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The Chicken as a Model Organism to StudyHeart Development

Johannes G. Wittig and Andrea Münsterberg

School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom

Correspondence: [email protected]

Heart development is a complex process and begins with the long-rangemigration of cardiacprogenitor cells during gastrulation. This culminates in the formation of a simple contractiletube with multiple layers, which undergoes remodeling into a four-chambered heart. Duringthis morphogenesis, additional cell populations become incorporated. It is important tounravel the underlying genetic and cellular mechanisms to be able to identify the embryonicorigin of diseases, including congenital malformations, which impair cardiac function andmay affect life expectancy or quality. Owing to the evolutionary conservation of develop-ment, observations made in nonamniote and amniote vertebrate species allow us to extrap-olate to human. This reviewwill focus on the contributions made to a better understanding ofheart development through studying avian embryos—mainly the chicken but also quailembryos. We will illustrate the classic and recent approaches used in the avian system,give an overview of the important discoveries made, and summarize the early stages ofcardiac development up to the establishment of the four-chambered heart.

EXPERIMENTAL APPROACHES USEDIN CHICKEN EMBRYOS

Studies in avian models have made significantcontributions to our understanding of em-

bryo development, including cardiac develop-ment. Both chicken (Gallus gallus) and Japanesequail (Coturnix japonica) embryos are easily ac-cessible from blastula stages onward. The chickis more commonly used as the eggs are largerand in ovo manipulations—including tissuegrafting, ablation, or injection—are performedthrough a small window cut into the shell, whichis then sealed before reincubating the egg forfurther embryo development (Bronner-Fraser2011). The complex events of heart develop-

ment and their timing in the chick embryo arewell documented and have been mapped ontothe Hamburger–Hamilton stages (Hamburgerand Hamilton 1951; Martinsen 2005). In addi-tion, quail embryonic stages (Ainsworth et al.2010) are extremely similar in size andmorphol-ogy to those in chick. This enables cross-speciesgrafting of quail donor tissue into a chick host tocreate chimeric embryos for the purpose of fatemapping (Le Douarin 1973). This classic tech-nique uses antibody staining to trace quail cellsand has provided fundamental insights, for ex-ample, into the contribution of neural crestcells (NCCs) to outflow tract (OFT) remodeling(Fig. 1; Kirby et al. 1983). The more recent avail-ability of transgenic chick embryos, where all

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cells are labeled with GFP, has facilitated thestraightforward detection of tissue graftedfrom chick to chick using fluorescence imaging(McGrew et al. 2008). Additional fluorescentreporter lines are now available (summarizedin Davey et al. 2018) and will enable evenmore sophisticated lineage analyses. For exam-ple, a Cytbow line uses random recombinationbetween loxP sites to generate clones of cellsindelibly labeled with either YFP, RFP, or mem-brane-blue FP. Although delivery of cre-recom-binase remains a challenge, cell labeling usingfluorescentmarks has the potential to expand onprevious clonal analyses, which used infectionwith replication-incompetent retrovirus (RCAS)(Mikawa et al. 1992; Cheng et al. 1999; Wei andMikawa 2000).

The use of cultured primordial germ cells(PGCs) has improved the efficacy of avian trans-genesis and should facilitate the generation ofgenome-edited transgenic birds (van de Lavoiret al. 2006; Macdonald et al. 2010) via modifi-cation of the PGC genome using CRISPR-Cas9

(Oishi et al. 2016). Different methods have beenused to deliver the CRISPR-Cas9 ribonucleo-protein complex. Examples include electropora-tion of inducible or constitutively expressed vec-tors encoding Cas9 and guide RNA (Véron et al.2015; Gandhi et al. 2017). Electroporation of thepreassembled CRISPR-Cas9 complex, as shownin cells (Schumann et al. 2015), should also bepossible in embryos and will reduce off-targeteffects. Furthermore, tools for CRISPR-Cas9 ge-nome engineering are continuously being opti-mized for the use in chicken (Williams et al.2018). The quail is also an attractive option fortransgenesis as its generation time is shorter(Poynter and Lansford 2008) and quail trans-genic lines have been generated and used forlive-cell imaging, for example, vasculogenesis(Sato et al. 2010; Davey et al. 2018). Further-more, adenoviral delivery of CRISPR-Cas9 di-rectly into the quail blastoderm generates chi-meras (Lee et al. 2019), which produce carriersof the targeted mutation; alternatively, quailPGCs can be transfected and are available for

M

C

T

V

Donor embryoAddition or

replacement

GraftingCrack egg and

inject inkAccess heart andinject compound

Lower embryo andseal egg

Cardiac injection

Electroporation

+

–

Figure 1.Experimental approaches in avian embryos. Schematic overview of experimental approaches commonlyused in avian species, namely grafting,microinjection, and electroporation. Grafting has, for example, identified asubpopulation of neural crest cells from the neural folds near somites 1–4 that contribute to the outflow tract.Various microinjection techniques have been described, including the delivery of anatagomirs into the myocar-dial wall to block microRNA function. This approach allows analysis of up to 3 days postinjection (∼HH27),which covers different phases of cardiac remodeling. Finally, chick embryo electroporation is widely usedand allows the targeted delivery of plasmid or viral constructs designed to interfere with cellular processes.(M) mesencephalic, (C) cardiac, (V) vagal, (T) trunk.

