MLC3F Transgene Expression in iv Mutant Mice Revealsthe Importance of Left-Right Signalling Pathways for theAcquisition of Left and Right Atrial But Not VentricularCompartment IdentityDIEGO FRANCO,1* ROBERT KELLY,2 ANTOON F.M. MOORMAN,1 WOUTER H. LAMERS,1

MARGARET BUCKINGHAM,2 AND NIGEL A. BROWN3

1Experimental and Molecular Cardiology Group, Academic Medical Center, University of Amsterdam, Amsterdam,The Netherlands2Department of Molecular Biology, Pasteur Institute, Paris, France3Department of Anatomy and Developmental Biology, St. George9s Hospital Medical School, London, United Kingdom

ABSTRACT Transcriptional differences be-tween left and right cardiac chambers are revealedby an nlacZ reporter transgene controlled by reg-ulatory sequences of the MLC3F gene, which isexpressed in the left ventricle (LV), atrioventricu-lar canal (AVC), and right atrium (RA). To examinethe role of left-right signalling in the acquisition ofleft and right chamber identity, we have investi-gated MLC3F transgene expression in iv mutantmice. iv/iv mice exhibit randomised direction ofheart looping and an elevated frequency of associ-ated laterality defects, including atrial isomerism.At fetal stages, 3F-nlacZ-2E transgene expressionremains confined to the morphological LV, AVC,and RA in L-loop hearts, although these appear onthe opposite side of the body. In cases of morpho-logically distinguishable right atrial appendageisomerism, both atrial appendages show strongtransgene expression. Conversely, specimens withmorphological left atrial appendage isomerismshow only weak expression in both atrial append-ages. The earliest left-right atrial differences in theexpression of the 3F-nlacZ-2E transgene are ob-served at E8.5. DiI labelling experiments confirmedthat transcriptional regionalisation of the 3F-nlacZ-2E transgene at this stage reflects futureatrial chamber identity. In some iv/iv embryos atE8.5, the asymmetry of 3F-nlacZ-2E expression waslost, suggesting atrial isomerism at the transcrip-tional level prior to chamber formation. These datasuggest that molecular specification of left andright atrial but not ventricular chambers is depen-dent on left-right axial cues. © 2001 Wiley-Liss, Inc.

Key words: iv/iv mice; atrial isomerism; handed-ness; myosin light chain; left-rightsignalling; transgenic mice; cardiacmorphogenesis; fate mapping

INTRODUCTION

Mesodermal cells acquire the potential to becomemyocardial cells soon after gastrulation. The primary

cardiomyocytes are arranged in a horseshoe-shapedcrescent that fuses in the midline area of the body axis,giving rise to a tubular heart (for a review see Fishmanand Chien, 1997). With further development, five dif-ferent morphological and functional compartments canbe distinguished in the embryonic heart: inflow tract,atrium, atrioventricular canal, ventricle, and outflowtract (Moorman and Lamers, 1994). As the heart be-gins to loop, myosin heavy chain (MHC) and myosinlight chain (MLC) isoforms become confined to distinctcardiac segments (Franco et al., 1998; Lyons, 1994;Moorman and Lamers, 1994). Regional specification,however, is likely to occur at an earlier stage sinceexpression of different atrial-specific and ventricular-specific myosin isoforms becomes regionalised in avianspecies prior to the morphological identification ofthese cardiac compartments (Yutzey and Bader, 1995;Yutzey et al., 1994). Furthermore, an iroquois-relatedhomeobox gene, cIrx-4, which modulates MHC expres-sion, is confined to the prospective ventricular myocar-dium at the linear heart tube stage (Bao et al., 1999;Bruneau et al., 2000).

