embryonic atrial function is essential for mouse embryogenesis, … · development and disease...

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IntroductionDuring mammalian development, the heart is the first organ todevelop and is essential for embryogenesis. Atrial functionincreases in parallel with morphogenesis during early heartdevelopment (Campbell et al., 1992). However, how the atriumcontributes to embryonic cardiovascular function has not beenevaluated.

Cardiac muscle expresses two major isoforms of myosinlight chain 2, MLC2v (MYL2 – Mouse Genome Informatics)and MLC2a (MYLC2A – Mouse Genome Informatics). Duringcardiogenesis, MLC2v is expressed exclusively in ventricularand atrio-ventricular junction myocardium. Ablation ofMLC2v results in disruption of ventricular function atembryonic day (ED) 11.5 and embryonic lethality at ED12.5(Chen et al., 1998). MLC2a is initially expressed throughoutthe heart at ED7.5 and becomes restricted to the atria afterED12.5 (Kubalak et al., 1994). Despite its early expressionthroughout the heart until ED12.5, MLC2a protein is onlyincorporated into myofibrillar structures in atria, not inventricle (Chen et al., 1998). As MLC2a is the only isoformexpressed in atria, we reasoned that disruption of MLC2awould interrupt embryonic atrial function, and would thereforeallow us to investigate the role of atrial function inembryogenesis. Our results have demonstrated that ablationof MLC2a disrupts the earliest functioning of the heart,demonstrating an early requirement for atrial function.Additionally, these results have allowed us to addresslongstanding questions in development concerning the role ofembryonic heart function in cardiac morphogenesis andangiogenesis.

Recently, it has been shown that the embryonic heart in earlychick embryos begins to beat substantially prior to arequirement for convective bulk transport to deliver oxygenand nutrients for growth (Burggren et al., 2000). Similarobservations have been made for zebrafish, clawed frog andsalamander embryos (Mellish et al., 1994; Pelster andBurggren, 1996; Territo and Burggren, 1998). These findingsare consistent with the hypothesis that early blood flow mayplay a role not only as a transportation fluid, but also as aphysical factor in development, perhaps influencing normalmorphogenesis of the heart and angiogenesis.

Hemodynamic parameters are known to be a determinant ofmyocardial growth, structure and function in the adult heart,and may be important in the etiology of heart failure (Hutchinset al., 1978; Rockman et al., 1994; Zak, 1974). It has beenpostulated that normal flow and hemodynamics are also crucialdeterminants of normal cardiac morphogenesis, and may playa causative role in congenital heart disease (Sedmera et al.,1998). Previously, this issue has been addressed by ligationexperiments to perturb blood flow in chick embryos (Hogerset al., 1997; Icardo, 1989; Sedmera et al., 1999). By technicalnecessity, these experiments perturbed blood flow in relativelylate stage embryos, between HH stages 21-24, a time whenconsiderable cardiac morphogenesis has already taken place.Because of the technically demanding nature of theseexperiments, small numbers of experimental embryos can beobtained, and from these variable phenotypes are observed(Broekhuizen et al., 1999). Additionally, mechanicalintervention to perturb blood flow may also restrict cellmigration, or may inflict non-specific tissue damage,

The requirement for atrial function in developing heart isunknown. To address this question, we have generated micedeficient in atrial myosin light chain 2 (MLC2a), a majorstructural component of the atrial myofibrillar apparatus.Inactivation of the Mlc2a gene resulted in severelydiminished atrial contraction and consequent embryoniclethality at ED10.5-11.5, demonstrating that atrial functionis essential for embryogenesis. Our data also address twolongstanding questions in cardiovascular development: theconnection between function and form during cardiacmorphogenesis, and the requirement for cardiac function

during vascular development. Diminished atrial function inMLC2a-null embryos resulted in a number of consistentsecondary abnormalities in both cardiac morphogenesisand angiogenesis. Our results unequivocally demonstratethat normal cardiac function is directly linked to normalmorphogenic development of heart and vasculature. Thesedata have important implications for the etiology ofcongenital heart disease.