J.G. Wittig and A. Münsterberg

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germline transmission (Shin et al. 2008; Serralboet al. 2019).

Transient transgenesis avoids the generationand maintenance of transgenic lines and is anextremely useful alternative, which to date hasprovided many insights into signaling pathwaysand gene regulatory networks governing cardiacdevelopment. This approach uses microinjec-tion of vectors followed by electroporation,either in ovo or in embryos cultured ex ovo (Ita-saki et al. 1999; Chapman et al. 2001; Sauka-Spengler and Barembaum 2008). A commonlyused ex ovo electroporation set-up is visualizedin Figure 1. Combining fluorescent reporterswith cardiogenic enhancers followed by electro-poration, live imaging, and quantitative analysisof cell migration trajectories can further increasethe depth with which cell lineages and their be-havior can be resolved (Dormann and Weijer2006; Song et al. 2014; Zamir et al. 2017).

To investigate the function of genes, their ac-tivity can bemanipulated. The targeted injectionand electroporationof vectors typically enhancesgene expression (Fig. 1). In addition, constitu-tively active or dominant negative forms of theprotein of interest can be expressed, for example,of transcriptional regulators, transmembrane re-ceptors, or intracellular signaling components(Song et al. 2014). The knockdown of gene activ-ity can be achieved by antisense morpholinos orRNAi oligos, which can be electroporated or de-livered using pluronic gel (Sauka-Spengler andBarembaum 2008; Rutland et al. 2009). Thissuite of tools also includes cholesterol-modifiedantisense-inhibitors of microRNA function,so-called AntagomiRs, which can be deliveredeffectively into the myocardial wall by targetedmicroinjection (Fig. 1; Wittig et al. 2019). Thelocal application of growth factors, pharmaco-logical agonists or antagonists, on beads or filterpaper, provideadditional opportunities for func-tional interference, for example, to study signal-ingpathways, andsimilarapproachescanbeusedtoexaminecardiac toxicityofdrugsorchemicals.Microsurgeryat later stagesofheartdevelopmentis possible after first removing the extraembry-onic membranes (Spurlin and Lwigale 2013).Approaches include atrial ligation, for example,to assess the role of blood flow on aortic arch

morphogenesis (Hu et al. 2009), electrocardio-gram (ECG) measurements (Shi et al. 2013), orcryoinjury to examine regenerative processes(Table 1; Palmquist-Gomes et al. 2016) .

EARLY EVENTS IN CARDIAC DEVELOPMENT

Cardiac Progenitor Cell Migration, FateSpecification, and Formation of the LinearHeart Tube

Gastrulation is a major event in early develop-ment when embryos undergo a dramatic trans-formation in shape to generate the three germlayers. This morphogenetic process has beenstudied extensively in chicken embryos and de-tailed reviews and book chapters are available.Because these early stages are easily accessible,the origin of cardiogenic cells in the blastula andgastrula have been mapped, and their specifica-tion toward the cardiac fate is well understood.Just before the start of gastrulation, the blastula—a flat concentric disc—consists of two layers:the epiblast, which generates all of the embryo,and the hypoblast beneath. Dye labeling wasused to map the presumptive heart territory inthe epiblast (Hatada and Stern 1994). Coculturesof tissue explants showed that posterior epiblastcells respond to an activin transforming growthfactor β (TGF-β) signal from the hypoblast toinduce cardiac myogenesis (Yatskievych et al.1997). Interestingly theTGF-β superfamilymem-bers bone morphogenetic protein 2 (BMP2) andBMP4 are inhibitory for cardiomyogenesis at thispregastrula stage (Ladd et al. 1998), although theyare important slightly later (see below).

The origin of cardiac mesoderm in primi-tive-streak gastrula stage embryos and their sub-sequent location in the lateral plate mesoderm,the so-called first heart field (FHF) (Fig. 2), hasbeen determined using a variety of methods in-cluding quail-chick chimeras, or labeling withdyes or carbon particles. These studies revealedthat cardiac mesoderm comes from the anteriormidprimitive streak between HH3 and HH3+,where at these early stages it is arranged in arostrocaudal sequence that reflects the organiza-tion of the straight heart tube (Garcia-Martinezand Schoenwolf 1993; Redkar et al. 2001; Wittig

Chickens as a Model for Heart Development

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and Münsterberg 2016, and references therein).A few hours later, by midgastrula stages HH4+,the contribution to the heart from the primitivestreak has ceased (Psychoyos and Stern 1996).Lineage tracing with RCAS indicates that atprimitive streak stages the premyocardial andpre-endocardial cells are distinct subpopula-tions and it has been suggested that they segre-gate before arriving in the primitive streak (Weiand Mikawa 2000). Given the rapid advances insingle-cell sequencing technologies it is likelythat the first appearance of discrete progenitorswill be reexamined before long; this may alsoreveal novel and potentially unique markersfor subpopulations.

