lsevier.com/locate/ydbio

Developmental Biology

Review

Left–right asymmetry and congenital cardiac defects: Getting to

the heart of the matter in vertebrate left–right axis determination

Ann F. Ramsdell *

Department of Cell and Developmental Biology and Anatomy, School of Medicine and Program in Women’s Studies,

College of Arts and Sciences, University of South Carolina, Columbia, SC 29208, USA

Department of Cell Biology and Anatomy and Cardiovascular Developmental Biology Center,

Medical University of South Carolina, Charleston, SC 29425, USA

Received for publication 9 June 2005, revised 21 July 2005, accepted 26 July 2005

Abstract

Cellular and molecular left– right differences that are present in the mesodermal heart fields suggest that the heart is lateralized from its

inception. Left– right asymmetry persists as the heart fields coalesce to form the primary heart tube, and overt, morphological asymmetry first

becomes evident when the heart tube undergoes looping morphogenesis. Thereafter, chamber formation, differentiation of the inflow and outflow

tracts, and position of the heart relative to the midline are additional features of heart development that exhibit left–right differences. Observations

made in human clinical studies and in animal models of laterality disease suggest that all of these features of cardiac development are influenced

by the embryonic left– right body axis. When errors in left– right axis determination happen, they almost always are associated with complex

congenital heart malformations. The purpose of this review is to highlight what is presently known about cardiac development and upstream

processes of left– right axis determination, and to consider how perturbation of the left– right body plan might ultimately result in particular types

of congenital heart defects.

D 2005 Elsevier Inc. All rights reserved.

Keywords: Cardiac development; Congenital heart defect; Heterotaxy; Left– right asymmetry; Situs inversus

Introduction

The significant morbidity and mortality of laterality disease

almost always are attributed to complex, congenital heart

defects (CHDs). This prevalence indicates that the developing

heart is extremely susceptible to disturbances in embryonic

left–right patterning. In attempt to define the cellular and

molecular mechanisms that underlie cardiac asymmetry, most

of the focus in the field has been on identifying genes and cell–

cell signaling interactions that establish and maintain global

left–right asymmetry of the vertebrate body plan. While this

line of investigation is certainly critical to unraveling the issue,

equally important is to understand how global left–right axial

patterning intersects with morphogenetic processes of heart

0012-1606/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.ydbio.2005.07.038

* Department of Cell and Developmental Biology and Anatomy, School of

Medicine and Program in Women’s Studies, College of Arts and Sciences,

University of South Carolina, Columbia, SC 29208, USA. Fax: +1 843 792

0664.

E-mail address: ramsdell@musc.edu.

development. In this review, I discuss how heart development

necessarily invokes three different types of asymmetric pattern

and summarize the upstream molecules and inductive signaling

processes that are central to current models of vertebrate left–

right axis determination. In addition, I propose how different

types of CHDs might arise when left–right axis defects impede

normal development of one or more of the three types of

asymmetric pattern in the heart.

Overview of left–right axis determination

Left–right development of the heart (and other organs) is

critically dependent upon upstream pathways that impose

asymmetry onto what is initially a bilaterally symmetric body

plan. These early-acting pathways are collectively known as

‘‘left–right axis determination’’ and involve not only the

breaking of bilateral symmetry, but also equally important,

the directional orientation of asymmetry relative to the

anteroposterior and dorsoventral body axes. Deviations in

left–right axis determination during embryogenesis result in a

288 (2005) 1 – 20

www.e

Fig. 1. Endpoints of left – right axial pattern. Ventral view of an MF-20

immunostained chick heart showing dextral (rightward) looping (A) and ventral

view of a Xenopus tadpole showing counterclockwise gut coiling (B). Nkx2.5

expression in spleen tissue detected by whole mount in situ hybridization in

Xenopus embryos (C–E, arrows). Although Nkx2.5 expression is initially

symmetric (C), it gradually becomes restricted to the left side only (D, E).

Normal asymmetry in left and right mouse lungs (F, ventral view) and

symmetric (abnormal) lobulation in an FGF8 null mouse (G, ventral view).

Left (L) and right (R) sides are indicated for all panels. See text for details.

Panel A courtesy of A. Wessels (Van den Hoff et al., 1999); panel B, A.

Ramsdell unpublished; panels C–E courtesy of P. Krieg (Patterson et al.,

2000); panels F and G courtesy of G. Martin (Meyers and Martin, 1999).

A.F. Ramsdell / Developmental Biology 288 (2005) 1–202

wide spectrum of abnormal laterality phenotypes that are

generally classified as either situs inversus or situs ambiguus.

Situs inversus is a condition in which the left–right axis is

reversed in alignment with the other two body axes, resulting in

a mirror image of normal body and organ situs (situs solitus).

Because of the concordant inversion of the body plan, one

common misperception is that situs inversus is not linked with

a higher than normal incidence of CHDs. However, the

estimated incidence of CHDs in situs inversus patients is

significantly higher than that in patients with situs solitus (i.e.,

¨3% vs. ¨0.08%) (Ferencz et al., 1985; Nugent et al., 1994;

Sternick et al., 2004). In addition, the risk for developing

laterality disease, and hence complex CHDs, is greatly

increased for progeny of individuals with situs inversus (Burn,

1991; Gebbia et al., 1997).