Key words: Myosin light chain 2a, MLC2a, Atria, Cardiacmorphogenesis, Angiogenesis, Embryogenesis

Summary

Embryonic atrial function is essential for mouse embryogenesis,cardiac morphogenesis and angiogenesisChengqun Huang, Farah Sheikh, Melinda Hollander, Chengleng Cai, David Becker, Po-Hsien Chu,Sylvia Evans and Ju Chen*

Institute of Molecular Medicine and Department of Medicine, School of Medicine, University of California-San Diego, 9500 GilmanDrive, La Jolla, CA 92093-0641, USA*Author for correspondence (e-mail: [email protected])

Accepted 27 August 2003

Development 130, 6111-6119© 2003 The Company of Biologists Ltddoi:10.1242/dev.00831

Research article Development and disease

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complicating the interpretation of any effects on growth ormorphogenesis. Genetic ablation of MLC2a has allowed us toselectively inactivate atrial function, altering hemodynamicsfrom the earliest stages of heart development. Prior to anygrowth retardation, MLC2a mutants exhibit consistentphenotypic abnormalities in embryonic cardiac morphology.Our results unequivocally demonstrate that normal growth andmorphogenesis of the heart are dependent on normal heartfunction from the earliest stages, and strongly suggest thataltered hemodynamics during embryonic development canhave significant morphologic consequences for heartdevelopment. Embryonic atrial insufficiency in MLC2amutants has severe consequences for multiple aspects ofchamber and looping morphogenesis, suggesting that a lesssevere perturbation could similarly affect these morphogeneticprocesses and give rise to congenital anomalies of the heart inhumans.

Many mouse mutants have been described thatsimultaneously affect early heart function and angiogenesis (Biet al., 1999; Gerety and Anderson, 2002; Li et al., 1999; Linet al., 1998; Lin et al., 1997; Lyons et al., 1995; Regan et al.,2002; Stanley et al., 2002; Tanaka et al., 1999; Yamagishi etal., 2000). However, each of these genes is expressed in bothdeveloping vasculature and heart, making it difficult to assessthe independent effects of embryonic heart malfunction on thedeveloping vasculature. The expression of MLC2a exclusivelyin developing myocardium allowed us to examine this questionin our MLC2a knockouts, and has clearly demonstrated arequirement for cardiac function in both extra- andintraembryonic angiogenesis.

Together, our data imply that alterations in blood flowduring early development, as a result of either genetic orenvironmental influences, may have an etiological role incongenital cardiovascular disease.

Materials and methodsGene targetingA 15 kb mouse MLC2a genomic DNA clone was isolated from a129SV library (Stratagene) and used to construct the MLC2a targetingvector by standard techniques. Briefly, a Cre-PGKNEOr cassette wasinserted into pBluescript BKII (targeting vector) flanked by a 4.5 kbBamH1 fragment, containing the 5′ untranslated sequence of MLC2aand a 4.0 kb Kpn1/Acc1 fragment, containing the 3′ untranslatedsequence of MLC2a, thereby replacing exon one through seven ofMLC2a. The 1.3 kb Cre recombinase gene (Gu et al., 1994) and the1.7 kb neomycin resistance (NEOr) gene were under the control ofthe MLC2a and phosphoglycerokinase (PGK) promoter, respectively.The targeting vector was subsequently linearized with XbaI andelectroporated into R1 ES cells. G418-resistant ES clones werescreened for homologous recombination by DNA blot analysis, asdescribed below. One positive clone that underwent properhomologous recombination was microinjected into blastocysts fromC57BL/6J mice at the Transgenic Core Facilities of the University ofCalifornia, San Diego. Male chimeras were bred with female BlackSwiss to test for germline transmission of the agouti coat phenotypefrom 129-derived ES cells.