After completing an epithelial-to-mesen-chymal transition (EMT) and emerging from

the primitive streak, cardiac progenitor cells(CPCs) undergo long-range migration. Fluores-cent labeling of CPCs at HH3 to HH3+, followedby live cell tracking, showed that CPCs movelaterally and anteriorly before converging to-ward the anterior intestinal portal (AIP)—theforming foregut (Yue et al. 2008; Song et al.2014). Interestingly, the AIP has been identifiedas a putative heart organizer; it shares a molec-ular signature with other organizer tissues andcan induce cardiac and ventricular identity innoncardiac mesoderm (Anderson et al. 2016).In gastrula stage embryos, the migration trajec-tories of CPCs are affected by exposure toBMP2/4 and wingless-type MMTV-integrationsite family member (WNT) 3a, with both sig-naling pathways converging on Sma and Mad-

Table 1. Experimental approaches used in avian embryos

Method (title) Intention References

Grafting of quail donor tissue into achick host

The fate of quail cells can be mapped inchimeric embryos

Le Douarin 1973

Ex ovo culture on filter paper ring indish, multiwell dish, or imagingchamber

Microinjection and electroporation of plasmidvectors, oligonucleotides, application ofgrowth factors, drugs, or chemicals—liveimaging

Chapman et al. 2001; Satoet al. 2010; Song et al.2014; Rozbicki et al.2015

Cardiac injections of AntagomiRs as anovel tool for knockdown (KD) ofmicroRNA (miRNA) during heartdevelopment

Best suitable between HH13–HH18.Depending on the stage, specific targetingof a subsection of the heart is possible tomanipulate expression of genes or KDmiRNA function. Also applicable formorpholinos, plasmid constructs, andothers

Wittig et al. 2019

A technique to increase accessibility tolate-stage chick embryos for in ovomanipulations

This approach allows accessibility of later stagechicken embryos (HH27–HH34); they areviable and can be used for manipulation

Spurlin and Lwigale 2013

An ex-ovo chicken embryo culturesystem suitable for imaging andmicrosurgery applications

The method describes a shell-less way toculture chicken embryos ex ovo for up to 14days and allows for various imaging andmicrosurgical applications, for example, leftatrial ligation

Yalcin et al. 2010

A chick embryo cryoinjury model forthe study of embryonic organdevelopment and repair

To examine regenerative and reparativecapabilities of embryonic tissues duringorganogenesis

Palmquist-Gomes et al.2016

Alterations in pulse wave propagationreflect the degree of outflow tract(OFT) banding in HH18 chickenembryos

Electrocardiogram (ECG) measurements ofchick hearts were conducted to studyhemodynamic changes that are linked tothe cardiac cycle; the latter can bemanipulated by various methods as OFTligation

Shi et al. 2013





related Protein (SMAD) 1 and promoting itsactivity (Yue et al. 2008; Song et al. 2014).

The identification of the source and natureof signals involved in specification of cardiacfate in the gastrula stage embryo was facilitatedby combining quail and chick tissues in explantcultures. These experiments used coculturesof candidate-inducing tissues with candidate-responding tissues (Schultheiss et al. 1995), orthe implantation of beads soaked in growth fac-tors to test their activity (Andrée et al. 1998).This showed that cardiac fate specification, in-dicated by expression of the transcription factorNk2 homeobox 5 (NKX2.5), is mediated byBMP2 from anterior lateral endoderm (Schul-theiss et al. 1997). In vivo,NKX2.5 is first detect-ed by in situ hybridization at HH5 in the lateralplate mesoderm. In chick, BMP-responsive cis-regulatory elements of NKX2.5—comprisingGATA-binding protein (GATA) and SMAD-binding sites—have been identified (Lee et al.2004). Additional work identified fibroblastgrowth factor 8 (FGF-8) together with inhibitorsof the canonical WNT pathway as importantsignals for cardiac fate acquisition (Marvinet al. 2001; Alsan and Schultheiss 2002). Howthese signaling networks integrate to regulateboth the migration of CPCs from the primitive

streak toward the FHF and their concomitantfate specification is unclear at present.

By HH7 a number of transcription factors,including Islet-1 (ISL1), GATA2/4, and NKX2.5,are expressed in the lateral splanchnic meso-dermwhere cardiogenic progenitors are located.At this stage, the bilateral heart-forming regionsare separated by the foregut endoderm in themidline, and posteriorly they extend to the levelof the first somite (Fig. 2). In quail, a contiguousarc of cardiogenic cells was detected as early asHH4 (Cui et al. 2009), before formation of theAIP. Fate maps using quail-chick grafts revealeda distinct population of cells medially adjacentto the HH7 cardiac crescent, which express en-dothelial markers and contribute to the endo-cardium (Milgrom-Hoffman et al. 2011). In ad-dition, tracking cells expressing a fluorescentreporter under the control of a mouse NKX2.5enhancer identified a population of positive cellsfrom outside the FHF, which also contributes tothe hemogenic epithelium of the endocardium(Zamir et al. 2017).