Situs ambiguus, also termed heterotaxy, is a much broader

category that refers to any combination of discordant normal

and abnormal left–right asymmetries that cannot be strictly

classified as situs solitus or situs inversus. Complex CHDs

almost always are present in individuals with situs ambiguus

with estimates reaching at or greater than 90% (Nugent et al.,

1994). Failure to establish asymmetry or errors in relay of axial

patterning information during development can cause situs

ambiguus, such that asymmetry in structure and placement of

organs still develops, albeit stochastically, due to the lack of

definitive positional information. Situs ambiguus also includes

isomerism, a condition in which normally lateralized organs

instead develop left or right symmetry. Cardiac defects

typically occurring with situs ambiguus include, but are not

limited to, atrial septal defects (ASDs), ventricular septal

defects (VSDs), transposition (or corrected transposition) of the

great arteries (TGA), double outlet right ventricle (DORV),

anomalous venous return, and aortic arch (AA) anomalies

(reviewed by Bowers et al., 1996).

Once the body plan is established and positional information

has been relayed throughout the embryo, three different

endpoints of left–right axial pattern are possible. The first is

directionally oriented looping that occurs in organs that

essentially begin as a tube (e.g., heart or gut) (Figs. 1A, B).

The series of bending and rotational movements in looping

morphogenesis are necessary to establish structural asymmetry

within organs as well as to establish proper organ placement

within the body. A second endpoint of left–right pattern is

unilateral regression and/or persistence of structure. One

example of this is during spleen development, in which two

organ fields are initially present to either side of the midline,

but under normal circumstances, only the left-side tissue

completes differentiation (Patterson et al., 2000) (Figs. 1C–

E). Third, as exemplified by left–right differences in lobulation

of the lungs and liver, some organs that first appear symmetric

go on to develop structural features that show ‘‘handedness’’ to

their asymmetry (Figs. 1F, G). This third endpoint is called

lateralization of structure, and it is preceded by cellular and

molecular left–right differences that are present even before

morphological asymmetry can be observed. As detailed below,

because the vertebrate heart must acquire all three endpoints of

left–right pattern during its formation, it is especially prone to

developing defects if any aspect of left–right axis determina-

tion is compromised.

Cardiac left–right development

Lateralization of the heart fields and the primary heart tube

Mesoderm cells appear to become specified to a cardiac

lineage quite early in development, either just prior to or during

their migration at gastrulation (Antin et al., 1994; Yatskievych

et al., 1997). As these cells gastrulate, they migrate anteriorly

and spread laterally to the left and right of the embryonic

midline (i.e., the primitive streak in chick or mouse, and the

dorsal midline in fish and frog), where they form two paired

fields of cardiac-fated mesoderm called the primary heart fields

(Fig. 2A). Lineage analysis of gastrulating cells in the chick

embryo has shown that very few cells cross the midline when

migrating through the primitive streak (Levy and Khaner,

1998). As discussed below, because left–right positional

information is present in the embryo during gastrulation stages,

this suggests that left and right mesoderm cells, including those

cells destined for the cardiac lineage, may be exposed to

Fig. 2. Overview of vertebrate heart development. Schematic view of the primary heart fields depicted in the chick embryo (A). As development proceeds, the

bilateral heart fields coalesce to form the heart tube, which begins the looping process as elongation of the tube continues. Prior to septation and completion of

looping, the heart tube (B) is comprised of a common atrium (A), atrioventricular canal (AVC), trabeculated left and right ventricle (VEN), outflow tract (OFT), and

aortic sac (AS). Endocardial cushions (yellow) form in the AVC and OFT regions, and line the inner curvature (stippled area). The myocardium of the inner curvature

is removed during remodeling (C) to allow completion of septal alignments and looping. This is accomplished by migration of myocardial cells into underlying

cushions, the latter which also become cellularized with mesenchyme cells derived from an epithelial–mesenchymal transformation of the endocardium in these

regions. Concomitant with these processes, the aorticopulmonary septum (APS), sinoatrial fold (SAF), and interatrial septum (IAS) form (C). Transformation of the

arterial pole into the adult (human) arterial pattern is depicted in panel D. The distal end of the OFT, called the truncus arteriosus, and the AS connect to the paired

dorsal aortae via a series of paired aortic arches (numbered and distinguished by colors). Structures derived from the different arches are color-coded to indicate

origin and dashed lines indicate regression. Transformation of the venous pole into the adult (human) venous pattern is depicted in panel E. Different veins are color-

coded to indicate contribution to different regions of the vena cava and dashed lines indicate regression. The paired cardinal veins drain into the sinus venosus of the

heart, and the right anterior cardinal and right common cardinal veins eventually contribute to the superior vena cava. The paired subcardinal veins form an

anastomosis and eventually contribute to part of the inferior vena cava (IVC). The IVC also derives from a portion of the right, but not left, supracardinal vein located

caudal to the kidneys as well as the right subsupracardinal vein. Panels B and C were redrawn and modified from Mjaatvedt et al. (1999).

A.F. Ramsdell / Developmental Biology 288 (2005) 1–20 3

different left–right patterning signals as they become arranged

in the newly formed mesodermal germ layer.