DNA analysis DNA was extracted from G418-resistant ES cell clones, yolk sac andmouse tails, as previously described (Moens et al., 1993). ES cellDNA was digested with SstI, electrophoresed on a 0.8% (w/v) agarosegel, and subsequently blotted onto nitrocellulose. A 400 bp fragment,

corresponding to the 5′ untranslated region of the MLC2a gene, wasgenerated by polymerase chain reaction (PCR) using mouse genomicDNA and specific MLC2a primers (forward primer, 5′-GTGA-GCCACTGAGAATGGTTGT-3′; reverse primer, 5′-CTTAGAACC-CCACTTCATCCCT-3′), and subsequently radiolabeled using[32P]dATP by random priming (Invitrogen, San Diego, CA). DNAblots were hybridized to the radiolabeled probe at 68°C, washed twicefor 15 minutes each time at 60°C in 0.1×SSC with 0.1% sodiumdodecyl sulphate (SDS), and visualized by autoradiography. DNAfrom yolk sac and mouse tails was also subjected to PCR, using bothCre (forward primer, 5′-GTTCGCAAGAACCTGATGGACA-3′;reverse primer, 5′-CTAGAGCCTGTTTTGCACGTTC-3′) andMLC2a (forward primer, 5′-GGAACAGAGACCAGCCACA-3′;reverse primer, 5′-GGTCTGATTTGCAGATGATC-3′) specificprimers, and products were visualized by ethidium bromide staining.

Whole-mount in situ hybridization analysis In situ hybridization was performed on MLC2a-null and somite-matched embryos for Mlc2a, Mlc2v, Nkx2.5, dHand, eHandand Tbx5,using digoxigenin-labeled antisense riboprobes, essentially aspreviously described (Wilkinson, 1992) with modifications. Briefly,plasmids containing Mlc2v, Nkx2.5and Tbx5cDNAs were linearizedwith NotI, HindIII and SpeI, respectively. T7 RNA polymerase(Invitrogen) was used to synthesize RNA ribprobes by in vitrotranscription in the presence of digoxigenin-11-dUTP (Roche). Fixedembryos were subsequently pre-hybridized in 50% formamide,5×SSC (pH 5), 50 ug/ml yeast RNA, 1% SDS and 50 µg/ml heparinat 68°C overnight, and subsequently hybridized with riboprobes at68°C for 18 hours. After vigorous washing, embryos were incubatedwith alkaline-phosphatase-conjugated anti-digoxigenin antibodies at4°C overnight (Roche), vigorously washed again, and incubated in aNTMT buffer (Promega), including freshly added nitrobluetetrazolium (NBT; 4.5 µl) and 5-bromo-4-chloro-3-indolyl phosphate(BCIP; 3.5 µl) per ml NTMT. Embryos were subsequently fixed in4% paraformaldehyde for 1-2 hours, until color development hadoccurred to the desired extent, and stored at 4°C in phosphate bufferedsaline (PBS) to terminate the color reaction. Whole embryos wereanalyzed and photographed on a ZEISS SV-5 dissecting microscopewith a Nikon C-mount 35 mm camera.

Protein blot analysisTotal protein extracts were prepared from hearts of MLC2a-null, aswell as wild-type, embryos, and protein analysis was performed aspreviously described (Chen et al., 1998). Immunodetection of MLC2a(28.2 kDa) and MLC2v (34.7 kDa) was performed in cardiac atria andventricular samples by using a rabbit polyclonal antibody to MLC2a(1:500), as previously described (Chen et al., 1998). The blot wassubsequently incubated with horseradish peroxidase-conjugated anti-rabbit Ig (1:1000; Sigma). Results were visualized by using enhancedchemiluminescence (Amersham).

Histology Embryos and yolk sacs from MLC2a-null and wild-type, somite-matched embryos were fixed, dehydrated and embedded in paraffinwax. Serial transverse sections were obtained at 10 µm intervals.Sections were stained with Hematoxylin and Eosin, and photographedas previously described (Zhou et al., 2001).

ImmunohistochemistryWhole embryos and yolk sacs were fixed in 4% paraformaldehydeovernight at 4°C, and then washed in a series of methanol washes andbleached in 5% H2O2/100% methanol for 4 hours at room temperature(RT). Subsequently, embryos were rinsed in 100% methanol,rehydrated and incubated in PBSMT (3% milk and 0.1% Triton X-100 in PBS) for 2 hours at RT. Embryos were then incubated with (1)platelet endothelial cell adhesion molecule (PECAM; 1:75; Sigma) or(2) fetal liver kinase 1 (FLK1; 1:400; Sigma) overnight at 4°C, washed

