
distal Tg(nkx2.5:ZsYellow)-expressing progenitorcellsto the OFT developmentacrossspecies(Bradeetal.,2007;Caietal.,2003;de
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RESEARCHARTICLE Development(2018)145,dev161497.doi:10.1242/dev.161497
Fig.2.NkxgenesdelimittheOFTprogenitorpool.(A-E)Dorsalview,anteriortowardsthetop,at26hpf.Insituhybridizationdepictsdiminishedexpressionof
mef2cb,whichlabelsaSHF-derivedCMprogenitors,inrepresentativenkx2.5−/−;nkx2.7+/+(n=6)(C),nkx2.5−/−;nkx2.7+/−(n=3)(D)andnkx2.5−/−;nkx2.7−/−(n=5)
(E)embryoscomparedwithwild-type(n=15)(A)andnkx2.5+/+;nkx2.7−/−(n=5)(B)embryos.(F,G)Ventralviewofthearterialpoleregionat36hpf.Confocal
projectionsofimmunohistochemistryforZsYellow(green)andEGFP(red)inembryoscarryingTg(nkx2.5:ZsYellow)andTg(myl7:EGFP)illustrate
decreasedcontributionofnkx2.5+aSHF-derivedCMprogenitors(ZsYellow+EGFP−)tothearterialpoleinnkx2.5−/−;nkx2.7−/−(G)comparedwithwild-type
(F)embryos(boxedareas).(H)QuantificationofZsYellow+EGFP−cellsatthearterialpoleinwild-type(n=9)andnkxmutant(n=8)embryosrevealsastatistically
significantdecreaseinthispopulationfollowingthelossofNkxgenefunction.Student’st-testwasusedtodeterminestatisticalsignificance.Meanands.e.m.of
eachdatasetareshown(**P<0.0001).
Pateretal.,2009;Panduretal.,2013;Witzeletal.,2012),delineates somitogenesis(Witzeletal.,2012).However,Isl2bappearstoplay
the SHFat both arterial and venous poles (Cai et al., 2003; Hami aroleinarterialpoledevelopment (Witzeletal.,2017)andIsl1in
et al., 2011; Snarret al., 2007a; Stoyeket al., 2015; Witzel et al., venouspoledevelopmentwithalossoflate-differentiatingCMsin
2012), and is required for proper pacemaker development and the IFT in isl1−/− embryos (de Pater et al., 2009). Thus, we
function (Liang et al., 2015; Tessadori et al., 2012; Vedantham postulated that Nkx genes inhibit isl1 expression at the SV. In
et al., 2015). In zebrafish, using an antibody that recognizes both support of our model, we found that Isl1 immunostaining is
Isl1andIsl2proteins(D’Amicoetal.,2007;HutchinsonandEisen, localized specifically to the IFT of wild-type embryos (Fig. 4A),
2006;Witzeletal.,2012),Isl1/2+cellsarefoundneartheOFTand while Isl1+ CMs extend throughout the atrium (S46+ region) in
IFT(dePateretal.,2009;Hamietal.,2011;Witzeletal.,2012),and nkx2.5−/−;nkx2.7+/+ embryos (Fig. 4C) and reach the OFT in
co-expression of Nkx2.5 in this population is evident during nkx2.5−/−;nkx2.7−/−embryos(Fig.4E).WequantifiedIsl1+nuclei
Fig.3.NkxgenesregulateOFTprogenitorproliferation
timinganddifferentiation.(A-D)Ventralviewofthearterialpole
regionat40hpf.Confocalprojectionsofimmunohistochemistry
forEdU(red),ZsYellow(green)andMF20(gray)inembryos
carryingTg(nkx2.5:ZsYellow)followingEdUincubationat18hpf
inwild-type(A,B)andnkx2.5−/−;nkx2.7+/+(C,D)embryos.White
arrowsindicateEdU+ZsYellow+cells.TheboxedareasinA,Care
shownathighermagnificationinB,D.(E-G)Proliferationindices
showingastatisticallysignificantincreaseinundifferentiated,
ZsYellow+MF20−progenitorsinnkxmutant(n=6)comparedwith
wild-type(n=6)embryos(G);theproliferationindexiscomparable
intheproximal,ZsYellow+MF20+CMpopulationcontributing
tothearterialpole(F)aswellasinthetotalOFT,ZsYellow+
MF20+/−CMpopulation(E).Student’st-testwasusedto
determinestatisticalsignificance.Themeanands.e.m.ofeach
datasetareshown(*P<0.005).