The earliest indication of cardiac molecular asymmetry is

observed after gastrulation, once cardiac cells are residents of

the primary heart fields. In the chick, there are genes and

proteins that are expressed either by only one heart field, or

genes that are expressed asymmetrically by both heart fields,

with expression being higher in one field compared to the

other. Three proteins that show relative asymmetry in the

primary heart fields are components of the extracellular matrix

and include fibrillin-2 (Rongish et al., 1998; Smith et al.,

1997), which is predominant on the right, and hLAMP-1

(Smith et al., 1997), and flectin (Tsuda et al., 1996), which are

predominant on the left. Pitx2c, a bicoid-related transcription

factor, is detected in cells only in the left, but not right, heart

field (Campione et al., 2001; St Amand et al., 1998). Flectin

and Pitx2c continue to be expressed asymmetrically as cells

become incorporated into the primary heart tube.

The asymmetric gene expression in the primary heart tube is

consistent with much earlier studies that demonstrated cellular

differences between the left and right heart fields. For example,

cardiomyocyte differentiation (Patten and Kramer, 1933) and

striated myofibril formation (Lindner, 1960) happen slightly

earlier in the right heart field compared to the left. The right

cardiogenic fold appears slightly more advanced than the left

(Stalsberg and DeHaan, 1969), and when cardia bifida is

A.F. Ramsdell / Developmental Biology 288 (2005) 1–204

elicited by preventing fusion of the two heart fields, two hearts

form that show left– right differences (Van Praagh and

DeHaan, 1967). The anterior portion of the heart tube is larger

in the right heart structure and the posterior portion of the heart

tube is larger in the left heart structure. Examination of the

relative contributions of the primary heart fields to the chick

heart indicates that the anterior and posterior regions of the

heart differ in their composition of cells that were derived from

each field. Specifically, a larger population of cells derived

from the right heart field contributes to the posterior (inflow)

region (Stalsberg, 1969). The cells that contribute to the

posterior region of the heart tube are the same subset of cells

that previously expressed nodal, lefty-2, and cSnr prior to their

incorporation into the primary heart tube. As discussed below,

nodal, lefty-2, and cSnr are left–right asymmetry genes that

are expressed specifically in the left or right (but not both)

lateral plate mesoderm, with their anterior expression domains

reaching into the posterior primary heart field. Although these

genes are no longer expressed once cardiac cells become

incorporated into the heart tube, this observation suggests that

this subset of posterior cardiac cells possesses a ‘‘history’’ of

molecular asymmetries that distinguishes it from other cells in

the heart. These observations suggest not only that the nascent

heart tube exhibits cellular and molecular laterality, but also

that laterality of the heart tube varies along the length of its

anterior–posterior axis.

Besides the primary heart fields, another source of

mesoderm cells, termed the ‘‘secondary’’, or ‘‘anterior’’, heart

field (hereafter called the secondary/anterior heart field, SAHF)

contributes to the heart tube (Franco et al., 2001; Kelly et al.,

2001; Mjaatvedt et al., 2001; Waldo et al., 2001,2005a,b;

Zaffran et al., 2004). The precise location of the SANF has

been debated, with some investigators defining this field as a

small area adjacent to the ventral pharynx (Waldo et al., 2001),

and others defining it as a broader area to include mesoderm

surrounding the aortic sac (AS) and extending into all of the

pharyngeal arches (Franco et al., 2001; Mjaatvedt et al., 2001).

Once cells from the left and right primary heart fields coalesce

to form the ventricular and inflow regions of the heart tube,

mesoderm cells from the SANF are added to the heart tube to

complete its formation. In chick (and perhaps in frog), the

SANF cells contribute to the conotruncus, or outflow tract

(OFT) of the heart (Martinsen et al., 2004; Mjaatvedt et al.,

2001; Waldo et al., 2001), in addition to the AS (Waldo et al.,

2005a,b). Moreover, there is evidence that the SANF con-

tributes to both the OFT and the right ventricle in the mouse

heart (Kelly et al., 2001; Zaffran et al., 2004). The successful

recruitment of cells from the SANF to the heart requires the

presence of a second cell type, the cardiac neural crest, which

migrates through the pharyngeal arches in order to eventually

populate the OFT, and to a lesser extent, the inflow region of

the heart (reviewed by Hutson and Kirby, 2003). The

importance of cardiac neural crest with respect to the SANF

is that the neural crest is thought to regulate ‘‘availability’’ of

inductive factors that mediate addition of SANF cells to the

OFT myocardium of the heart (Farrell et al., 1999, 2001;

Waldo et al., 2005a,b; Yelbuz et al., 2002, 2003). Whether this

regulation is direct or indirect is not known. Nevertheless,

neural crest ablation experiments result in hearts that have

shortened OFTs, in addition to several other types of defects

that are discussed below (Farrell et al., 2001; Martinsen et al.,

2004; Yelbuz et al., 2002, 2003). Similar to the primary heart

fields, the SANF exhibits asymmetric Pitx2c expression that is

detected in the left, but not right, AS mesoderm and left

pharyngeal arch mesenchyme (Liu et al., 2002), suggesting that

molecular and cellular laterality is present also in the SANF. In

addition, Pitx2c plus two Pitx2 isoforms (Pitx2a and Pitx2b)

are expressed–albeit symmetrically–in cardiac neural crest

cells as they migrate into the heart (Hamblet et al., 2002;

Kioussi et al., 2002).