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in PBSMT and incubated with anti-mouse horseradishperoxidase (1:1000) overnight at 4°C. Subsequently,embryos were washed in PBSMT and briefly rinsed inPBT (0.2% BSA and 0.1% Triton X-100 in PBS). Forcolor development, embryos were incubated in 0.2mg/ml DAB (Sigma) and 0.35% NiCl2 in PBT at RT,until the desired intensity was achieved. The reactionwas stopped with the addition of 0.03% H2O2. Theembryos were rinsed several times in PBT andPBS, and fixed in a 2% paraformaldehyde/0.1%gluteraldehyde solution overnight at 4°C. The nextday, embryos were rinsed several times in PBS, andequilibrated at RT in 50% glycerol and then 70%glycerol for one hour each. Samples were analyzedand photographed on a ZEISS SV-5 dissectingmicroscope with a Nikon C-mount 35 mm camera.

Transmission electron microscopy Embryos were fixed in 0.1 M cacodylate, containing2% paraformaldehyde and 2% gluteradehyde,overnight at RT, and subsequently processed fortransmission electron microscopy as previouslydescribed (Chen et al., 1998).

Assessment of heart rateED9.5 embryos (with deciduas and fetal blood vesselsleft attached) were dissected from anesthetizedmothers (n=3) in 37°C DMEM containing 5% fetalbovine serum (Invitrogen), and placed within a closed35 mm dish on the stage of a dissecting microscopeconnected to an image processor. Light wastransmitted through a region of the atrioventricularjunction and was detected by the image processor.Using the computer monitor, both atrial andventricular beating rates were determined three times,simultaneously by two people within a three minuteperiod.

ResultsTargeted disruption of the MLC2a geneThe MLC2a gene replacement targeting construct is depictedin Fig. 1A (middle panel). The targeting construct wasdesigned to replace the entire coding sequence (exons one toseven) of the MLC2a gene, with Cre cDNA and the neomycincytidine deaminase gene cassette (NEO, Fig. 1A, middlepanel), thereby inactivating the gene. Targeted ES cells wereidentified by Southern blot hybridization analysis,characteristic of the targeted allele, giving the expected 7.5 kbmutant fragment compared with the wild-type 9.5 kb fragment(Fig. 1B). This strategy resulted in a lack of MLC2a proteinexpression within homozygous MLC2a hearts, as detected byprotein blot analysis using specific MLC2 antibodies (Fig.1C),when compared with wild-type littermates.

The MLC2a-null mutation results in embryoniclethality Heterozygous MLC2a-mutant offspring were viable, fertileand appeared normal in all respects. However, no viableMLC2a-null offspring were obtained in litters from MLC2aheterozygote intercrosses (Table 1), indicating that theMLC2a-null mutation was embryonic lethal. To assess thetiming of lethality, Mendelian ratios of viable embryos fromMLC2a heterozygote intercrosses were assessed at early

embryonic developmental stages (ED7.5-ED11.5; Table 1).All MLC2a-null embryos were viable up to ED10.5 (Table 1).However, at ED10.5, null embryos were observed to havesevere chest edema, indicative of heart failure, and growtharrest at approximately somite pair (sp) 24-28 (Fig. 2A,G).Assessment of MLC2a-null embryos at earlier stages ofdevelopment revealed that growth retardation became evidentat approximately ED 9.0-9.25 (sp 15-17). No viable MLC2a-null embryos were observed at ED11.5 (Table 1), suggestingthat embryonic lethality occurred between ED10.5 and 11.5.As no embryos of normal appearance and growth werehomozygous null for MLC2a, the observed phenotype of

Fig. 1.Targeted generation of MLC2a-null mice. (A) Targeting strategy. A restrictionmap of the relevant genomic region of MLC2a (top), the targeting vector for MLC2a(middle) and the mutated/targeted locus after recombination (bottom) are shown. Thetranslational start site is indicated by ATG. Orientations of Creand neomycinresistance genes (Neo) are indicated by arrows. A, Acc1; B, BamH1; H, HindIII; K,Kpn1; S, Sst1; X, Xba1. (B) Detection of wild-type and targeted alleles by Southernblot analysis. DNA from electroporated MLC2a ES cells was digested with Sst1 andanalyzed by Southern blot analysis with a probe as shown in A. The 9.5 and 7.5 kbbands represent wild-type and targeted alleles, respectively. (C) Detection of MLC2aby protein analysis. Proteins were prepared from E9.5 MLC2a-null (–/–) and wild-type (+/+) embryos, and analyzed with MLC2a antibodies.