Within the fetal heart, left and right atrial andventricular chambers are morphologically different(Brown and Anderson, 1999; Webb et al., 1996). Somegenes such as MCK, connexin40, connexin43, and anumber of MHC and MLC isoforms display transientleft and right differences in ventricular expression pat-terns (Kelly et al., 1998a; Lyons, 1994; Van Kempen etal., 1991, 1996; Zammit et al., 2000). Transgenic micecarrying regulatory sequences of sarcomeric geneshave also revealed transcriptional differences between

Grant sponsor: British Heart Foundation; Grant number: RG98004; Grant sponsor: EU Biomed; Grant number: PL 964004; Grantsponsor: NWO; Grant number: 902-16-219; Grant sponsor: DutchHeart Foundation; Grant number: 97206.

*Correspondence to: Diego Franco, Department of ExperimentalBiology, Faculty of Health and Experimental Sciences, University ofJaen, 23071 Jaen, Spain.E-mail: dfranco@ujaen.es

Received 28 September 2000; Accepted 8 February 2001Published online 25 April 2001

DEVELOPMENTAL DYNAMICS 221:206–215 (2001)

© 2001 WILEY-LISS, INC.

the left and right atrial and ventricular compartments(Biben et al., 1996; Kelly et al., 1995; Kelly et al.,1998b; Kuisk et al., 1996; Ross et al., 1996; ), providingpotential molecular markers to examine the acquisi-tion of left and right identity during cardiogenesis(Franco et al., 1997; Kelly et al., 1999). More recently,Pitx-2 has been reported to show left/right differencesin the atrial myocardium (Franco et al., 2000; Campi-one et al., 2001; Schweickert et al., 2000).

The heart is the first embryonic structure to displaymorphological left-right asymmetry. Under normalconditions (situs solitus), the cardiac tube loops to-wards the right side of the embryo (D-loop). Severalgenes (nodal, lefty-2, Pitx-2) are expressed asymmetri-cally in the lateral plate mesoderm (LPM) prior tocardiac looping in mice (Campione et al., 1999; Col-lignon et al., 1996; Logan et al., 1998; Meno et al., 1996;Piedra et al., 1998; Yoshioka et al., 1998; for a reviewsee Burdine and Schier, 2000). Interestingly, Pitx-2 ispreferentially expressed in the left side of the cardiactube just before cardiac looping (Campione et al., 1999,2001; Logan et al., 1998; Ryan et al., 1998). It has beenproposed that Pitx-2 plays a role in processing embry-onic left-right signalling, contributing to the determi-nation of the asymmetries of the heart and gut (Cam-pione et al., 1999; Logan et al., 1998; Piedra et al.,1998; Ryan et al., 1998).

Abnormal specification of cardiac laterality has beendescribed in several mutant mice strains such as iv(Hummel and Chapman, 1959), inv (Yokoyama et al.,1993), legless (Singh et al., 1991), and brachyury (Kinget al., 1998). Hummel and Chapman (1959) showedthat embryos homozygous for the iv gene present in-verted cardiac looping in approximately 50% of cases.More recently, it has been shown that several cardiacmalformations such as double-outlet right ventricle(DORV), persistent truncus arteriosus (PTA), andatrial isomerism are observed with a relatively highincidence in the iv/iv background (Icardo, 1990; Icardoand Sanchez de Vega,1991; Seo et al., 1992). Evidencefrom a number of studies of mice carrying null allelesfor components of the left/right cascade suggests thatleft-right situs and cardiac looping are independentevents (King et al., 1998; see for a review Burdine andSchier, 2000). However, the precise timing of theevents that underlie the specification of left and rightatrial situs remain unknown due to the lack of left/right molecular markers.

Transgenic mice in which regulatory sequences ofthe myosin light chain 3F (MLC3F) gene drive nlacZexpression in the right atrium and left ventricle pro-vide the first marker that permits molecular distinc-tion of left and right cardiac compartments (Franco etal., 1997; Kelly et al., 1995). We have investigatedwhether the specification of abnormal handedness af-fects the transcriptional potential of each cardiac com-partment in normal and congenitally malformedhearts. We have also examined the earliest stage atwhich left and right differences can be detected during

atrial specification. For this purpose, we have crossed3F-nlacZ-2E transgenics with iv mice. We provide ev-idence that transcriptional differences between the leftand right atrial appendages are first observed soonafter cardiac looping, as revealed by expression pat-terns of the MLC3F transgenes and fate mapping ex-periments. Our data also demonstrate that transcrip-tional specification of the cardiac chambers isindependent of the direction of heart looping.