Experimental manipulations made in gastrula and neurula

stage embryos can alter left–right development of the heart,

including asymmetric gene expression in the heart fields and

directionality of looping morphogenesis. In the African clawed

frog, Xenopus laevis, perturbation of the ectodermal extracel-

lular matrix in the blastocoel roof causes reversed heart looping

(Yost, 1992). Treating embryos at early neurula stages with

heparan sulfate proteoglycan synthesis inhibitors prevents heart

tube looping (Yost, 1990), and extirpations of midline tissues

causes inverted or bilateral expression patterns of normally

asymmetric genes in the lateral plate mesoderm, in addition to

reversed heart tube looping if performed prior to closure of the

neural tube (Danos and Yost, 1996; Lohr et al., 1997).

Likewise, treatments that cause left–right axis perturbations

in chick embryos cause both abnormal heart looping and

inverted expression of the genes and proteins that are normally

asymmetric in the heart fields. When explants of chick

(Stalsberg, 1970) or Xenopus (Danos and Yost, 1996; Yost,

1990) precardiac mesoderm and its associated ectoderm are

cultured in isolation, these tissues form a tube-shaped structure

that somewhat resembles the primary heart tube including

looping. Depending on the stage of the embryo from which the

tissue is harvested, the direction of looping is either normal or

stochastic. In Xenopus, the ability of the explant structures to

consistently loop in a normal direction increases as the age of

the donor embryo reaches late neurulation stages (Danos and

Yost, 1996; Yost, 1990). Collectively, the observations made in

Xenopus and chick demonstrate not only that cellular and

molecular laterality is present in the heart from its inception,

but also that some aspects of cardiac left–right asymmetry are

regulated by processes that precede the appearance of the heart

fields and the primary heart tube. This means that as cardiac

mesoderm becomes established and as it contributes to the

heart tube, these cells are already specified, at least to some

extent, for left–right identities.

Looping morphogenesis is necessary for septation and

chamber and vessel concordance

Even before the heart tube has completed its formation, it

starts to undergo looping morphogenesis. Because cardiac

looping is a highly conserved process that occurs quite early in

vertebrate development, the directionality of the heart loop is

commonly used as a ‘‘readout’’ of body situs. However, despite

Fig. 3. Congenital heart defects that are frequently associated with laterality

disease. A schematic depiction of a normal heart is shown in panel A. Red and

blue indicate oxygenated and deoxygenated blood, respectively, and purple

shading in panels B–G indicates mixing of oxygenated and deoxygenated

blood. A ventricular septal defect (VSD) is shown in panel B. An atrial septal

defect is shown in panel C. Double-inlet left ventricle (DILV) is shown in panel

D. Double-outlet right ventricle (DORV) is shown in panel E. Transposition of

the great arteries (TGA) is shown in panel F. Persistent truncus arteriosus (PTA)

is shown in panel G. All hearts are drawn in ventral view.

A.F. Ramsdell / Developmental Biology 288 (2005) 1–20 5

it being one of the most widely recognized aspects of cardiac

asymmetry, it is often less well appreciated that looping is a

complex process involving both bending and rotational move-

ments (reviewed by Manner, 2000). As the heart tube continues

to elongate, it first develops a dextral loop (‘‘d’’ loop) that

results from the coordinated activities of ventral bending and

rightward rotation. Dextral looping occurs concomitantly with

increased growth at the outer vs. inner curvature, a process that

accompanies chamber formation and that ultimately causes the

heart tube to take on a ‘‘C’’-shaped appearance (Christoffels et

al., 2000; Rumyantsev, 1977; Thompson et al., 1990). Under

the influence of the left–right body axis, the dextral loop

normally orients to the right side of the embryonic midline,

aligning the primordial cardiac chambers to face the outer

curvature (Christoffels et al., 2000), and it is this aspect of

looping that most investigators equate with ‘‘rightward’’

looping. Thereafter, the dextrally looped heart transitions to

an ‘‘S’’ shape, a process that shifts the ventricular bend caudally

toward the atria. The final phase of looping morphogenesis is

characterized by the ‘‘wedging’’ of the distal OFT toward the

right atrium. At this point of development, the heart has lost its

tubular character, and the anterior (arterial) and posterior

(venous) poles of the heart are brought together in close

proximity. Current models of the ‘‘biomechanics’’ of looping

suggest that forces that are present on both the left and right

sides of the heart tube drive looping morphogenesis. Impor-

tantly, the left- and right-side forces are thought not to be

equivalent and are thought to differ at the cranial vs. caudal

aspects of the heart, such that the cranial portion of the tube

undergoes a rightward rotation and the caudal portion of the

tube undergoes a leftward rotation (Manner, 2004; Voronov et

al., 2004). The opposing polarity of rotations at the two ends of

the tube implies that asymmetry genes that control looping may

be expressed in opposite left–right patterns at the distal ends of

the heart tube (Manner, 2004; Voronov et al., 2004).

It should be emphasized that both the process of looping per

se and the directionality of the loop are important for normal

heart development. Because most cardiac structures arise from

cells derived from more than one area of the heart tube, the

significant outcome of looping is to rearrange regions of the

heart tube so they are appropriately positioned for proper

formation and alignment of chambers, valves, and septa (Fig.

2B). Thus, although left–right differences in the ventricles are

established by anteroposterior, rather than left–right patterning

processes (Franco et al., 2001; Thomas et al., 1998), the

directionality of looping determines whether the morphological

left ventricle underlies the left atrium, and the morphological

right ventricle beneath the right atrium. When the topological

situs of the ventricles is correct, the heart is said to have atrio-

ventricular (A-V) concordance.