Table 1. Genotypic frequency of embryos from MLC 2a+/–

intercrossesStage Litters +/+ +/– –/– Total

E8.5 4 7 23 6 36E9.5 20 33 115 35 183E10.5 8 12 32 14 58E11.5 3 4 15 6* 25P1 7 14 33 0 57

*Embryos found dead.

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growth retardation and embryonic lethality was fullypenetrant.

MLC2a-null embryos display aberrant cardiacmorphogenesis from the linear heart tube stageA comparison of MLC2a-null mutants and their wild-type or

heterozygous littermates revealed no morphologically evidentdifferences at the early cardiac crescent stage ED7.75 (5-7sp)(data not shown). However, aberrant cardiac morphogenesis inMLC2a mutants was apparent at the early linear heart tubestage ED8.5 (8-10 sp) (Fig. 3A-F). Relative to the linear hearttubes of wild-type or heterozygous littermates, mutant heart


Fig. 2.Microscopic and histologicalassessment of physical defects inMLC2a-null embryos. Whole embryoassessment of wild-type (A) andMLC2a-null (G) mouse embryos atsomite pairs 24. Note that the nullmutant displays severe chest edema.Paraffin wax-embedded serialsections of wild-type (B-F) andMLC2a-null (H-L) mouse embryoswere stained for nuclei and cytoplasmwith Hematoxylin and Eosin,respectively. White arrows point outthe lack of mesenchymal cell seedingin atrioventricular cushions ofMLC2a mutant (K), in contrast tonormal mesenchymal cell seeding inatrioventricular cushions of wild-typecontrol (E). Black arrows in D and J indicate dilated ventricle. A, atria; OT, outflow tract; RA, right ventricle; LA, left atria; RV, right ventricle;LV, left ventricle; SV, sinus venosus.

Fig. 3.Assessment of cardiac defects in MLC2a-null embryos. Left (A,D,G,J,M,P,S,V), front (B,E,H,K,N,Q,T,W) and right (C,F,I,L,O,R,U,X)views of wild-type (+/+) and MLC2a-null (–/–) embryos at somite pairs 10 (A-F), 13 (G-L), 16 (M-R) and 22 (S-X). Relative to the linear hearttubes of wild-type littermates (A-C), mutant heart tubes appeared to be enlarged and amorphous, with no clear distinction between the bulbusarteriosus and future left ventricle of the heart (D-F). With progression through looping morphogenesis, mutant hearts exhibited aberrantmorphologies in each cardiac chamber, accompanied by overall abnormalities in looping architecture (J-L,P-R,V-X).


tubes appeared to be enlarged and amorphous, with no cleardistinction between the bulbus arteriosus and the future leftventricle of the heart. With progression through loopingmorphogenesis, mutant hearts exhibited aberrant morphologiesin each cardiac chamber, accompanied by overall abnormalitiesin looping architecture (Fig. 3G-R). By sp 22, enlarged atriawere present in the mutant embryos relative to littermatecontrols (Fig. 3S-X). By sp 24-28, both enlarged atria andenlarged outflow tracts were apparent (Fig. 2). Morphologicalabnormalities in hearts from mutant embryos were consistentover a large number of mutants analyzed.

Histological analysis of MLC2a mutant embryos and theircontrol littermates revealed a number of striking differencesbetween them in myocardium and endocardium. Mutantventricular myocardium was relatively thin walled, withunderdeveloped trabeculae and left ventricular dilation whencompared with controls, at ED10.0-10.5. At early stagesof atrioventricular valve formation, the atrioventricular

cushion becomes seeded by mesenchymal cells that haveundergone an epithelial to mesenchymal transformation(Markwald et al., 1999). No seeding by mesenchymal cellswas apparent in MLC2a mutant embryos at sp 24 (Fig. 2).Similar results were also obtained at sp 22 (data not shown).