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Fig.4.NkxgenesinhibitIsl1expressionatthesinusvenosus.(A-E)Lateralview,anteriortowardsthetop,at54hpfinwild-type(A),nkx2.5+/+;nkx2.7−/−(B),
nkx2.5−/−;nkx2.7+/+(C),nkx2.5−/−;nkx2.7+/−(D)andnkx2.5−/−;nkx2.7−/−(E)embryos.RepresentativeconfocalimagesofimmunofluorescenceforIsl1(green)
andS46(red)demonstratethatIsl1isrestrictedtothevenouspoleofwild-type(A)andnkx2.5+/+;nkx2.7−/−(B)embryos.However,Isl1expressionexpands
throughouttheatriuminnkxmutants(C-E).(F)QuantificationofIsl1+S46+CMsisdepictedshowingagradualincreaseincellnumberwithaprogressivelossof
nkxalleles.Meanands.e.m.ofeachdatasetareshown(*P<0.005)inwild-type(n=16),nkx2.5+/+;nkx2.7−/−(n=2),nkx2.5−/−;nkx2.7+/+(n=6),nkx2.5−/−;nkx2.7+/−
(n=10)andnkx2.5−/−;nkx2.7−/−(n=4)embryos.Student’st-testwasusedtodeterminestatisticalsignificance.
anddetectedaprogressiveincreaseinthenumberofcellsexpressing NkxgenesregulatedifferentiationofpSHFprogenitors
thistranscriptionfactorasNkxgenedoseisdiminished(Fig.4F). Although recent work has shown the contribution of late-
Notably,thenumberofIsl1+CMsinthenkx2.5−/−;nkx2.7−/−heart differentiating CMs to the venous pole in zebrafish (de Pater
isapproximatelyequivalenttothetotalnumberofCMsinwild-type et al., 2009; Mosimann et al., 2015), little is known about the
embryos (Targoff et al., 2013). Next, we investigated Nkx2.5 and transcriptional regulatory networks guiding accretion and
Isl1 expression patterns employing immunohistochemistry in differentiation of this pSHF progenitor population. Given the
embryos carrying Tg(nkx2.5:ZsYellow) at 52hpf. This transgene ability of nkx2.5 to restrict Isl1 to the IFT (Fig. 5N-Q), we
faithfullyrecapitulatestheendogenousnkx2.5expressionobserved hypothesize that Nkx genes are essential in patterning the pSHF-
byinsituhybridization(datanotshown).WefindthatNkx2.5and derived CMs in this region. To test this hypothesis, we first
Isl1arevirtuallymutuallyexclusiveatthevenouspoleinwild-type performedadevelopmentaltimingassaytodelineatethekineticsof
embryos(Fig.5A-A″).Nkx2.5isexpressedinadecreasinggradient pSHFprogenitoraccumulationusingembryosexpressingTg(myl7:
fromtheOFTtotheinflowoftheatrium,whereasIsl1expressionis EGFP)andTg(-5.1myl7:nDsRed2)(asinFig.1A-I). Intriguingly,
confinedtoSVwithonlyafewcellsco-expressingNkx2.5andIsl1 our data reveal a statisticallysignificant increase in pSHF-derived
(Fig. 5A″). Alternatively, in nkx mutants, Isl1 expression is CMs, or EGFP+ DsRed− CMs, at the venous pole in nkx2.5−/−;
derepressed in all atrial CMs that normally express Nkx2.5 nkx2.7+/+ embryos along with further enhancement in nkx2.5−/−;
(Fig. 5B-E″). Taken together, our studies strongly support the in nkx2.7−/− embryos when compared with wild-type embryos
vivo suppressionof Isl1+cell fate by Nkx genes in the SV during (Fig. 6A-I,N). However, the source of the increased late-
cardiacdevelopment. differentiating CMs at the IFT remains unknown. This finding
We then explored the timing of the Nkx-Isl1 interaction in may reflect either increased proliferation of pSHF progenitors or
further detail. We evaluated isl1 expression during earlystages of enhancedaccrualofpSHF-derivedCMs.
cardiac differentiation in the ALPM. Our data illustrate that Consistent with our data illustrating normal CM production
specification of isl1+ progenitors occurs normally at 10 somites at the arterial pole following the loss of Nkx gene function
and15somites(Fig.5F-Ianddatanotshown),butisl1expressionis (Fig. S1), we observed no difference in proliferation indices at
enhancedat26hpfinnkxmutantcomparedwithwild-typeembryos the venous pole following EdU incorporation at 24hpf
(Fig. 5J-M). These findings indicate that Nkx genes are first between nkx mutant and wild-type embryos (data not shown).
required to inhibit isl1 at the heart tube stage. Using a novel Thus, we hypothesized that Nkx genes are required for pSHF
transgenegeneratedinourlabforubiquitousnkx2.5overexpression, progenitor proliferation prior to accretion to the IFT. We
Tg(hsp70l:nkx2.5-EGFP) (George et al., 2015), we tested the performed an EdU incorporation assay at 18hpf in embryos T
N
sufficiency of nkx2.5 to inhibit isl1 expression (Fig. 5N-Q). carrying Tg(-5.1myl7:nDsRed2) to detect dividing CMs at the
E
Following heat-shock activation of this inducible transgene at 21 venous pole. Quantification of the proliferation indices for M
somites, isl1 expression was repressed specifically in the inflow EdU+ DsRed+ MF20+ cells in the IFT region at this time point P
region at both 24hpf and 52hpf (Fig. 5P,Q). Altogether, these also revealed no statistically significant difference between O
resultsdemonstratethatNkx2.5isnotrequiredforisl1+progenitor wild-type and nkx mutant embryos (Fig. 6J-M,O). These data L
E
specification, but activation of Nkx2.5 transcription prior to heart support the conclusion that the IFT expansion in nkx mutant V
tubeelongationissufficienttorepressisl1atthevenouspoleduring embryos is most likely due to enhanced pSHF-derived E
D
chamberemergence. CM differentiation.