DiI labeling of cardiac mesoderm at HH7–8combined with microincisions to probe tissuestress followed by time-lapse imaging and com-putational modeling, showed that the bilateralprimordia converge to form a linear heart tube

HH9 HH11HH7 HH13HH5

Figure 2. From heart fields to early looping. The major steps of early heart formation are illustrated here incartoon form. The bilateral cardiogenic mesoderm comprises progenitors of the first heart field (green) andsecond heart field (red), which are organizedmediolaterally (HH5). Together they contribute to outflow tract andright ventricle (red) and left ventricle and atria (green). During fusion, which is closely associated with foregutmorphogenesis (see text), themediolateral organization converts into an anteroposterior organization generatingthe early heart tube byHH10. At that stage, the heart is still linear and shortly after undergoes bulging and dextral(rightward) bending, which initiates the C-shape (HH11–HH13). Subsequent remodeling leads to compartmen-talization of the heart.





by HH9+, which shortly after begins to bulge(Fig. 2). This process is mediated by convergentextension and treatment with pharmacologicalinhibitors showed that cellular rearrangementsrequire cell proliferation and Rho-associatedcoiled-coil-containing protein kinase (ROCK)-dependent myosin contractions (Aleksandrovaet al. 2015; Hosseini et al. 2017; Kidokoro etal. 2018). Cells converge toward the midlineand epithelialize and form a contractile tubewith endocardial and myocardial cell layers sep-arated by an acellular layer of cardiac jelly (CJ)(HH9). The jelly is composed of extracellularmatrix (ECM) components including hyalu-ronan (HA), versican (VCAN), and collagens,which are synthesized by cells of the myocardi-um in response to BMP2 and WNT6 signals(Person et al. 2005). Through direct labeling, ithas been shown that ECM components such asfibronectin and fibrillin-2 translocate into theforming heart tube (Aleksandrova et al. 2012).Furthermore, heart tube formation is tightly co-ordinated with foregut morphogenesis and isaccompanied by large-scale tissue deformationsand folding of the endoderm (Cui et al. 2009;Varner and Taber 2012). Computational mod-eling illustrates how differential growth rates inendoderm and mesoderm can drive these dra-matic morphological changes (Hosseini et al.2017).

After formation of the early heart tube, thebilateral symmetry is broken by rightward loop-ing beginning at HH10 leading to a C-shape(Fig. 2). At this time, the dorsal mesocardium,which connects the heart tube to the foregut andsecures it in the pericardial cavity, breaks down.This allows the anterior and posterior ends of thetube—the arterial and venous poles, respectively—to converge, leading to the so-called S-shape(HH14–18). In a final phase, the position ofthe OFT relative to the atria is rearranged andlooping is complete by HH24 (Männer 2000,2004, additional references in Wittig and Mün-sterberg 2016). This process was first describedin chick and depends on extrinsic and intrinsicmechanisms including mechanical constraints,asymmetric cell growth, and proliferation andingression of precursor cells at the posteriorend of the tube. Recent work also implicates

the EMT inducer paired-related homeobox(PRX) 1, which is regulated by left–right asym-metry signals expressed in the lateral plate, in theinitiation of the looping process (Ocaña et al.2017). Importantly, any disturbances can leadto various laterality defects described in a recentreview, which also provides detailed compari-sons of the process in fish, chick, andmice (Des-grange et al. 2018, and additional referencestherein).

ADDITIONAL CELL POPULATIONSCONTRIBUTING TO THE HEART

Additional Heart Fields

Another seminal discovery was the identifica-tion of a secondary heart field (SHF) and ananterior heart field (AHF), which comprise pop-ulations of cells in the pharyngeal mesodermthat make an essential contribution to the OFT(Mjaatvedt et al. 2001; Waldo et al. 2001; Yutzeyand Kirby 2002). The origin of AHF and SHFprogenitors has been mapped to the primitivestreak. Dye labeling showed that AHF precur-sors are located immediately adjacent and an-terior to CPCs that form the FHF and SHF,respectively. ByHH7 the cardiogenic mesodermis organized such that FHF progenitors, contrib-uting to the atria and left ventricle (LV), arelocated lateral to SHF progenitors, which gener-ate the right ventricle (RV) and OFT (Fig. 2;Abu-Issa and Kirby 2008). This mediolateralorganization converts into an anteroposteriororganization during the fusion process that gen-erates the primitive heart tube described above(Hosseini et al. 2017; Kidokoro et al. 2018). AHFcells located in the cranial paraxial mesoderm(CPM) at HH8 are found in pharyngeal meso-derm of the first branchial arch and the OFT byHH15. CPCs emerging from the primitivestreak slightly posteriorly express the transcrip-tion factor ISL1 when located in the splanchnicmesoderm (HH8) and are later found in OFTand endo- and myocardium of the heart tube(HH12) (Camp et al. 2012). This is consistentwith classic quail–chick chimera experimentsshowing that CPM-derived cells contribute toangioblasts in the OFT (Noden 1991) and to





OFT myocardium and endocardium (Tirosh-Finkel et al. 2006).

Interestingly, the CPM also contributes tohead muscles (Noden 1991) and expressionanalyses revealed a close relationship betweenthe heart and craniofacial muscle programs withoverlapping molecular signatures in splanchniclateral plate mesoderm and CPM. In particular,the transcription factors T-box 1 (TBX1) andpaired-like homeodomain 2 (PITX2) are in-volved in both cardiogenesis and craniofacialmyogenesis (for review, see Tzahor 2009). Inthe CPM, cardiac differentiation is blocked byneural tube-derived canonical WNT signals.This inhibition can be mimicked by electropo-ration of vectors encoding WNT ligands; con-versely, WNT antagonists promote cardiogene-sis (Marvin et al. 2001; Nathan et al. 2008).Activation of WNT signaling at this stage in de-velopment led to inhibition of both cardiac andskeletal muscle differentiation markers, indicat-ing a genetic program that is at least partiallyoverlapping (Nathan et al. 2008). Althoughwork from the same group, using overexpressionin chick embryos, also showed that ISL1 is in-volved in specification of cardiac versus skeletalmyogenic progenitors (Harel et al. 2009).