RESULTS

We crossed 3F-nlacZ-2E transgenic mice with iv mu-tant mice. The presence of the transgene had no ob-servable effect on the iv/iv mutant phenotype, nor didthe presence of the mutation modify overall expressionof the transgene (see Fig. 1). Within the iv/iv back-ground, approximately 50% of the embryos display in-verted cardiac looping (Hummel and Chapman, 1959).Of 212 embryos examined from iv/3F-nlacZ-2E back-crossed with iv/iv mice, 47 (22%, expected 25%) hadinverted heart loops and 116 expressed the transgene(55%, expected 50%). Similarly, of 198 embryos exam-ined from iv/3F-nlacZ-2E intercrossed with iv/3F-nlacZ-2E, 24 (12.1%, expected 12.5%) had invertedheart loops and 162 expressed the transgene (82%,expected 75%). Transgene expression was analysed un-der three different conditions, normal cardiac looping(D-loop), inverted cardiac looping (L-loop), and atrialisomeric hearts, including specimens with either D-loop or L-loop phenotypes.

Expression Pattern of the 3F-nlacZ-2ETransgene in Normal and Inverted Hearts

In D-loop hearts (E10.5), the expression pattern ofthe 3F-nlacZ-2E transgene is predominantly confinedto the right atrial appendage, the atrioventricular ca-nal, and the left ventricle (Franco et al., 1997; Fig. 1).Within the left ventricle, a patch of nonexpressing cellscan be observed in the dorsal aspect of the ventricularfree wall adjacent to the atrioventricular junction (seeFig. 2A). In L-loop hearts, the nlacZ expression patternis a mirror-image, i.e., transgene expression is confinedto the morphological right atrial appendage (located onthe left side of the embryo), the atrioventricular canal,and the morphological left ventricle (located on theright side of the embryo) (Fig. 1).

Transgene Expression in Isomeric Hearts

The arrangement of the atrial appendages can bedetermined in fetal stages by morphological examina-tion of the junction between the appendage (left andright) and the sinus venosus-derived component ofeach atrium (Seo et al., 1992; Webb et al., 1996). Withinthe iv background, a small proportion (4.8%) of micedisplay atrial isomerism (Seo et al., 1992). In thisstudy, our aim was to examine whether differential leftand right transgene expression in the atrial append-ages correlates with left and right appendage identity.For this purpose, we analysed 220 specimens ranging

207ACQUISITION OF LEFT/RIGHT CARDIAC IDENTITY

Fig. 1. b-galactosidase expression in iv/3F-nlacZ-2E embryos at E10.5 (A–D) and E8.5 (E,F).The embryos in A,C, and E correspond to normalD-looped hearts. The embryos in B and F haveinverted turning, and the heart in D is also inverted(L-loop). Expression of b-galactosidase is confinedto the right atrium and left ventricle, and skeletalmuscle regardless of the direction of embryonicturning (A–F). In E and F, a patch of b-galactosi-dase nonexpressing cells is observed in the mor-phological left sinus horn (arrows) regardless of thetype of cardiac looping. RA, right atrium; LA, leftatrium; RV, right ventricle; LV, left ventricle.