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RESEARCHARTICLE Development(2018)145,dev161497.doi:10.1242/dev.161497
Fig.5.Nkx2.5isnotrequiredforisl1+progenitorspecification,butissufficienttorepressisl1atthevenouspole.(A-E″)Representativeconfocalimages,
lateralview,anteriortowardsthetop,at52hpf.ImmunostainingforIsl1(red)inembryoscarryingTg(nkx2.5:ZsYellow)demonstratesthatIsl1isrestrictedtothe
venouspoleandNkx2.5expressionisabsentinthisregioninwild-type(whitearrow)(A-A″)andnkx2.5+/+;nkx2.7−/−(B-B″)embryos.However,Isl1expressionis
derepressedintheatriumofnkx2.5−/−;nkx2.7+/+(C-C″)andnkx2.5−/−;nkx2.7+/−(D-D″)embryos,andexpandstowardstheoutflowtractinnkx2.5−/−;nkx2.7−/−
(E-E″)embryos.(F-I)Dorsalview,anteriortowardsthetop,at10somites.InsituhybridizationshowsnovariationinALPMisl1expressionbetweenwild-type(F)and
nkxmutant(G-I)embryos.(J-M)Lateralview,anteriortowardstheleft,at26hpf.Insituhybridizationdemonstratesthatisl1expressionisrestrictedtoaring
atthevenouspoleofthehearttubeinwild-type(J)andnkx2.5+/+;nkx2.7−/−(K)embryos.However,isl1expressionexpandstotheatriuminnkx2.5−/−;nkx2.7+/+
embryos(L)andthroughouttheentirehearttubeinnkx2.5−/−;nkx2.7−/−embryos(M).Blackarrowsindicatetheringofisl1expressionneartheIFT(J,K).(N-Q)Lateral
view,anteriortowardstheleft,at24hpf(N,P)andventralview,anteriortowardsthetop,at52hpf(O,Q)innon-transgenic(N,O)andTg(hsp70l:nkx2.5-EGFP)
(P,Q)embryos.Insituhybridizationdemonstratesthatisl1expressionisrestrictedtoaringatthevenouspole(blackarrows)at24hpf(n=9)(N)and52hpf(n=7)(O),
andthatoverexpressionofnkx2.5inhibitsisl1expressionspecificallyinthisregion(dottedarrows)at24hpf(n=9/11)(P)and52hpf(n=8/9)(Q).
Nkxgenespatternspecializednodaltissueinthesinus (AVC)andIFTat52hpf(Fig.7A,F,K).Intriguingly,allthreegenes
venosusviaisl1 are expressed throughout the myocardium in nkx mutant embryos
Althoughrecentstudieshaveelucidatedtheexistenceofspecialized (Fig.7C-E,H-J,M-O).Weobserveasimilarexpression patternfor
conductiontissueinthezebrafishheart(Arrenbergetal.,2010;Chi shox2whencomparingwild-typeandnkxmutantembryos(Fig.S2).
etal.,2008;Milanetal.,2006)andhaveidentifiedisl1+asmolecular Taken together, our findings illustrate that Nkx genes regulate
marker of this population (Tessadori et al., 2012), the pathways regionalizationofSANmarkerstotheIFT.