Fluorescent dye injections have been used tomap the origin of pacemaker cells. In an HH8embryo, these progenitors are found outside theNKX2.5 and ISL1-positive regions, posterior tothe FHF and SHF in lateral plate mesoderm ad-jacent to somite 3. This region has been referredto as a tertiary heart field (Bressan et al. 2013).Pacemaker fate is specified byWNT8c mediatedcanonical WNT signals and inhibited by Cres-cent, a WNT antagonist, which promotes car-diogenesis more anteriorly (Marvin et al. 2001).

Formation of the Epicardial Cell Layer

The epicardium is the outermost layer envelop-ing the heart, it forms relatively late in heartdevelopment from the proepicardial organ(PEO). The PEO is a transient structure and firstdetected at HH14 by electron microscopy (EM)as a group of cells located at the back of the heartbetween the sinus venosus and the liver bud(Männer 2000). From this site, the proepicardial

cells migrate via a cellular bridge to colonize themyocardium of the looping heart. In addition,proepicardial cells integrate with pacemakermyocardial cells to mediate the remodelingand functional maturation of the cardiac pace-maker region into the sinoatrial node (SAN)(Bressan et al. 2018). In chick, the PEO developsasymmetrically on the right side from coelomicsomatic lateral plate mesoderm (Schlueter andBrand 2013). This process depends on left–rightsignals that determine sidedness in the earlyembryo, in particular FGF-8 and snail familytranscriptional repressor (SNAI) 1. The lattertargets Twist-family basic helix–loop–helixtranscription factor (TWIST) 1 to regulate mo-bilization of cells form somatic mesoderm(Schlueter and Brand 2013). A residual PEOinitially forms on the left but is lost by apoptosis(Schlueter and Brand 2009). The PEO can beidentified by strong expression ofWilms TumorProtein (WT) 1 and TBX18 and is induced bythe liver bud (Ishii et al. 2007). Although thePEO generates the majority of the epicardium,transplantations showed that epicardium cover-ing the distal OFT originates from elsewhere(Pérez-Pomares et al. 2003). Proepicardial iden-tity and the growth of the epicardium are medi-ated by BMP signaling. BMP triggers PE cellmigration outward over the heart; this also re-quires the transcription factor TBX5 (Hatcheret al. 2004; Schlueter et al. 2006; Ishii et al.2010). By HH17–18, villous protrusions of thePE reach the atrioventricular canal (AVC) andmigrate further by HH20 when they surpass theinner curvature of the heart. By stage HH21–23,the first cells start penetrating the dorsal myo-cardium of the ventricles. Formation of the epi-cardium is completed by HH27, but quail chickchimeras determined that subsequently the epi-cardium contributes to various cell populationsuntil HH31 (day 7 of development). These in-clude the AV-endocardial cushions, the endo-thelial and smooth muscle cells of the coronaryvessels and perivascular and intramyocardial fi-broblasts (Männer 1999). A recent comparativereview discusses the evolutionary origin of theproepicardium and the potential contribution ofsingle-cell sequencing approaches, which maylead to a better understanding of the lineage





relationships of epicardial cell types (Simões andRiley 2018).

CARDIAC DEVELOPMENT POST-HEARTTUBE FORMATION

Following heart tube formation, extensive re-modeling is necessary to achieve the four-cham-bered anatomy of the heart. This complexmorphogenesis has been studied in detail inchicken embryos and several processes have totake place including (1) cushion formation andvalve development, (2) septation and chamberseparation, (3) trabeculation and compaction,and (4) the formation of the cardiac conductionsystem (Martinsen 2005).

Cushion Formation and Valve Development

The first signs of CJ deposition at the OFT andAVC can be observed just after initiation oflooping. This marks the beginning of endocar-dial cushion formation. EMT triggers themigra-tion of endocardial cells into the CJ where

they proliferate and further contribute to ECMsynthesis expanding the cushions. In chick em-bryos, EMT takes place between stages HH14and HH19 (Mercado-Pimentel and Runyan2007).

Cushion formation occurs at two mainlocations: the AVC and theOFT. The AVC cush-ions emerge at stage HH16; they soon get pop-ulated by mesenchymal cells originating fromthe endocardium, which have undergone myo-cardium-induced EMT causing the endothelialcells to loosen cell–cell adhesions to becomemi-gratory to invade the ECM (Harris and Black2010). In vivo labeling experiments in chickhave shown that these early AVC cushions—adorsal and ventral endocardial cushion, willfuse and form the septum intermedium (SIM)(Fig. 3), which later contributes to the aorticleaflet of the mitral valve and the posterior infe-rior and septal leaflets of the tricuspid valve (dela Cruz et al. 1977; de la Cruz and Markwald1998). Further contributions to the mitral andtricuspid valve come from lateral cushions,which develop on both sides of the heart later