In addition to A-V relations, looping also affects septation of

the heart. As looping occurs, cushion tissues (progenitor

valvuloseptal tissues) that have formed in the atrioventricular

canal (AVC) and the OFT regions of the heart are brought

together at the inner curvature of the looped heart tube. This

repositioning allows two important processes to occur (Fig. 2C).

First, it facilitates septation by bringing AV and OFT cushion

tissues together for formation of the atrioventricular septum

(AVS) and the OFT septum. If either end of the heart tube is

delayed or somehow impaired in its looping, then this would

cause cushion tissues to be out of alignment at the inner curvature

of the heart, increasing the chance for septal defects to occur

(Figs. 3B, G). Second, remodeling of the myocardium at the

inner curvature is necessary in order for the OFTand the AVC to

merge to form the future mitroaortic continuity. One potential

mechanism bywhich this remodeling occurs is through a process

termed myocardialization, in which the OFT cushion tissues at

the level of the inner curvature become invaded by overlying

myocardial cells (van den Hoff et al., 1999). Abnormal

myocardialization in hearts of trisomy 16 and other mouse

mutant models is correlated with incomplete looping and failure

to properly remodel the inner curvature, suggesting that

myocardialization plays an important role in facilitating the final

A.F. Ramsdell / Developmental Biology 288 (2005) 1–206

aspects of looping morphogenesis (Waller et al., 2000; Bartram

et al., 2001; Boot et al., 2004). One probable outcome of

myocardialization, therefore, is that it allows completion of

wedging, which in turn, is necessary to establish properly aligned

inflow and outflow tracts. In addition, this process also creates

the muscular outflow septum below the level of the valves.

Differentiation of the inflow tract (IFT) and OFT occurs

during wedging. In the IFT, the common AVC must become

divided into left and right components by an AVS that shifts

rightward during the final phase of looping to become

positioned directly atop the ventricular septum. This results

in alignment of the AVC with the ventricles. If the IFT does not

fully undergo this rightward shift, a condition known as

double-inlet left ventricle (DILV) persists (Fig. 3C). DILV

results in persistence of blood flowing into the left ventricle

from both the left and right atriums.

Meanwhile, at the OFT region of the heart, the conotruncus

shifts leftward and simultaneously twists 180- to become

positioned atop the AVS. This repositioning of the conotruncus

serves two purposes. First, it brings proximal conal cushion

tissues into alignment with AV and ventricular cushions so that

septation can finish. Second, the rotational movement of the

conotruncus is necessary to position the future base of the aorta

and pulmonary artery (which form from the distal conotruncus

and the AS) with the appropriate ventricles. Failure to correctly

align the conotruncus during looping morphogenesis can result

in double-outlet right ventricle (DORV), a condition in which

the right ventricle communicates with both the aorta and the

pulmonary artery and the left ventricle has no outlet (Fig. 3D).

This is different from the type of defect that would occur if the

rotational aspect of conotruncal wedging was disturbed.

Defects specifically in its rotational component would misalign

the base of the aorta and the pulmonary artery with the left and

right ventricles, resulting in a condition called transposition of

the great arteries (TGA) (Fig. 3E).

In instances where normal conotruncal wedging and rotation

happen, an outflow defect known as persistent truncus arterious

(PTA) still can occur (Fig. 3F). The base of the aorta and the

pulmonary artery form from the distal portion of the conotruncus

plus the AS. Both the AS and the conotruncus must become

divided in order to separate systemic and pulmonary blood flow

through this region. When this septation process does not occur

normally, PTA is the result. It has been appreciated for many

years that aorticopulmonary septation requires the population of

this region by the cardiac neural crest that pass through the

pharyngeal arches to form the APS, which divides the AS

(Nishibatake et al., 1987). Specifically, neural crest arising from

between the fourth and sixth pharyngeal arches extends into the

AS and ultimately merges with the fused OFT cushion tissues.

Perturbation of this process in vivo, by neural crest cell ablation,

results in PTA (Nishibatake et al., 1987).

Lateralization and differentiation of the inflow tract and AV

canal

Unlike the ventricles, which form in ‘‘series’’, the atria derive

from a common progenitor, the common atrium, which must

become divided into two chambers with distinct, left–right

features (Anderson, 1992; Min et al., 2000). In Xenopus, this

process of division may be related to differences in atrial cells

that derive from the left vs. right primary heart field (Gormley

and Nascone-Yoder, 2003). Once differentiated, the morpho-

logical right atrium contains pectinate muscles in its atrial

appendage and receives the IVC, and the morphological left

atrium contains a trabeculated appendage that lacks pectinated

muscle and receives the pulmonary vein. Failure to achieve one

or the other of these lateralized differences during division of

the common atrium is the basis of atrial isomerism.

Studies of the interatrial septum (IAS) suggest that the left–

right differences that arise during lateralization of the common

atrium also can be related to the existence of two different cell

populations that are present in this region. The IAS is derived, in

large part, from a myocardial infolding of the left atrial wall,

followed by transformation of some IAS cells to mesenchyme

(Wessels et al., 2000). Myocardial cells of the IAS share

common characteristics, such as creatine kinase B and Pitx2c

expression with cells in the left, but not right, atrial wall,

indicating a molecular asymmetry that is present in the common

atrium prior to its differentiation (Franco and Campione, 2003;

Liu et al., 2002; Wessels et al., 2000). Because the IAS

originates from the cell population specified for ‘‘leftness’’, in

instances where left-side signaling pathways are impaired (e.g.,

right isomerism), it would be predicted that structures derived

from the ‘‘left’’ cell population will not form. Consistent with

this prediction, there is a significant deficiency, if not complete

absence, of the IAS in hearts of many individuals afflicted with

right atrial isomerism (Bowers et al., 1996).