controlling the development of the sinoatrial node (SAN) are still Next,wepostulatedthatIsl1actingdownstreamofNkxgenesis
underinvestigation.Therefore,fromevidenceofconductionsystem responsibleforpatterningtheSANandestablishingthepacemaker
abnormalitiesinindividualswithNKX2-5mutations(Bensonetal., activity of the zebrafish heart. Using a previously validated
1999; Elliott et al., 2003; Gutierrez-Roelens et al., 2002; Schott morpholino (MO) to knockdown Isl1 function (Hutchinson and T
N
etal.,1998)andfromourfindingsrevealingthatNkxgenesregulate Eisen,2006),weobservedthatbmp4expressionisabsentinwild-
E
isl1 expression (Figs 4 and 5), we posited that Nkx genes are type and nkx2.5+/+;nkx2.7−/− embryos specifically at the IFT M
fundamentalinpatterningthepSHF-derivedCMsthatcontributeto following islet1E2 MO injection, whereas normal AVC and OFT P
the specialized nodal tissue in the SV. To address this issue, we expressionofbmp4persists(compareFig.8A,BwithF,G).These O
exploredtheexpressionofanumberofpacemakercellgenes:bmp4, findingsareconsistentwithevidenceofselectiveinhibitionofbmp4 L
E
tbx2b,hcn4andshox2(Blaschkeetal.,2007;Espinoza-Lewisetal., expressionintheIFToftheisl1sa29mutant (dePateretal.,2009). V
2009; Tessadori et al., 2012). In wild-type embryos, bmp4, tbx2b Interestingly, inhibition of isl1 splicing in nkx mutant embryos E
D
andhcn4expressionisrestrictedtotheOFT,atrioventricularcanal reveals specific downregulation of ectopic atrial and IFT bmp4
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Fig.6.Nkxgenesregulatecardiomyocyteaccretiontotheatrium.(A-I)Lateralimages,anteriortowardsthetop,at52hpf.Representativereconstructionsof
10z-stacksfromadevelopmentaltimingassayhighlightthevenouspoleofwild-type(A-C),nkx2.5−/−;nkx2.7+/+(D-F)andnkx2.5−/−;nkx2.7−/−(G-I)embryos
carryingTg(myl7:EGFP)(green)andTg(-5.1myl7:nDsRed2)(red).AsterisksindicateEGFP+DsRed−cells.(J-M)Ventralview,anteriortowardsthetop,at40hpf
ofwild-type(J,K)andnkx2.5−/−;nkx2.7+/+(L,M)embryoscarryingTg(-5.1myl7:nDsRed2).Confocalprojections(J,L)ofimmunohistochemistryforEdU
(red),DsRed(green)andMF20(gray)followingEdUincubationat18hpfandfourz-stackreconstructions(K,M)wereusedtocountEdU+DsRed+MF20+CMsin
theatrium.TheboxedareasinJ,LareshownathighermagnificationinK,M,respectively. WhitearrowsindicateEdU+DsRed+CMs.(N,O)Quantificationof
EGFP+DsRed−cellsatthevenouspoleforwild-type(n=12),nkx2.5−/−;nkx2.7+/+(n=5)andnkx2.5−/−;nkx2.7−/−(n=4)embryos,andproliferationindicesin
wild-type(n=5)andnkxmutant(n=3)embryos.Student’st-testwasusedtodeterminestatisticalsignificance.Meanands.e.m.ofeachdatasetareshown
(**P<0.0001).ThereisnostatisticallysignificantdifferencebetweenproliferationindicesintheDsRed+MF20+populationsofwild-typeandnkxmutantembryos.
expression(compareFig.8H-JwithC-E)inthecontextofotherwise the atrial and ventricular conduction velocities in wild-type and
normal development. Together, our results demonstrate that the nkx2.5+/+;nkx2.7−/− embryos (Fig. 9E,F,I). In contrast, disruption
misregulation of bmp4 expression in nkx mutant embryos is ofnkx2.5genefunctionaffectsmultiplecomponentsoftheaction
mediated by isl1 overexpression, ultimately placing isl1 potential with significant slowing of the upstroke phase in both
downstream of Nkx genes in defining the pSHF-derived chambers(Fig.9A-C),markeddiminutionofthemaximumslopeof
specializedconductioncellsoftheIFT. the action potential upstroke (Fig. 9C), as well as a substantial
prolongationofatrialactionpotentialdurationandaccentuationof
Nkxgenesregulateelectrophysiologicalpatterning phase 4 depolarization in atrial CMs (Fig. 9A,D). Moreover,
Finally,weexploredtheeffectsofthederepressionofSANgeneson homozygous loss of nkx2.5 function leads to severe slowing in
the electrophysiological phenotype in nkx mutant hearts. The actionpotentialpropagation,asdemonstratedbycloserspacingof
establishment of electrical gradients enables the transition from theisochronesandsignificantlylowermeanconductionvelocities
peristaltic to sequential contraction in the developing heart and this measured across both cardiac chambers (Fig. 9G-I). Interestingly,
patterningisregulatedbydifferentialcontributionsofFHFandSHF althoughthereisfailureofthenormalspecificationofphysiological
progenitorpopulations(Mosimannetal.,2015).Wehypothesizedthat AVdelayintheheartsofnkx2.5−/−;nkx2.7+/+embryos(Fig.9K,L),
thelossofNkxgenefunctioncouldleadtoadisruptioninregional thereremainsignificantdifferencesinactionpotentialcharacteristics
patternsofintercellularcouplinggivenboththeabsenceofventricular between the ventricle and atrium (Fig. 9J,K and Fig. S3A-C,E-G).