HH38

RV

LVTV MV

M

ET

RA OFTVLA

ENC

HH20

PV

PA

C

M

MC

CJ

DEC

VECEC

ENC

NCC

T

HH30

ENC

RVLV

RA LASIM

ME

T

RLCLLC

VS

Figure 3. Formation of cushions and septa. Schematic illustrations of sectioned hearts that depict important stepsof cardiac cushion maturation to septa and valves in the chicken embryo. By HH20, the heart consists of aprimitive atrium and a primitive ventricle; endocardial cushions have formed in the outflow tract (OFT) and atthe atrioventricular canal (AVC) junction. Mesenchymal cells have entered the cushions, originating fromepithelial-to-mesenchymal transition (EMT) induced by the endocardium. By HH30, the dorsal endocardialcushion and ventral endocardial cushion have fused to form the septum intermedium (SIM), which is joinedwiththe interatrial septum. More cushions have developed, namely, the left and right lateral cushions, which con-tribute to the formation of the tricuspid andmitral valves later in development. Growth of the ventricular septum(VS) toward the SIMhas notfinished by this stage but is completed byHH38 separating the ventricles. By then, allvalves have matured. (C) conus, (CJ) cardiac jelly, neural crest cell (NCC), (DEC) dorsal endocardial cushion,(EC) endocardial cushions, (ENC) endocardium, (E) epicardium, (LA) left atrium, (LLC) left lateral cushion,(LV) left ventricle, (M) myocardium, (MV) mitral valve, (OFTV) outflow tract valves, (PA) primitive atrium,(PV) primitive ventricle, (RA) right atrium, (RLC) right lateral cushion, (RV) right ventricle, (T) trabeculae, (TV)tricuspid valve, (VEC) ventral endocardial cushion.





in development starting at around HH26 in thechick embryo (Fig. 3; Moreno-Rodriguez andKrug 2015).

The second site of cushion formation is theOFT, which initiates slightly later than the AVCcushions at around HH18 (Person et al. 2005).By HH21 pairs of distinctive cushions can beobserved within OFT (Fig. 3), located proximal-ly (left and right) and distally (dorsal and rightventral). In fact, microscopy of chicken heartsections showed that those cushions are notcompletely separate units. The left proximalcushion stretches from the ventricular openingto the top of theOFTwhere it is connected to thearterial segment and becomes the distal dorsalcushion (Qayyum et al. 2001). The third distalOFT cushion forms left to the distal right ventralcushion at around HH25–26. These five OFTcushions contribute to a temporary aorticopul-monary septum that is replaced later and to thesemilunar valves, which prevent blood flow backinto the heart (Martinsen 2005; Snarr et al.2008). Importantly, OFT cushion maturationdepends not only on EMT-derivedmesenchymebut also on cardiac NCC, whichmigrate into thedistal OFT (Kirby and Waldo 1995) as early asstage HH18. The NCCs are essential for the for-mation of the interatrial septum (IAS). Overall,it takes up to stage HH36 (10 days) to completeformation of valves and septa in the chickenheart (Fig. 3; Martinsen 2005; Person et al.2005). Interestingly, quail and chick transplan-tation experiments showed that the develop-mental potential of NCC is already defined byHH8–10. Quail NCC were grafted from mesen-cephalic, cardiac, or truncal regions into the car-diac NCC region of host chick embryos (Fig. 1).Grafts of noncardiac NCC lead to severe OFTdefects as these cells were not able to contributeto its proper development, suggesting that bythis stage fate is restricted (Kirby and Waldo1995). Interestingly, NCCs also contribute toventricular cardiomyocytes, as recently revealedusing RCAS-mediated lineage analysis in chick.This contribution had previously been over-looked but was also confirmed in mouse byWnt1-cre lineage analysis. Furthermore, theNCC molecular program was up-regulated inthe regenerating zebrafish myocardium, thus

highlighting its potential importance for heartrepair (Tang et al. 2019).

Atrial Chamber Separation

Early on in heart development the four cham-bers are not separated from each other and thenecessary septa for their division arise duringremodeling of the heart. The IAS arises approx-imately at HH16 in the chicken heart. It origi-nates as septumprimum from the cephalodorsalwall (Quiring 1933; Hendrix and Morse 1977).In contrast to higher vertebrates, which have aseptum secundum, there is only the septum pri-mum in avian species (Morse et al. 1984). How-ever, a common characteristic among avian andmammalian development is that the IAS doesnot completely close until after birth to allow forinteratrial blood flow. This is necessary for op-timal oxygen supply, which is later ensured bythe pulmonary system. In the chick embryo, thefusion of the IAS with the ventral and dorsalcushions of the AVC occurs by HH24. Duringthis fusion, a secondary foramen-like structurefor interatrial blood flow arises in the form ofmultiple perforations in the middle of the IAS.This structure stays open until 2 days afterhatching when it closes (Martinsen 2005). Thedynamic changes of blood flow, which beginduring atrial septation, can be measured easilyin chicken embryos (for review, see Kowalski etal. 2014). Unidirectional blood flow is alreadyestablished before cushion formation in theprimitive heart tube at HH12 (Hu and Clark1989). With the appearance of the AVC cush-ions the backflow of blood is minimized, as theycontract together with the myocardium andthus reduce the opening to the atrium (Boselliet al. 2015). Subsequently the AVC cushions be-come cellularized and lose their flexibility, mostlikely in preparation to fusion. This loss in flex-ibility reduces their valve-like function, butmyocardial contraction velocity decreases aswell as the associated suction effect. As atrialcontraction becomes the dominant factor forpumping, AV blood flow velocity increases(Butcher et al. 2007). Optical coherence tomog-raphy and ultrasound imaging of avian embryoswere used together with modeling to determine





the pumping mechanisms that lead to pulsatingblood flow. This implicated both peristaltic andimpedance pumping mechanisms (Butcheret al. 2007; Kozlovsky et al. 2016). Better under-standing of the biomechanics underlying bloodflow has important clinical implications, for ex-ample, for surgery after aortic dissection ormyocardial infarct (for review, see Furst 2015).