We also performed whole-mount in situ hybridizationanalysis of MLC2a-null embryos and control littermates, usingdigoxigenin-labeled antisense riboprobes for a number ofmyocardial markers, including TBX5 (left ventricle and atria),MLC2v (ventricle) and NKX2.5 (entire myocardium). Nodifferences were observed between MLC2a mutants and theircontrol littermates (data not shown).

MLC2a-null embryos display lack of atrialmyofibrillar organization and function Ultrastructural analysis revealed that MLC2a-null atrialcardiomyocytes have a complete absence of myofibrillarorganization, a lack of normal parallel alignment of thick

and thin filaments when comparedwith somite-matched wild-typecontrols (Fig. 4A,C). By contrast,cardiomyocytes from the left ventricleof MLC2a-null embryos exhibitednormal parallel alignment of thick andthin filaments, and Z-line formation,similar to somite-matched wild-typecontrols (Fig. 4B,D). In terms offunction, severely diminished atrialbeating was observed in MLC2a-nullembryos (Fig. 5). By contrast, nosignificant difference in the rate ofventricular beating was observedbetween MLC2a-null and somite-matched wild-type controls (Fig. 5).

MLC2a-null embryos displaydefects in both extraembryonicand intraembryonic vasculatureMacroscopic analysis of yolk sacs fromMLC2a-null embryos revealed thatthey lack both large and small vessels,as compared with wild-type orheterozygous littermate controls(Fig. 6A-J). Histological analysesdemonstrated that the endodermal andmesodermal layers within the yolksac of MLC2a-null embryos werecompletely dissociated, which was incontrast to findings with controllittermates (Fig. 6E,J). Lack of vesselformation in yolk sacs of MLC2a-nullembryos was also confirmed by whole-mount immunostaining using theendothelial cell specific markerPECAM (Fig. 6K-P).

MLC2a-null mutants also exhibiteddefects in formation of intraembryonicvasculature, evident by sp 24, asrevealed by PECAM staining (Fig. 6Q-T). In the mutants, cranial vessels wereless complex, and intersegmental

Fig. 4.Ultrastructural analysis of atrial and ventricular myocytes from MLC2a-null and wild-type somite matched embryos. Representative images of cardiac atria and left ventricles ofwild-type (+/+) and MLC2a-null (–/–) embryos at sp 22, as assessed by transmission electronmicroscopy. Black arrowhead indicates the lack of myofibrillar organization in atrial sectionsfrom MLC2a-null mutants (C), in contrast to the organized myofibrillar structure observed inatrial sections from wild-type littermates (A). White arrows indicate the normal Z lineformation seen in left ventricular sections from both wild-type (B) and MLC2a-null (D)embryos.

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vessels were disorganized relative to those observed in controllittermates.

DiscussionIn the present study, we have investigated the role of atrialfunction during embryogenesis by generating a mouse modelbearing a null mutation of MLC2a, a structural protein presentin cardiac atria. The requirement for atrial function duringembryogenesis has remained unexplored (Campbell et al.,1992).

MLC2a-null mice died at approximately ED10.5-11.5 ofcardiovascular insufficiency, as indicated by pericardial edema.The proximal cause of death was atrial malfunction. Consistentwith the ablation of the atrial specific isoform of myosin lightchain 2, the atrial chamber of null embryos contractedinfrequently and sporadically, and displayed a lack ofmyofibrillar assembly. By contrast, the left ventricle contractednormally, and displayed normal myofilament assembly. Theseresults are consistent with previous studies from our laboratory,which demonstrated that although MLC2a is initiallyexpressed throughout the linear heart tube before becomingrestricted to the atria at ED12.5 (Kubalak et al., 1994), MLC2aprotein is not incorporated into the sarcomeric structure of theventricles (Chen et al., 1998).

We have utilized the fact that atrial function is disruptedfrom the beginning of cardiogenesis to investigatelongstanding questions as to the role of early heart function incardiac morphogenesis, and in both extra- and intraembryonicvasculogenesis. Our studies clearly demonstrate that disruptionof embryonic heart function has severe consequences oncrucial aspects of morphological development of the heart, andon angiogenesis. These results have important implications forthe etiology of congenital heart disease.