Fig. 2. Dorsal views of D-loop iv/3F-nlacZ-2E embryos at E14.5 (A,B).Ventral (C,D) and dorsal (E,F) views in D-loop iv/3F-nlacZ-2E embryos atE12.5. A: b-galactosidase expression is mainly confined to the left ven-tricle (LV) and right atrium (RA), with almost no expression in the rightventricle (RV), left atrium (LA), or caval vein myocardium (CV), in anormal D-looped heart. Note the presence of a b-galactosidase negativeregion in the dorsal aspect of the left ventricle. B: b-galactosidase ex-pression in a right isomeric heart is confined to the left ventricle (LV) andboth atrial appendages (mRA), while no expression of b-galactosidase

can be observed in the caval vein myocardium. C,E: Embryo with normalatrial situs. D,F: Embryo with right atrial isomerism. Note that the expres-sion in the heart with normal atrial situs is confined mainly to the leftventricle (LV) and right atrium (RA), with a continuum of expression at thelevel of the atrioventricular canal, whereas in the right atrial isomericheart, expression is observed in both atrial appendages (mRA), as wellas in the left ventricle (LV). Note that expression of b-galactosidasebifurcates at the level of the atrioventricular canal (arrows, F). mRA,morphological right atrium; LA, left atrium; RA, right atrium.

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from E14.5 to E18.5, of which 28 showed morphologicalevidence of right atrial appendage isomerism (RAI) andfour showed morphological evidence of left atrial ap-pendage isomerism (LAI). The presence of right andleft atrial appendage isomerism was observed indepen-dently in both D-looped and L-looped hearts.

Transgene expression in RAI hearts was confined tothe morphological left ventricle, atrioventricular canal,and both atrial appendages, as illustrated in Figure 2.No expression of the transgene was observed in themyocardium of the symmetrically branched superiorcaval veins (Figs. 2 and 3). Such an expression patternwas also observed at earlier stages (E10.5/E12.5), priorto detectable differences in left and right atrial append-age morphology (Fig. 3). Interestingly, an extensiveb-galactosidase positive area was observed in the dor-sal region of the atrioventricular canal, which bifur-cated to both morphological right atrial appendages(Fig. 2F).

In LAI hearts, nlacZ expression was mostly confinedto the morphological left ventricle and atrioventricularcanal (Fig. 3C). Scattered b-galactosidase positive cellswere observed in the most caudal aspect of the atrialappendages, but the overall atrial appendage wasmainly negative for nlacZ expression (Fig. 3C). Nobifurcation of transgene expression was observed in thedorsal aspect of the atrioventricular junction (data notshown).

Within the 220 specimens analysed, in no case wasabnormal expression of the transgene documented inthe ventricular chambers, independently of whetherthe specimens were D-looped or L-looped or whetherisomerism of the atrial appendages was observed.

Transgene Expression During Early CardiacLooping

The expression of the 3F-nlacZ-2E transgene inhearts with atrial isomerism at fetal stages is high inboth atrial appendages in RAI and low in both atrialappendages in LAI. Moreover, the fact that such aregionalised expression pattern is seen at earlierstages suggests that the expression of 3F-nlacZ-2E pro-vides a molecular marker for investigating the earliestsigns of left and right atrial appendage identity. Tolook further at the early onset of left and right atrialappendage specification, we investigated nlacZ expres-sion in wild type 3F-nlacZ-2E and in iv/3F-nlacZ-2Eembryos. These embryos were compared, at similarstages, to 3F-nlacZ-9 embryos, which show expressionin both left and right atria and ventricles but not in theembryonic outflow tract or inflow tract myocardium(Franco et al., 1997, 2000).

In the early symmetrical cardiac tube of 3F-nlacZ-9embryos, b-galactosidase positive cells are distributedthroughout the visible tube, including the left and rightsinus horns (Fig. 4E). At this stage, 3F-nlacZ-2E ex-pression is broadly similar, also extending bilaterallywithin the sinus horns, but with a small nonexpressingregion medially, particularly in the most caudal area,

overlying the foregut (Fig. 4A). As looping begins, 3F-nlacZ-9 expression is observed throughout most of theheart, with the outflow tract becoming visible as anonexpressing region (Fig. 4G,H). The outflow tractis similarly b-galactosidase negative in early 3F-nlacZ-2E looping hearts: expression of this transgene,however, is weaker in the left than in the right sinushorn (Fig. 4C,D). In these hearts, a b-galactosidasenegative area stretches from the most caudal portion ofthe left sinus horn to a more medial region of theprospective atrioventricular canal. At this stage, thepresumptive ventricular area is present whereas nomorphological indications of the primary atrium can bedetected.