Ventricular Trabeculation and Separation

Trabeculae are temporary structures that form ataround HH16–17 in the chicken embryo wherethey initiate at the level of the greater curvatureof the looped primitive ventricle (Sedmera et al.2000). During the course of development, theyundergo a process called compaction, which re-duces their surface area and ultimately contrib-ute their mass to the myocardial wall. Theirfunction during development is to mediate nu-trient and oxygen exchange before coronaryvascularization, routing of blood flow, increas-ing the myocardial mass, and facilitation of in-traventricular conduction (Martinsen 2005).Trabeculae are a part of the myocardium, whichcan be separated into two distinctive layers: ahighly proliferative layer—the compact myocar-dium, which is closer to the epicardium and amitotically inactive layer on the inside closer tothe endocardium (MacGrogan et al. 2018).Studies on the chicken embryo have been instru-mental for our understanding of trabeculaemorphogenesis during development, character-ized in detail by an extensive series of EM images(Sedmera et al. 1997). Soon after initiation oftrabeculation at HH16–17, a primitive ventric-ular septum (VS) can be observed by HH19–20.This structure grows until HH27 before fusingwith the SIM. Ventricular septation is completeby HH34 (8 days) (Waldo et al. 1998); at thisstage, myocardial proliferation and compactionincrease. Proliferation of the compact myocar-dium and the concomitant compaction process,which continue until embryonic day 14, result inthe ingression of the basal portions of the tra-beculae into the ventricular wall. This leads to anincrease in its thickness and a multilayered or-ganization, which is required for contraction ofthe adult heart (Martinsen 2005). Trabeculae

development is affected by hemodynamicchanges in the heart (Sedmera et al. 1999).Left atrial ligation drastically impacted bloodflow, which resulted in faster compacting trabec-ulae in the LV, a pressure overload and increasedcardiomyocyte proliferation in the RV. Further-more, a higher incidence of septation defectswas observed in the ligated hearts. More recentstudies regarding trabeculae development andfunction were conducted in mouse using vari-ous genetic tools (reviewed by MacGrogan et al.2018); however, our understanding of their im-portance remains incomplete.

Formation of the Cardiac Conduction System

Voltage-sensitive dyes have been used to detectaction potentials and pacemaking activity asearly as the 7–8 somite stage chick embryos(HH9) (Kamino et al. 1981). However, ataround HH11–12, the first sign of a more spe-cialized conduction system can be observed asthe faster conduction segments of the primitiveatrium and ventricle are now separated by amore slowly conducting AV canal (Gourdieet al. 2003). The AV-ring forms shortly after atHH14–15, it is centrally located in the heart andrequired for the later conduction cascade thatoriginates from the SAN. Electrical impulse isconducted across the atrial chambers to the AV-node and the AV-ring, then along the AV-bun-dle to finally arrive at the Purkinje fibers in theventricle completing the signal cascade (Martin-sen 2005). Most important for this specializedconduction system is the Purkinje fiber network,which develops in response to vessel-derivedendothelin between days 10–20 and spreadsacross the subendocardium of the left and RVsin the adult chicken heart. Linage tracing inchick has shown that Purkinje fibers originatefrommyocytes (Cheng et al. 1999). During theirconversion from cardiomyocytes and terminaldifferentiation, Purkinje fibers down-regulatecardiac transcription factors and up-regulate aset of genes associated with skeletal muscle,including the transcription factor myoblastdetermination protein 1 (MYOD) (Takebaya-shi-Suzuki et al. 2001). They also express thegap junction protein Connexin 40 (CX40)





(Gourdie et al. 1993). Interestingly CX40 is notexpressed in the slowAV conduction system andspecifically labels the fast conduction systempresent in atrioventricular bundles, the left andright bundle branches, and the peripheral ven-tricular conduction system (Christoffels andMoorman 2009).

SIMILARITIES AND DIFFERENCES BETWEENCHICK AND HUMAN HEARTS

The chick and human hearts are both four-chambered consisting of the right atrium (RA)and ventricle (RV), and the left atrium (LA) andventricle (LV) (Fig. 4). Chamber separation inthese amniote species ensures separation of oxy-gen-deprived blood (RA/RV) and oxygenatedblood (LA/LV). From the RV, the pulmonaryartery transports oxygen-poor blood to the

lungs, and from the LV the aorta delivers reoxy-genated blood to the body. Heart valves ensurethe direction of blood circulation and there arefour valves in both species. Within the heart thetricuspid valve separates the RA and RV, and themitral valve is located between the LA and LV.In addition, two valves are situated in theOFT toregulate blood flow where it exits from the heart:the pulmonary valve for pulmonary bloodflow from the RV and the aortic valve for aorticblood flow from the LV. Thus, the fundamentalcircuitry is very similar between chick and hu-man (Lo et al. 2010). But there are some phys-iological and anatomical differences, which isnot unexpected. These differences include theaverage body temperature and heart rate, whichare both higher in chicken. Furthermore, theventricles have more muscle mass relative tochamber size, and the LV is significantly bigger