Whether lateralized processes can influence other aspects of

cardiac septation has not been investigated; however, several

features of cardiac cushion formation (progenitor valvuloseptal

tissue) in the AVC region suggest that this is possible. Despite

their dorsal and ventral anatomic positions in the looped heart,

the original superior (dorsal) and inferior (ventral) AV

endocardial cushions form from the original left and right

sides of the AVC (Lamers and Moorman, 2002; Moreno-

Rodriguez et al., 1997). Consistent with their initial left–right

origins, the inferior and superior cushions exhibit distinct

properties throughout septation morphogenesis, including

differences in temporal proliferative rates, spatial distribution,

and absolute amounts of mesenchymal tissue formed (Lamers

and Moorman, 2002; Moreno-Rodriguez et al., 1997). Addi-

tionally, myocardium in the AV canal exhibits left, but not

right, side Pitx2c expression (Campione et al., 2001). These

types of cellular and molecular left–right differences suggest

that similar to the common atrium, the AVC region also is

lateralized. Laterality disturbances in the AVC would be

predicted to affect endocardial cushion tissue formation,

ultimately increasing risk for valvuloseptal defects. By analogy

to the AVC and the common atrium, it is tempting to speculate

that the AS and conotruncus similarly develop a cellular and

molecular laterality that influences their subsequent division

into the base of the outlet vessels. If so, this could represent a

process that, if affected by abnormalities in left–right pattern,

could result in OFT defects such as PTA and/or TGA.

A.F. Ramsdell / Developmental Biology 288 (2005) 1–20 7

Asymmetric tissue regression patterns the aortic arches and

venous return

In addition to lateralization and looping, the third type of

asymmetry that occurs during normal heart development is

unilateral regression of blood vessels that connect with the

inflow and outflow portions of the heart. This feature is the

basis for the complex patterned regression and persistence of

the six pairs of aortic arch (AA) arteries and the aorta (Davies

and Guest, 2003). Initially, the AA arteries develop as a series

of six bilaterally paired vessels that connect with the paired

dorsal aortae (Fig. 2D). During AA artery remodeling, the first

two pairs of AA arteries regress into capillary beds. The third

pair of AA arteries persists and eventually becomes the paired

common carotid arteries. In contrast to the symmetric fates of

AAs 1–3, the left artery of the fourth pair of AA arteries

contributes to the aortic arch (in mammals), and the right artery

contributes to the right subclavian artery. The fifth pair of AA

arteries completely regresses or fails to form, and the sixth pair

of AA arteries persists only on the left side to contribute to the

pulmonary artery and the truncus arteriosus. At the inflow

region of the heart, similar mechanisms of regression/persis-

tence operate to pattern the paired posterior cardinal, sub-

cardinal, and supracardinal veins, which ultimately give rise to

the right-side inferior vena cava (Fasouliotis et al., 2002) (Fig.

2E). Aberrant regression/persistence patterns in the AA arteries

and the cardinal veins therefore can result in many types of AA

anomalies or abnormal venous return, depending on which

particular vessels are affected (Ruscazio et al., 1998).

Left–right patterning events upstream of cardiac

development

Because cardiac laterality defects nearly always occur in

conjunction with laterality defects in one or more other organs,

this indicates that the causative perturbation is one that occurs

early in development and that precedes organogenesis. Awidely

held view of left–right development is that it proceeds as a three

‘‘step’’ process. The first is left–right axis determination, which

establishes initial asymmetry in the embryo that is in correct

alignment with the other two body axes. Thereafter, left–right

positional information emanating from this axis must propagate

throughout the embryo over a wide range of developmental

stages, so that this information becomes relayed to each

emerging cell and tissue type. Finally, once organogenesis

begins, cells then must interpret and respond to the global left–

right ‘‘blueprint’’ in order to translate this information into

anatomical asymmetries. Errors in any of these three steps can

result in cardiac (and other) laterality defects. The nature of the

upstream signaling molecules that convey left–right patterning

information in each of the three ‘‘steps’’ remained for the most

part unknown until the pivotal 1995 discovery that sonic

hedgehog and activin can function as left–right asymmetry

genes in the chick (Levin et al., 1995). Since this time, the field

of vertebrate left–right development has rapidly grown to

recognize dozens more genes, as well as many types of cell–cell

signaling interactions, that act in concert to establish left–right

asymmetries in the embryo. Molecules and inductive signaling

processes that are central to current models of vertebrate left–

right development are discussed below.