We examined 3F-nlacZ-2E transgene expression iniv/iv embryos during the looping phase, in comparisonwith wild-type 3F-nlacZ-2E and 3F-nlacZ-9 embryos.We found a subset of iv/3F-nlacZ-2E embryos (,10%)in which b-galactosidase staining extended bilaterallyand evenly within the sinus horns, without any areas ofnon-expression (Fig. 4I). This staining pattern is re-markably similar to that seen in wild-type 3F-nlacZ-9embryos at the same stage (Fig. 4H). This b-galactosi-dase pattern in iv/iv embryos corresponds to whatwould be expected of RAI hearts, suggesting that theearliest distinction between left and right atrial ap-pendages takes place at the early looping stage. Therest of iv/3F-nlacZ-2E embryos display a b-galactosi-dase expression pattern comparable, or mirror-image,to wild type 3F-nlacZ-2E.

Fate Mapping of the Presumptive Left AtrialPrecursor Region

The observation of nonstaining regions in early 3F-nlacZ-2E hearts poses two questions: 1.) are the non-staining medial cells displaced leftwards as loopingstarts, to become the left-sided unstained atrial region?and 2.) what is the fate of the left-side area?

To answer these questions, we labelled five differentearly hearts by DiI injection on each experimental set-ting, then cultured embryos to postlooping stages. Inall cases in which dye was injected medially into asymmetrical heart tube, the labelled cells were locatedin the left ventricle after looping (Fig. 5A,A’). In con-trast, dye injected into the left horn region that doesnot express the 3F-nlacZ-2E transgene was found inthe left side of the atrium, postlooping (Fig. 5C,C’).When hearts were injected at intermediate positionsbetween these two regions, labelled cells were found inthe area of the atrioventricular canal (Fig. 5B,B’). Fromthese experiments, we conclude that the early medialnonexpressing cells are fated to be left ventricle andare not related to the nonstaining left sinus horn myo-cardium, which will contribute to the left atrium. Inline with these findings, the most medial part of thelinear heart tube, constituted of a cluster of b-galacto-sidase negative cells gives rise to the patch of negativecells in the most dorsal aspect of the left ventricularfree wall in wild-type 3F-nlacZ-2E transgenics.

210 FRANCO ET AL.

DISCUSSION

We have analysed mice showing compartment-spe-cific transgene expression in the left ventricle, atrio-ventricular canal and right atrium (3F-nlacZ-2E trans-

genic mice; Kelly et al., 1995) in the iv background(Hummel and Chapman, 1959). This cross permits usto examine the emergence of transcriptional differ-ences between left and right components of the devel-

Fig. 3. Ventral views of b-galactosidase transgene expression inD-loop (A,B) and L-loop (C) iv/3F-nlacZ-2E embryonic hearts at E10.5.Transverse sections of E10.5 iv/3F-nlacZ-2E embryos from a normalatrial situs heart (D) and RAI heart (E). A: Heart with normal atrial situsheart. B: Right atrial isomeric (RAI) heart. C: Left atrial isomeric (LAI)heart. Observe that symmetrical intense expression of b-galactosidase isobserved in both atrial appendages (mRA) of the RAI heart (B), whereasonly weak and patchy expression is observed in both atrial appendages(mLA) in LAI heart (C), in accordance with the differential transgeneexpression levels of the right (RA) and left (LA) atrial appendages in the

normal atrial situs heart (A). mLA, morphological left atrium; mRA, mor-phological right atrium, OFT, outflow tract. D: Expression of b-galactosi-dase is mostly confined to the left ventricle, atrioventricular canal, and theright atrial appendage myocardium in normal D-loop hearts. E: b-galac-tosidase expression within a right atrial isomeric heart is seen in the leftventricle and both atrial appendages, but does not extend into the myo-cardium of the caval veins (RSCV). RA, right atrium; LA, left atrium; RV,right ventricle; LV, left ventricle; mRA, morphological right atrium; AVC,atrioventricular canal; RSCV, right superior caval vein.