identity (Targoff et al., 2013, 2008) and the expanded SAN gene Although the hearts from nkx2.5+/+;nkx2.7−/− embryos did not
expressiondomaininnkxmutantcomparedwithwild-typeembryos exhibit any detectable electrophysiological phenotype (Fig. 9F),
(Fig.7). combined loss of function of both nkx homologs in nkx2.5−/−;
To explore these issues, we measured action potential nkx2.7−/− embryos leads to complete loss of electrophysiological
characteristicsandconductionpropertiesinisolatedwild-typeand chamberidentityandfurtherslowingoftheactionpotentialupstroke
nkx mutant hearts using high-resolution optical mapping (Fig. 9, phaseinthe atrium (Fig.9A,C,Jand Fig.S3D).The lossofaction T
N
Fig.S3).Wedidnotdetectanyelectrophysiologicalphenotypesin potential duration differences between chambers is also evident in
E
embryoscarryingsingle-alleledeficienciesinoneorbothnkxgenes nkx2.5−/−;nkx2.7−/−embryos(Fig.9D,KandFig.S3H),includingthe M
(nkx2.5+/−;nkx2.7+/+,nkx2.5+/+;nkx2.7+/−andnkx2.5+/−;nkx2.7+/−). emergenceofa pacemakerphenotype in ventricularcells(Fig.9B) P
When comparing wild-type and nkx2.5+/+;nkx2.7−/− embryos, and slowingoftheconductionvelocity (Fig.9H,I,L). Interestingly, O
there is no significant difference in the detailed action potential therearesubtle,butsignificant,paradoxicaleffectsonabsoluteatrial L
E
morphology (Fig. 9A,B), maximum upstroke velocity, [dF/dt]max and ventricular conduction velocities in nkx2.5−/−;nkx2.7−/− when V
(Fig. 9C), or atrial and ventricular action potential durations compared with nkx2.5−/−;nkx2.7+/+ embryos. In the atrium, the E
(Fig.9D).Furthermore,therearenoobserveddifferencesbetween nkx2.5−/−;nkx2.7−/−embryosexhibitevenslowerconductionthanthe D
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RESEARCHARTICLE Development(2018)145,dev161497.doi:10.1242/dev.161497
Fig.7.NkxgenesregulateSANmarkers.(A-O)Ventralview,anteriortowardsthetop,at52hpf.Dottedwhitelinesillustratethecardiacsilhouettes.Insitu
hybridizationforbmp4(A-E),tbx2b(F-J)andhcn4(K-O).Expressionofbmp4isrestrictedtotheOFT(whitearrow),AVC(yellowarrow)andIFT(blackarrow)in
wild-type(n=7/7)(A)andnkx2.5+/+;nkx2.7−/−(n=4/4)(B)embryos.However,itsexpressionisexpandedthroughoutthecardiacchambersinnkx2.5−/−;nkx2.7+/+
(n=3/4)(C),nkx2.5−/−;nkx2.7+/−(n=3/3)(D)andnkx2.5−/−;nkx2.7−/−(n=3/3)(E)embryos.Similarfindingsareobservedinthewild-type(n=6/7)(F),nkx2.5+/+;
nkx2.7−/−(n=3/4)(G),nkx2.5−/−;nkx2.7+/+(n=3/3)(H),nkx2.5−/−;nkx2.7+/−(n=2/2)(I)andnkx2.5−/−;nkx2.7−/−(n=2/2)(J)embryosusingthetbx2bprobe,andin
wild-type(n=8/8)(K),nkx2.5+/+;nkx2.7−/−(n=5/6)(L),nkx2.5−/−;nkx2.7+/+(n=9/11)(M),nkx2.5−/−;nkx2.7+/−(n=3/3)(N)andnkx2.5−/−;nkx2.7−/−(n=3/3)(O)
embryosusingthehcn4probe.
nkx2.5−/−;nkx2.7+/+embryos.However,intheventricle,thereisan CMparametersthroughoutearlycardiacdevelopmentandchamber
increase in the conduction velocity in the nkx2.5−/−;nkx2.7−/− identitymaintenance.
embryos, although conduction remains markedly slowed and
chamber identity is lost from an electrophysiological standpoint. DISCUSSION
Although these electrophysiological attributes of the nkx mutant Together,thesefindingsoffernewinsightsintotherolesofNkxgenes
embryosreflectseveredefectsinintercellularcoupling,theindividual in SHF development, highlighting the differential regulation at the
CM properties exhibit a phenotype that is more advanced arterial and venous poles. We show that Nkx genes promote the
developmentally than that observed in the primitive heart tube. establishmentoftheSHFCMprogenitorpoolanddifferentiationof
Altogether,thesedatahighlighttheindependentregulationofdiscrete thispopulationattheOFT.OurstudiesalsorevealthatNkxgenesare
T
N
E
M
P
Fig.8.isl1actsdownstreamofNkxgenesatthevenouspole.(A-J)Ventralview,anteriortowardsthetop,at52hpf.Dottedwhitelinesillustratethecardiac O
silhouettes.Insituhybridizationforbmp4(A-J)inwild-type(A,F),nkx2.5+/+;nkx2.7−/−(B,G),nkx2.5−/−;nkx2.7+/+(C,H),nkx2.5−/−;nkx2.7+/−(D,I)andnkx2.5−/−; L
nkx2.7−/−(E,J)embryos.Inhibitionofisl1expressionusingislet1E2MOinwild-type(n=4/4)(compareAwithF)andnkx2.5+/+;nkx2.7−/−(n=4/4)(compare E
BandG)embryosleadstodownregulationofbmp4expressionspecificallyatthevenouspoleoftheheart(blackarrow)withoutaffectingtheAVC(yellowarrow) V
andOFT(whitearrow)expression.Innkx2.5−/−;nkx2.7+/+(n=2/2),nkx2.5−/−;nkx2.7+/−(n=5/6)andnkx2.5−/−;nkx2.7−/−(n=3/3)embryos,isl1inhibitionrescues E
D
themisregulationofbmp4expressionintheatrium(dottedarrows),yetAVCandOFTexpressionpatternsremainunchanged(compareC-EwithH-J).