Breaking bilateral symmetry: models of left – right axis

determination

In Xenopus, there is a patch of tissue located in the dorsal part

of the blastopore, called the dorsal lip, that is capable of initiating

gastrulation and directing complete secondary axis formation

when transplanted to a ventral region in a host embryo. Because

of the unique inductive properties of this tissue, the dorsal lip is

historically known as the ‘‘organizer’’. Functionally equivalent

structures exist in mammals (the node), avians (Hensen’s node),

and zebrafish (shield). Implicit in the patterning properties

observed in grafted organizer/node tissue is that the organizer/

node is a source of positional information for the different cell

types that it induces. Direct evidence for node involvement in

left–right development was first derived from studies in chick

and mouse, in which a series of grafting and extirpation

experiments indicated that the node is both necessary and

sufficient to direct left–right asymmetry of the body plan

(Davidson et al., 1999; Pagan-Westphal and Tabin, 1998). In

chick, the node is not the first source of left–right asymmetry

information; but rather, tissues adjacent to the node impart left–

right pattern that is in turn relayed by the node to surrounding

tissues as development proceeds (Pagan-Westphal and Tabin,

1998). In the mouse embryo, node ablation during late

gastrulation results in embryos with normal anterior–posterior

and dorsoventral development, but abnormal left–right devel-

opment, highlighting its important function in propagating left–

right patterning information (Davidson et al., 1999).

The earliest molecular aspect of left–right axis determina-

tion that is clearly conserved among all vertebrates is the

asymmetric, left-side expression of nodal. Nodal is a TGFhfamily member that is expressed in the left half of the node,

followed by the onset of a wide domain of expression in the

left, but not right, lateral plate mesoderm. Normal, left-side

only nodal expression is required for normal left–right

development in all species thus far examined (Collignon et

al., 1996; Hyatt et al., 1996; Levin et al., 1995; Lohr et al.,

1997; Lowe et al., 1996; Rebagliati et al., 1998b; Sampath et

al., 1997). Bilateral, absent, or right-side nodal expression

patterns are observed in iv/iv mice, a classic animal model of

heterotaxy (Collignon et al., 1996; Lowe et al., 1996). In inv/

inv mice, which exhibit situs inversus, inverted (right-side)

nodal expression is observed (Collignon et al., 1996).

Consistent with these findings, direct perturbation of nodal

expression in mouse results in situs ambiguus, indicating that

left– right development of the heart and visceral organs

requires restricted, left-side nodal activity (Brennan et al.,

2002). Studies of nodal homologs and components of the nodal

signaling pathway in Xenopus and zebrafish have corroborated

its central role in establishing vertebrate left–right asymmetries

(Ahmad et al., 2004; Hashimoto et al., 2004; Lohr et al., 1997,

1998; Rebagliati et al., 1998a,b; Sampath et al., 1997; Schier,

2003; Schier and Shen, 2000).

A.F. Ramsdell / Developmental Biology 288 (2005) 1–208

Experimental evidence derived from mouse, chick, and

Xenopus has led to a number of models that seek to explain the

steps of left–right axis determination that operate upstream of

nodal expression. In the mouse, ‘‘nodal flow’’, a mechanism

that involves ciliary function in node cells, is the prevailing

model. Null mutations in genes that are necessary for cilia

formation and/or function (e.g., iv (a.k.a. left–right dynein), inv,

Kif3-A, Kif3-B, HFH-4, RFX3, D2LIC) result in pronounced

laterality defects (Bonnafe et al., 2004; Brody et al., 2000; Chen

et al., 1998; Marszalek et al., 1999; Morgan et al., 1988; Nonaka

et al., 1998; Rana et al., 2004; Supp et al., 1997; Watanabe et al.,

2003). A role for ciliary genes was not necessarily unexpected,

since much earlier studies of Kartegener’s syndrome (a situs

inversus phenotype) had revealed an association between

human laterality defects and ultrastructural defects in cilia

(reviewed by Palmblad et al., 1984). Stunning experiments

performed with video microscopy have shown that motile cilia

present in the center of the mouse node propagate directional

fluid flow, and furthermore, that this flow is abnormal in several

mouse models bearing null mutations in ciliary motor proteins

(Nonaka et al., 1998, 2002; Okada et al., 1999; Watanabe et al.,

2003). One widely held interpretation is that nodal flow directs

left–right axis formation by causing asymmetric accumulation

of a diffusible morphogen that, in turn, launches widespread

asymmetric gene expression of factors such as nodal. In support

of this model, it has been shown that nodal cilia in mouse,

rabbit, zebrafish, and medakafish exhibit a posterior tilt that is

thought to result in much deeper ciliary contact with nodal fluid

in the right-to-left portion of ciliary rotational beating, a

phenomenon that in turn could account for asymmetric

Fig. 4. Nodal flow models proposed for mouse and zebrafish. In mouse (A), there ar

express left – right dynein (lrd) (green) and polycystin-2 (red), propagate directional

but not left – right dynein. In response to the directional fluid flow, Ca2+ levels beco

regulate left-side nodal expression (B). In zebrafish (C), dorsal forerunner cells expr

right defects, as do mutations in genes that are necessary for these cells to form K

develop cilia that propagate directional fluid flow, which in turn, is proposed to