211ACQUISITION OF LEFT/RIGHT CARDIAC IDENTITY

oping heart. Transgene expression in inverted hearts(L-loop) demonstrates that the transcriptional identityof left and right atrial and ventricular cardiac seg-ments correlates with the morphological identity ofeach segment.

We show that 3F-nlacZ-2E embryos are differen-tially expressed in the right and left atrial as well asventricular components regardless of the direction ofcardiac looping; transgene expression remains confinedto the morphological right atrial appendage myocar-dium independent of its position relative to the embry-onic left-right axis. Similarly, embryos that displayisomeric atrial appendages show transgene expressionaccording to the morphological identity of the atrium.This is the first study in which a marker that molecu-larly distinguishes left and right atrial appendages hasbeen used to investigate early chamber identity. Inter-estingly, transgene expression in isomeric hearts isalways confined to the trabeculated atrial appendagesbut is not observed in the venous myocardial compo-nent (caval veins or pulmonary veins myocardium).These observations support the notion that the atrialappendages and the venous components are distinctmorphological and transcriptional entities (Franco etal., 2000).

Regionalized expression of the 3F-nlacZ-2E trans-gene is observed from an early stage of cardiac looping(E8.5) onwards. Analysis of transgene expression in asubset of iv/3F-nlacZ-2E embryos demonstrates bilat-eral expression in the left and right sinus horns. Wesuggest that these embryos would have developed rightatrial isomeric hearts. In support of this conclusion,injection of DiI into the most posterior (caudal) regionof the left sinus horn labels the left atrial appendagemyocardium. Left and right atrial appendages, there-fore, appear to be established by the early loopingstage.

During embryogenesis, the first morphological evi-dence of asymmetry is observed as a rightward loopingof the cardiac tube. Mutations in the left-right dyneingene generate randomization of embryonic laterality iniv mutant mice (Supp et al., 1997), in which asymmet-ric expression of genes such as nodal, lefty-2, and Pitx-2is randomized (Campione et al., 1999; Lowe et al.,1996; Piedra et al., 1998). Our observations suggestthat the acquisition of left and right atrial myocardialidentity precedes chamber morphogenesis. The nodal/lefty-2 signalling pathway may, therefore, be involvedin the specification of the left and right atrial append-ages. Recently, it has been reported that null mutants

Fig. 4. Whole-mount revelation of b-galactosidase activity in wild type3F-nlacZ-2E (A–D), 3F-nlacZ-9 (E–H) and iv/3F-nlacZ-2E (I) embryosduring early cardiac looping stages (E8.0–8.5). Note that b-galactosi-dase expression between 3F-nlacZ-2E and 3F-nlacZ-9 embryos is similarat the linear cardiac tube stage (A and E). The first clear difference inexpression can be observed in the early looping stage (asterisk, C), andbecomes clearly visible in later stages (asterisk, D). At this stage (E8.5;D), expression of 3F-nlacZ-2E transgene is largely confined to the rightsinus horn (RSH) myocardium; expression in the left sinus horn (LSH)and midline of the cardiac tube (arrowheads) is weak and patchy. Ex-pression of b-galactosidase in the heart of 3F-nlacZ-9 embryos is sym-metrically distributed in both left and right sinus horns, including the

midline region (arrows) of the cardiac tube (H). D*: Transverse section ofan E8.5 3F-nlacZ-2E embryo at the level depicted in D (dotted line). Notethat b-galactosidase positive cells are observed in both left and rightsides of the looping heart but not in the ventral midline. These b-galac-tosidase nonexpressing cells are myocytes as revealed by SERCA2immunohistochemistry. H*: Transverse section of an E8.5 3F-nlacZ-9embryo at the level depicted in H (dotted line). Note that all myocardialcells in the ventral midline display b-galactosidase staining. I: A subset ofiv/3F-nlacZ-2E embryos displayed symmetrical expression of b-galacto-sidase, similar to the pattern observed in the 3F-nlacZ-9 embryos at thesame stage. This embryo is a case of presumptive right atrial isomerism.R, right side; L, left side; nf, neural fold.