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RESEARCHARTICLE Development(2018)145,dev161497.doi:10.1242/dev.161497
Fig.9.Nkxgenesregulateelectrophysiologicalpatterning.(A,B)Representativeatrial(A)andventricular(B)actionpotentialsshowingthetimecoursesof
cellulardepolarizationandrepolarizationinwild-type,nkx2.5+/+;nkx2.7−/−,nkx2.5−/−;nkx2.7+/+andnkx2.5−/−;nkx2.7−/−hearts.(C)Meanmaximumslopeofthe
atrialandventricularactionpotentialsmeasuredasthemaximumofthetimederivativeoftheactionpotentialupstrokephase,[dF/dt] ,inwild-type,nkx2.5+/+;
max
nkx2.7−/−,nkx2.5−/−;nkx2.7+/+andnkx2.5−/−;nkx2.7−/−hearts.(D)Actionpotentialduration(APD)inwild-type,nkx2.5+/+;nkx2.7−/−,nkx2.5−/−;nkx2.7+/+and
nkx2.5−/−;nkx2.7−/−hearts.(E-H)Representativeisochronalmapsillustratingthepositionsofthedepolarizingwavefrontin5mstimeintervalsforwild-type(E),
nkx2.5+/+;nkx2.7−/−(F),nkx2.5−/−;nkx2.7+/+(G)andnkx2.5−/−;nkx2.7−/−(H)hearts.(I)Meanatrialandventricularactionpotentialpropagationvelocitiesinwild-
type,nkx2.5+/+;nkx2.7−/−,nkx2.5−/−;nkx2.7+/+andnkx2.5−/−;nkx2.7−/−hearts.(J-L)Absolutedifferencesinthemaximumslopeoftheupstrokephase,[dF/dt]
max
(J),APD(K)andtheconductionvelocity(L)betweentheventricleandtheatriuminwild-type,nkx2.5+/+;nkx2.7−/−,nkx2.5−/−;nkx2.7+/+andnkx2.5−/−;nkx2.7−/−
hearts.Student’st-testwasusedtodeterminestatisticalsignificance.Themeanands.e.m.ofeachdatasetareshown(*P<0.05)forwild-type(n=5),nkx2.5+/+;
nkx2.7−/−(n=5),nkx2.5−/−;nkx2.7+/+(n=6)andnkx2.5−/−;nkx2.7−/−(n=3)hearts.
essentialfortheproperpatterningofthevenouspolebyregulatingthe and isl1 (Fig. 5F-I) in nkx mutant embryos. However, nkx2.5 has
maintenanceofworkingmyocardialpropertiesthroughrepressionof beenshowntoregulateSHFprogenitorproliferationattheproximal
isl1.FollowingthelossofNkxgenefunction,theentiremyocardium OFT via ltbp3 (Guner-Ataman et al., 2013). Through detailed
adoptsanIsl1+identityassociatedwithactivationoftheSANgenes dissection of the proliferative potential of the proximal and distal
bmp4, tbx2b, hcn4 and shox2. Misregulation of these SAN genes OFT progenitors, our results further elucidate the role that Nkx
is associated with electrophysiological evidence of expansion of genes play in limiting the aSHF-derived CM population by
the pacemaker phenotype across both chambers, with cellular regulating the timing of progenitor differentiation at the arterial
characteristics suggesting diminished sodium channel availability at pole.Inviewofstudiesilluminatingacommonprogenitorofheart
themembranes,featuresofcalcium-dependentactionpotentials,and and head muscle derivatives in other models (Harel et al., 2012;
diastolic phase 4 potentials. Together, our findings implicate Nkx Lescroart et al., 2015, 2010; Nagelberg et al., 2015; Wang et al.,
genes in combinatorial roles for anterior-posterior patterning of 2013),wepostulatethataSHFprogenitorscouldalsoadoptahead
chamber identity, regional integration of FHF and SHF territories, musclefateintheabsenceofNkxgenefunction.Futureexperiments
andmaturationofdiscretecassettesofphysiologicalCMdifferentiation wouldberequiredtotestthishypothesis,whichmayofferadditional
withinthehearttube.Moreover,thesecellularabnormalitiesinduced insightsintothediminishedlate-differentiatingcellsattheOFTof
bythelossofNkxgenefunctioninthetwo-chamberedzebrafishheart nkx mutant embryos. Taken together, although other mechanisms
offerapotentialwindowintothemorphologicalandconductionsystem may play a role, our findings highlight a novel function of Nkx T
N
defectsinindividualswithCHDthatharborNKX2-5mutations. genes in establishing an aSHF progenitor pool necessary to
E