Chicken heart Human heart

RA

RV

RV

RALA

LVLV

LA

Tricuspidvalve

Tricuspidvalve

Mitral valve

Mitral valve

Common carotid

Right subclavian

artery

Leftsubclavian

artery

Rightbranchiocephalic

arteryLeft

branchiocephalicartery

Mainpulmonary

artery

Rightaortic arch

Ascendingaorta

Left aortic arch

Pulmonary artery

Right superiorvena cava

Inferiorvena cava

Pulmonary valve

Aorticvalve

Figure 4. Chick versus human heart. Shown are illustrations of the chick and human heart to depict their strongsimilarity and a few species-specific differences. Among these differences are a smaller right ventricle (RV) in thechick, different directions of the ascending aorta, right in chick and left in human, and a thicker myocardium inthe chick to facilitate higher cardiac load together with higher heart rates. (LA) left atrium, (LV) left ventricle,(RA) right atrium.





compared with the RV allowing for up to fivetimes higher systemic pressure (Dzialowski andCrossley 2015). Outside the heart, the details ofpharyngeal arch remodeling during embryogen-esis vary between species (for details, see Poel-mann et al. 2017). As a result, the right branch ofthe aortic arch remains in the chick; therefore,the ascending aorta bends toward the right be-fore it descends. Conversely in human the as-cending aortic arch bends toward the left side(Fig. 4; Lo et al. 2010; Poelmann et al. 2017).

Implication for Congenital Heart Defects

Despite the differences in final form andphysiology, there are many examples in whichstudies in avian embryos have contributed fun-damental insights into developmental mecha-nisms as illustrated throughout this chapter.These, in turn, have been instrumental to en-hance our understanding of the origin of con-genital heart defects (CHDs). In particular,work from the Kirby laboratory (Kirby et al.1983) has informed on the importance of thecardiac NCC, as well as the SHF (Waldo et al.2001), for common OFTmalformations such aspersistent truncus arteriosus, transposition ofthe great vessels, and double-outlet RV. Removalof the cardiac NCC in chick can also lead toanomalies of the aortic arch or cardiac inflow.All of these heart defects have also been seen inhuman and they are often associated with struc-tural defects in other organs to which NCCscontribute (for review, see Kirby and Waldo1990).

CONCLUDING REMARKS

The chicken embryo has made seminal contri-butions to our current understanding of heartdevelopment and approaches used in avian of-ten work hand-in-hand with genetic manip-ulations that are more easily performed inthe mouse model. In particular, microsurgeryis straightforward in avian embryos, which rep-resents a distinct advantage. Multiple popula-tions of cells, which together build the heart,have been discovered and their origin mapped.This includes the FHF and SHF, the cardiac neu-

ral crest, the PEO, and pacemaker cells. Further-more, in vivo functional interference experi-ments have determined some of the geneticpathways and signals that control their differen-tiation. With new genetic tools, deep sequenc-ing, and genomics approaches as well as furtheradvances in imaging becoming available, there isno doubt that studies in chick embryos will con-tinue to reveal novel insights into cardiac devel-opment and the mechanisms that contribute tocongenital heart malformations, to cardiovascu-lar conditions, and to cardiac repair.

ACKNOWLEDGMENTS

J.G.W. was funded by a studentship from theBritish Heart Foundation (BHF FS/15/41/31564). Research in A.M.’s laboratory was support-ed by a BHF Project Grant (PG/15/77/3176).

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published online November 25, 2019Cold Spring Harb Perspect Biol Johannes G. Wittig and Andrea Münsterberg The Chicken as a Model Organism to Study Heart Development

Subject Collection Heart Development and Disease

Heart DiseaseIn Vivo and In Vitro Genetic Models of Congenital

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Cardiac Neural CrestHiroyuki Yamagishi

Development of the Cardiac Conduction SystemSamadrita Bhattacharyya and Nikhil V. Munshi

The Endocardium and Heart ValvesBailey Dye and Joy Lincoln

Understanding Ventricular Septation3D Anatomy of the Developing Heart:

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Long Noncoding RNAs in Cardiac DevelopmentMichael Alexanian and Samir Ounzain

Four-Chambered HeartCardiac Morphogenesis: Specification of the

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Genetic Basis of Human Congenital Heart DiseaseShannon N. Nees and Wendy K. Chung

DevelopmentThe Chicken as a Model Organism to Study Heart

Johannes G. Wittig and Andrea MünsterbergMultiple Roads to the Heart and Head MusclesCardiopharyngeal Progenitor Specification:

Benjamin Swedlund and Fabienne Lescroart

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Richard C.V. Tyser and Shankar SrinivasDevelopmentGenetic and Epigenetic Control of Heart

Brynn N. Akerberg and William T. Pu

Cardiovascular Heart-Defect ModelingDevelopment, Regeneration Discovery, and

: Experimental Access to CardiovascularXenopus

Stefan Hoppler and Frank L. ConlonRegeneration, and Diseaseduring Embryonic Development, Heart Formation and Growth of Cardiac Lymphatics

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DevelopmentReptiles as a Model System to Study Heart

Bjarke Jensen and Vincent M. ChristoffelsDevelopment and DiseaseThe Formation of Coronary Vessels in Cardiac

Lingjuan He, Kathy O. Lui and Bin Zhou

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