212 FRANCO ET AL.

of the lefty-1 gene, which reveal bilateral expression oflefty-2, nodal, and Pitx-2 in the LPM, show left thoracicisomerism (Meno et al., 1998). Gene targeting experi-ments have demonstrated the role of the activin recep-tor IIB and Pitx-2 on the determination of right pulmo-nary isomerism (Oh and Li, 1997; Gage and Camper,1999; Lu et al., 1999; Kitamura et al., 1999). Thesedata, in conjunction with our findings on the earlyacquisition of left and right atrial appendage identity,

support the hypothesis that bilateral expression of thenodal/lefty-2/Pitx-2 signalling pathway can give rise toleft atrial appendage isomerism whereas absence ofnodal/lefty-2/Pitx-2 signalling results in right atrialappendage isomerism.

In contrast to our observations of molecular isomer-ism of the atrial compartments, we detected no exam-ples of ventricular isomerism (out of 220 iv/iv trans-gene positive embryos). Since isomeric ventricles havenot been described in the literature (Becker and Ander-son, 1983), these findings suggest that the acquisitionof right (pulmonary) and left (systemic) ventricularidentity is not dependent predominantly on left-rightpatterning. Indeed, it has been previously postulated,based on fate mapping experiments (De la Cruz et al.,1989; Brown and Anderson, 1999), that anteroposteriorpatterning plays a role on the acquisition of right andleft ventricular chamber identity. Furthermore, ex-pression of transcription factors dHAND and eHAND,which are initially regionalised along the anteroposte-rior axis of the tubular heart and later in the left andright embryonic ventricles, has been shown to definesystemic and pulmonary ventricular identity indepen-dent of situs in inv mutant mice (Thomas et al., 1998).The myocardium composing the early symmetrical tu-bular heart appears, therefore, to be predisposed togive rise to the systemic and pulmonary ventricularcompartments. Consistent with data from cell-labellingexperiments in the chick (de la Cruz et al., 1898), ourDiI labelling data in the mouse suggest that prospec-tive atrial myocardial cells have yet to become incorpo-rated into the cardiac tube. This difference in timing ofdifferentiation suggests that the atrial precursors maystill be susceptible to right-left signalling pathways.This hypothesis is underscored by our fate mappingexperiments, which show that only the systemic andpulmonary ventricles are present at early stages ofcardiac tube formation, whereas only after cardiaclooping has started, do the most caudal myocardialcells map to the atrial components.

EXPERIMENTAL PROCEDURESTransgenic Mice

MLC3F transgenic lines carry the nlacZ reportergene under the transcriptional control of regulatoryelements of the MLC3F gene. 3F-nlacZ-2E mice (Kellyet al., 1995) harbour a construct with 2 kilobases (kb) ofupstream sequence in front of the MLC3F promoter,driving expression of an nlacZ reporter gene and the 39MLC1F/3F enhancer element placed 39 to the polyade-nylation site. 3F-nlacZ-9 contains a 9-kb fragment ofDNA upstream of the MLC3F transcription initiationsite (Franco et al., 1997). A second muscle-specific en-hancer (approximately at 24 kb from the MLC3F tran-scription initiation site) is present within this 9-kbsequence (Kelly et al., 1997). Details of transgene con-struction and the characterisation of skeletal and car-diac muscle expression during development and in the

Fig. 5. DiI labelling experiments carried out at different cardiacstages. A–C: Frontal view of different stages labelled on the 3F-nlacZ-2Ebackground; the star indicates where the site of DiI injection was located.A’–C’: correspond to the localization of the DiI label obtained after 24 hr.A’ and B’ are left side views. C’ is a cranial view. Labelling of the middleline of the cardiac tube maps to the left ventricle (A, A’). Labelling of theregion anterior to the left sinus horn at an early looping stage maps to theatrioventricular canal (B, B’). Labelling of the most posterior region of theleft sinus horn leads to labelling of the left atrium (C,C’). R, right side; L,left side; RA, right atrium; LA, left atrium; OFT, outflow tract.