Despite our demonstration that myofibrillogenesis in extra-atrial myocardium was intact in MLC2a-null mutants, defectsin chamber morphogenesis were observed throughout theheart, beginning at the early linear heart tube stage. Just priorto this stage, cardiac contraction has initiated, and embryonicand extraembryonic circulations have amalgamated (Kaufman,

1998). Linear heart tubes of MLC2a mutants appearedrelatively large and amorphous relative to control littermates.At subsequent stages of heart development, loopingmorphogenesis occurred in mutant hearts, but was aberrant ina number of respects, including the length, shape and size ofeach cardiac segment, and their geometrical relationship toeach other. Histological analysis revealed multipleabnormalities within myocardial and endocardial lineages ofMLC2a homozygous-knockout embryos. The ventricularmyocardium of MLC2a mutants was relatively thin, with fewertrabeculae, and left ventricular (LV) dilation may haveoccurred secondary to diminished cardiac function.

Similar secondary effects on ventricular morphogenesisconsequent to atrial dysfunction have been made in a zebrafishline carrying a mutation in an atrial myosin heavy chain, weakatrium (wea) (see Berdougo et al., 2003). As with the leftventricle in MLC2a mouse mutants, ventricles in wea mutantsexhibit a decreased ventricular lumen.

A crucial aspect of cardiac morphogenesis is the formationof septa, which divide the mature heart into four chambers. Asan initial stage in the division of the atrial and ventricularchambers, endothelial cells in the region of the atrioventricularcanal become activated to invade the cardiac jelly (Markwaldet al., 1999). The seeding of cardiac jelly by endocardial cellsoccurs at approximately ED10 in the mouse. In MLC2ahomozygous-knockout embryos, this seeding was notobserved. This observation suggests that septation and valveformation, among the most common manifestations ofcongenital heart disease (Epstein and Buck, 2000), can becritically affected by aberrations of early myocardial function.

Our observations have demonstrated that absence of atrialcontraction can impact multiple aspects of cardiacmorphogenesis, and can affect both myocardial andendocardial lineages. These effects may be secondary toalterations in hemodyamic fluid forces, or secondary tooxygen or nutrient delivery. We favor the explanation that thefirst alterations in cardiac morphology observed in MLC2amutants can be attributed to changes in hemodynamic forcesfor the following reasons. Initial aberrations in mutant heartmorphologies are observed shortly after embryonic andextraembryonic circulations have merged. Oxygen-carryingred blood cells are not present within the bloodstream of themouse embryo until the 20 to 25 somite stage (Palis et al.,1999), rendering it unlikely that insufficient circulation ofblood affects tissue oxygenation prior to this stage. Growthretardation in MLC2a embryos is not apparent untilapproximately 16 somites, making it unlikely that nutrientsare limiting at early heart tube stages. Consistent with theseobservations, experiments in a number of vertebrate specieshave demonstrated that heart beat and circulationconsiderably precede the demand for oxygen or nutrientdelivery (Burggren et al., 2000). Alterations in fluid forces,distinct from effects on oxygen or nutrient delivery, havepreviously been shown to be crucial for kidneymorphogenesis (Serluca et al., 2002). Our observationssuggest that fluid forces also play a crucial role at the earlieststages of cardiac morphogenesis.

A number of previous studies have demonstrated thatalterations in fluid forces experienced by endothelial cells,including those of the endocardium, can result in changes intheir alignment and changes in gene expression (Icardo, 1989;


Fig. 5.Baseline atrial and ventricular heart rates of MLC2a-null andwild-type littermate embryos. Mean heart rates at baseline are shownfor both cardiac atria and ventricles of wild-type (WT) and MLC2a-null (KO) mouse embryos at ED 9.5. Mean heart rates, measured inbeats per minute (bpm), were 51±6 and 4±4 for WT and KO atria,and 53±10 and 48±7 for WT and KO ventricles, respectively.