ThroughillustrationofthedifferentialfunctionsofNkxfactorsin assembleanarterialpoleofthepropersizeandmorphology. M
the aSHFand pSHF, our studies complement and extend work in Ourdataalsoillustratesthat,intheabsenceofnkx2.5,theatrial P
other model organisms. Previous studies have revealed a role for myocardium adopts a venous pole identity characterized by O
Nkx2-5 in limiting cardiac progenitor specification and favoring misexpression of isl1 and SAN genes associated with failure of L
E
SHF proliferation (Prall et al., 2007). Yet in zebrafish, early specification of the cardiac conduction system (CCS) tissue at V
specification of SHF progenitors appears normal given the the AV boundary. Further graded expansion of this abnormal CM E
D
expression patternsofnkx2.5,nkx2.7,hand2 (Targoffet al.2013) differentiation, including manifestation of increased cellular
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RESEARCHARTICLE Development(2018)145,dev161497.doi:10.1242/dev.161497
automaticity and pacemaker-like behaviors throughout the entire the different potential for cardiac regeneration between zebrafish
ventricle,isobservedfollowingthecombinatoriallossofnkx2.5and andhighervertebrates.
nkx2.7.Knockdownofisl1issufficienttorescuetheeffectsofnkx
mutationsonSANgeneexpression,suggestingthatIsl1repressionby
MATERIALSANDMETHODS
Nkx genes patternsthe venous pole of the heart and delineatesthe
Zebrafishmutationsandtransgenes
specialized CCS tissue. However, the molecular mechanisms by Weusedzebrafishcarryingthefollowingpreviouslydescribedmutations
whichNkxgenescontrolIFTdevelopmentthroughrefinementofisl1 andtransgenes:nkx2.5vu179(Targoffetal.,2013),nkx2.7vu413(Targoffetal.,
expressionhaveyettobedetermined.Specifically,theroleofbmp4in 2013),Tg(-5.1myl7:nDsRed2)f2(Mablyetal.,2003),Tg(myl7:EGFP)twu277
establishingelectrophysiologicalCMcharacteristicsrequiresfurther (Huang et al., 2003), Tg(nkx2.5:ZsYellow)fb7 (Zhou et al., 2011) and
investigation. Yet our results offer a potential explanation for the Tg(hsp70l:nkx2.5-EGFP) (Targoff et al., 2013). All zebrafish work
followedInstitutionalAnimalCareandUseCommittee-approvedprotocols.
electrophysiological malfunction associated with the loss of fast-
conductingpropertiesanddefectsinspecificationoftheventricular
Injection
conduction system lineage in Nkx2-5-deficient models (Moskowitz
Embryos were injected at the one-cell stage with 6ng of the validated
etal.,2007;Pashmforoushetal.,2004).Furthermore,weproposethat
morpholino (MO) islet1E2 (5′-TTAATCTGCGTTACCTGATGTAGTC-
thepivotalrolefornkxgenesinestablishingavenouspolephenotype 3′) (Hutchinson and Eisen, 2006). Uninjected sibling embryos were
andSANidentitycouldexplainprogressiveAVconductiondefects
processedascontrols.
into adulthood that are exhibited in individuals with NKX2-5
mutations(Jayetal.,2003). Heatshockconditions
WehavepreviouslyshownthatventricularCMstransdifferentiate Embryosfromoutcrosses offish carrying Tg(hsp70l:nkx2.5-EGFP) were
into atrial CMs, demonstrating plasticity of the differentiated maintainedat28.5°Candexposedtoheatshockat37°Catdesiredstages(21
myocardium in nkx mutant embryos (Targoff et al., 2013). It is somitesto24somites).Toimplementheatshock,50embryoswereplacedin
possible that flexibility of the differentiated CMs results in 2.5mlofembryomediumina35mmPetridishontopofacoveredheat
block for 1h. Three hours after the initiation of heat shock, transgenic
sarcomeric rearrangement that allows for fate transformation in
the nkx2.5−/−;nkx2.7−/− embryos. In support of this concept, a embryoswereidentifiedbyvisualizationofubiquitousEGFPexpression.