213ACQUISITION OF LEFT/RIGHT CARDIAC IDENTITY

adult have been reported elsewhere (Franco et al.,1997; Kelly et al., 1995, 1997).

Generation of 3F-nlacZ-2E Mice in the ivBackground

Heterozygous 3F-nlacZ-2E mice were crossed withhomozygous iv/iv mice. The first generation of offspring(F0) was selected for transgene expression by b-galac-tosidase coloration of tail tip preparations. F1 3F-nlacZ-2E positive mice (iv/3F) were backcrossed withhomozygous iv/iv mice, or intercrossed with each other,and embryos ranging from embryonic day (E) 7 to E18were collected. The day of vaginal plug was taken asE0.5. Embryos were excised from the uterus and thethoracic wall was removed (E12 to E16) exposing theheart to allow maximal penetrance of fixatives andreagents. Specimens were briefly fixed in 4% freshlyprepared formaldehyde (30 min to 1 hr) and rinsedtwice in phosphate-buffered saline (PBS).

In Toto X-Gal Histochemical Staining

Incubation in X-gal solution at 37°C was performedfor periods of 30 min to overnight (Sanes et al., 1986;Franco et al., 2001). Subsequently, embryos were post-fixed in 4% freshly made formaldehyde for 4 hr toovernight and conserved in 70% glycerol in PBT (PBSwith 0.1% Tween-20).

X-Gal Histochemical Staining on TissueSections

Embryos were rinsed in increasing concentrations ofsucrose (10, 20, and 30% in PBS) for 2 hr at each step,embedded in OCT (Miles Inc.) and frozen. Freeze cryo-stat serial sections of 7–10 mm were cut, mounted ontogelatin-coated slides and stored at 220°C until use.Incubation in X-gal solution at 37°C was performed forperiods of 30 min to overnight as detailed elsewhere(Sanes et al., 1986; Franco et al., 2001). Sections werecounterstained with azofloxine for 5 min, dehydrated ina graded ethanol series, and mounted in Entellan(Merck).

X-Gal Histochemical Staining andImmunohistochemistry

Cryostat sections were allowed to warm to room tem-perature for 30 min. Incubation in X-gal solution at37°C was performed for periods of 30 min to overnightas described elsewhere (Franco et al., 1997). Severalsections were processed for SERCA2 immunohisto-chemical detection as described in Franco et al. (2001).Briefly, sections were hydrated in decreasing ethanolconcentrations, rinsed in PBS, and incubated overnightwith a primary antibody against SERCA2. Subse-quently, sections were thoroughly washed in PBS,incubated with an alkaline phosphatase-coupledsecondary antibody overnight. Coloration of alkalinephosphatase activity was revealed with incubation inNBT/CIBP solution for approximately 20 min. Sectionswere dehydrated and mounted in Entellan.

DiI Labeling

Embryos ranging from 3 to 15 somites were collected,transferred to Hank9s solution, and injected with DiIusing a needle drawn from glass capillary tubing. Foreach experimental timepoint, 5 embryos were used.Labelling experiments were video-taped during injec-tion. Embryos were cultured for 24 hr in vitro in 75%rat serum, 25% T6 medium in 5% CO2, 20% O2/75% N2in rolling bottles. DiI labelled tissues were photo-graphed in unfixed embryos.

ACKNOWLEDGMENTS

We are indebted to Hata Zavrelova and Corrie deGier-de Vries for technical support, and to Dr. FrankWuytack for his kind supply of SERCA2 antibody.

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215ACQUISITION OF LEFT/RIGHT CARDIAC IDENTITY