Resnick et al., 2000; Shyy and Chien, 2002; Topper andGimbrone, 1999). Genes that are regulated within endotheliumin response to hemodynamic sheer stress include thoseinvolved in extracellular matrix remodeling and growth factorpathways. In this light, it is interesting to note the changesobserved in both cardiac jelly and extra-atrial myocardium inthe MLC2a mutant, where alterations in hemodyamic sheerstress experienced by the endocardium would be expected tooccur owing to the lack of atrial function.

In addition to defects in cardiac architecture, MLC2a-nullmice also displayed defects in both yolk sac andintraembryonic angiogenesis. In MLC2a mutants, the initialvascular plexus of the yolk sac forms, but does not remodelinto a vascular network of larger vessels. Similar secondaryeffects of cardiac malfunction on yolk sac angiogenesis havebeen observed in a mouse knockout of the sodium calciumexchanger, and in a cardiac specific rescue of an N-cadherin-null mouse, where a previous defect in yolk sac angiogenesiswas rescued by expression of either N- or E-cadherin in heart(Koushik et al., 2001; Luo et al., 2001; Wakimoto et al., 2000).

However, in addition to observed effects on yolk sacangiogenesis, MLC2a mutants also show defects inintraembryonic vasculogenesis. Initial stages of angiogenesis

Fig. 6.Microscopic andhistological assessment ofextra- and intraembryonicvascular formation in MLC2a-null mutants and wild-typelittermates. (A-I) Whole-mount assessment of wild-type (+/+) and MLC2a-null(–/–) yolk sacs at ED8.5(A,F), ED9 (B,G), ED9.5(C,H) and ED10 (D,I).(E,J) Paraffin wax-embeddedsections from wild-type (E)and MLC2a-null (J) yolk sacsat ED10 were stained fornuclei and cytoplasm withHematoxylin and Eosin,respectively. Separation ofmesodermal and endodermallayers was observed in theMLC2a-null yolk sac (J).(K-P) Whole-mountimmunostaining of wild-type(+/+) and MLC2a-null (–/–)yolk sacs for Flk1 at ED8.5(K,N), and PECAM at ED9(L,O) and ED9.5 (M,P).(Q-T) Whole-mountimmunostaining of wild-type(Q,R) and MLC2a-null (S,T)embryos for PECAM atsomite pair 24. White arrowspoint out the lack oforganization of cranial andintersegmental vessels inMLC2a-null embryos, relativeto controls.

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appear normal, but subsequent remodeling of vessels does notoccur normally in the MLC2a homozygous-null embryos.These results demonstrate aberrations in intraembryonicangiogenesis in MLC2a mutants, which do not reflect anintrinsic defect in endothelial or smooth muscle cells, but ratherare secondary to cardiac malfunction. Similar defects invasculogenesis have been reported in a number of knockoutmice that are mutant for genes that are expressed both inmyocardium and in vascular cells, or in vascular endotheliumand endocardium (Puri et al., 1999). Our results uniquelydemonstrate that cardiac function is required for normalmaturation of intraembryonic vessels. Our results also cautionthat an intrinsic requirement for gene function in vascular celltypes cannot be inferred in cases where defects in vascularmaturation are accompanied by defects in myocardial function.The latter could arise owing to defects within myocardiumitself, as seen here with the MLC2a knockout, or within celllineages that affect the myocardium, including theendocardium (Puri et al., 1999).

In summary, our analysis of the MLC2a mutant hasdemonstrated that ablation of atrial function can have drasticsecondary consequences on cardiac morphology and vasculardevelopment. They also suggest that more minor perturbationsof atrial or cardiac contractile function, including perturbationsof flow during development, could have less drastic but seriousconsequences for normal cardiovascular development in thehuman fetus. In particular, our findings suggest that normalalignment of cardiac chambers and cushion formation, crucialaspects in septation of the normal heart that are perturbed inthe majority of cases of congenital cardiac disease, can beperturbed consequent to aberrant cardiac function.

This work was supported by grants from NIH (J.C. and S.E.). F.S.is a recipient of the CIHR/Heart and Stroke Foundation of Canadapartnership program postdoctoral fellowship. P.-H.C. is supportedby grants NHRI-EX91-9108SC and NHRI-EX92-9108SC from theNational Health Research Institute, Taiwan.

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embryonic atrial function is essential for mouse embryogenesis, … · development and disease...

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