Non-transgenicsiblingembryosexposedtoheatshockservedascontrols.
recent report demonstrates that ablation of ventricular CMs in the
zebrafish embryos leadsto atrial-to-ventricular transdifferentiation
Depigmentationofembryos
withassociatedsarcomeric restructuring(Zhang etal.,2013). Our
Whennecessary,embryosweredepigmentedafterovernightfixationin4%
electrophysiological studies further substantiate this hypothesis
paraformaldehyde at 4°C. They were washed twice in PBT (PBS-0.1%
throughtheirdemonstrationthatgradedlossofnkxallelesregulates Tween)thenincubatedin3%H O ,0.5%KOHand0.1%Tweenfor8min
2 2
multiple elements of the functional identity of the atrial and at 26h post fertilization (hpf) and 18min at 52hpf. Subsequently, the
ventricular myocardium. Although atrial and ventricular action embryoswerewashedtwiceagaininPBTandstoredinmethanolat−20°C.
potential features and conduction properties are distinct in wild-
type, nkx2.5+/+;nkx2.7−/− and nkx2.5−/−;nkx2.7+/+ hearts, each of Insituhybridization
the individual null alleles exhibits discrete effects on resting Weperformedwhole-mountinsituhybridizationaspreviouslydescribed
membranepotentials,CM activation, actionpotential morphology (Yelonetal.,1999)formef2cb(ZDB-GENE-040901-7),isl1(ZDB-GENE-
980526-112), bmp4 (ZDB-GENE-980528-2059), tbx2b (ZDB-GENE-
and conduction velocity. Moreover, the electrophysiological
990726-27), hcn4 (ZDB-GENE-050420-360) and shox2 (ZDB-GENE-
differences between atrial and ventricular CMs are completely
abolishedinnkx2.5−/−;nkx2.7−/−embryos.Despitethesefindings, 040426-1457).
there are discordances in the effects of individual genes on
Immunofluorescence
electrophysiology in the atrium and ventricle that implicate Whole-mountdoubleimmunofluorescencewasconductedusingaprotocol
additional regulatory partners in the specification of the complex described previously (Alexander et al., 1998) for the following primary
regionalelectricalfunctionnecessaryforavertebrateheart. antibodies:anti-Islet1/2(1:50;39.4D5,DevelopmentalStudiesHybridoma
Takentogether,thesestudiesonthefunctionsofNkxgenesinthe Bank),anti-atrialmyosinheavychain(1:20;S46,DevelopmentalStudies
aSHFandpSHFhighlightimportantconceptsinourunderstanding Hybridoma Bank), anti-RCFP (1:50; Clontech, 632475) and anti-GFP
of CM differentiation. Changes in the expression of individual (1:100; Life Technologies, A11122). When employing the following
primaryantibodies,anti-Eln2(1:500;Miaoetal.,2007)andanti-sarcomeric
transcription factors, signaling molecules and ion channels reveal
myosin heavy chain (1:20; MF20, Developmental Studies Hybridoma
discretephysiologicalmodulesandcanremodelnuancedfunctional
Bank), another published protocol was used (Zhou et al., 2011). The
characteristicsinCMs.Giventhismalleability,thenotionofastable
following secondary antibodies were employed: goat anti-mouse IgG2b
differentiated state does not fully reflect the resolution of modern
AlexaFluor488or568,goatanti-mouseIgG1AlexaFluor488or568,goat
cellularbiology,whichhasrevealedphenotypesthatarearesultof anti-mouseIgG2aAlexaFluor488,goatanti-rabbitIgGAlexaFluor488or
combinationsofdiscreteandoverlappingproteinrepertoires.These 568,andgoatanti-mouseIgGCy5(allat1:200;Invitrogen).
dataparallelinvitrostudiesiniPS-deriveddifferentiatedcelltypes.
Despite a physiological resemblance to mature CMs, these Proliferationassay
populations demonstrate remarkable cellular heterogeneity in CardiomyocyteproliferationinembryosexpressingTg(-5.1myl7:nDsRed2)f2 T
detailed electrophysiological phenotypes and transcriptional orTg(nkx2.5:ZsYellow)fb7wasassessedusingtheClick-iTEdUImagingKit N
E
(LifeTechnologies)withreportedprotocols(Cheesmanetal.,2011;Mahler
profiles that more closely resemble one of the parent fibroblast M
etal.,2010;Schindleretal.,2014).FollowingEdUincubationfor30min
than of the CM being studied. Our data suggest that individual P
on ice, embryos were chased for 24h. Embryos were then fixed in 2%
proteins may be sufficient and necessary to induce and maintain O
formaldehyde, washed once in PBS and washed twice in PBS-0.2%
plasticstateswheretheregenerativepotentialintheheartmightbe L
saponin.Subsequently,immunostainingwasperformedasdescribedabove E
realized. Thus, it would be valuable to address the fundamental followedbytheClick-iTstainingreaction.EdU+ZsYellow+MF20+/−and V
nature of cardiomyocyte differentiation at the level of individual EdU+ DsRed+ MF20− cells were counted using ImageJ software, going E
D
physiologic functions in order to obtain a better understanding of throughallz-stacks.Wecalculatedtheproliferationindicesasthepercentage
10