mesoderm. Panels A and B courtesy of M. Brueckner (McGrath et al., 2003) and p

deposition of components swept by nodal flow (Kramer-Zucker

et al., 2005; Okada et al., 2005). Direct evidence for the ability

of nodal cilia to generate an asymmetric gradient was

demonstrated by the introduction of fluorescently conjugated

dextran particles (comparable to protein ¨40 kDa in size),

which were found to distribute preferentially on the left side of

the rabbit node (Okada et al., 2005), and FGF, SHH, and

retinoic acid are factors that are proposed to be involved in

generating the morphogen gradient in the mouse node (Tanaka

et al., 2005). However, as detailed elsewhere (Hornstein and

Tabin, 2005; Levin, 2004; Wagner and Yost, 2000), there are

some inconsistencies between predictions of the nodal flow

morphogen model and the nature of the laterality defects present

in mice null for genes that are needed for ciliary formation or

function. With the discovery of a second population of nodal

cilia, the so-called ‘‘mechanosensory cilia’’, an alternative

model has been put forth. As reported by Brueckner and

colleagues (McGrath et al., 2003), the mouse node also contains

non-motile cilia that are located on its periphery and that detect

flow generated by the central, motile cilia (Fig. 4A). The net

result of this detection is a transient spike in intracellular Ca2+

levels in cells to the left of the node, which in turn culminates in

left-side nodal expression (Fig. 4B). In this alternative, ‘‘two-

cilia’’ model, defects in either or both types of cilia would cause

defective left–right axis determination. Consistent with this,

mice null for polycystin-2 –a gene that is mutated in polycystic

kidney disease (Mochizuki et al., 1996) and that also is

expressed by the non-motile, sensory cilia in the node (McGrath

et al., 2003)–exhibit abnormal left–right development in

addition to renal and pancreatic cysts (Pennekamp et al.,

e two populations of cilia located in the node. The centrally located cilia, which

fluid flow that is detected by peripherally located cilia that express polycystin-2

me elevated in cells located to the left of the node, which in turn is proposed to

ess lrd. Genetic mutations (oep, sur, ntl) that inhibit lrd expression cause left –

uppfer’s vesicle (ntl, spt). Once organized into Kuppfer’s vesicle, these cells

regulate asymmetric gene expression (nodal, lefty, pitx2) in the lateral plate

anel C courtesy of H. J. Yost (Essner et al., 2005).

A.F. Ramsdell / Developmental Biology 288 (2005) 1–20 9

2002). Exactly how left–right differences in Ca2+ signaling

regulate nodal asymmetry still needs to be defined, and it has

been suggested that decreased levels on the right side of the

node might function to repress nodal expression on this side

(McGrath et al., 2003). The recent findings that inversin (the

protein encoded by inv) blocks canonical Wnt signaling and

that fluid flow can cause increased levels of inversin in ciliated

cells suggest that important roles for these two factors in ciliary

function and ultimately, regulation of nodal asymmetry, might

also be found (Simons et al., 2005).

As details unfolded with the nodal flow model in the mouse,

efforts weremade to determine whether this mechanism operates

in other vertebrates. ‘‘Nodal’’ cilia were soon discovered in

chick, frog, and zebrafish (Essner et al., 2002). Functional

studies in zebrafish indicate that similar to the nodal cilia models

in mouse, directional fluid flow is a critical aspect of left–right

asymmetry determination in this species (Essner et al., 2005;

Kramer-Zucker et al., 2005). In the zebrafish, a small population

of cells known as dorsal forerunner cells migrate at the leading

edge of the shield (‘‘node’’). Near the end of gastrulation, these

cells involute and form a structure of undefined function called

Kuppfer’s vesicle. Defects in dorsal forerunner cell migration or

interference with Kuppfer’s vesicle formation causes left–right

defects (Amack and Yost, 2004) and ciliated cells in Kuppfer’s

vesicle express LRD, which is necessary for their ability to

generate directional fluid flow in this region (Essner et al., 2005)

(Fig. 4C). As in mouse, it is proposed that unidirectional fluid

flow (in Kuppfer’s vesicle) regulates asymmetric nodal expres-

sion in lateral tissue via directed accumulation of an unknown,

left-side determinant (Essner et al., 2005).

Fig. 5. Models of axis initiation in Xenopus and chick. (A) A summary of process

Beginning with the one-cell stage embryo, cortical rotation affects formation of bo

proteins and physiological processes become restricted in expression and/or activity

mesoderm is regulated by left-side Vg1 signaling, and an opposing, right-side B

functional relationships but do not necessarily imply direct interactions; question ma

for details. (B) A summary of processes and genes that function upstream of left-side

node drives a transient, left-side increase in extracellular calcium, which in turn ac

hedgehog expression, is required for left-side nodal expression; however, the relation

side and right-side pathways, as well as midline influences, reinforce/repress nodal e

but do not necessarily imply direct interactions. See text and references cited withi

In Xenopus, there is abundant evidence that molecular

asymmetry is established at stages of development that precede

detection of ‘‘nodal’’ cilia (Fig. 5A). In the cleavage stage

Xenopus embryo, two molecular asymmetries are present: a

fusicoccin receptor, called the 14-3-3E protein, is expressed in

right, but not left, blastomeres at the 2–4 cell stage (Bunney et

al., 2003), and an H+/K+-ATPase pump is transiently asym-

metrically expressed in the right, but not left, ventral cell of the

4-cell stage embryo (Levin et al., 2002). Whether there is

functional overlap between these two proteins is not clear (14-3-

3 proteins control a variety of H+ pumps and ion channels in

other systems); but, regardless, assays employing inhibitors of

either the 14-3-3E protein or the H+/K+-ATPase pump result in

situs ambiguus as well as abnormal nodal expression patterns

(Bunney et al., 2003; Levin et al., 2002). The asymmetric

expression of the H+/K+-ATPase pump results in differential

left– right pH and voltage gradients; these gradients are

proposed to set up left-side accumulation of small molecule

morphogen(s)–serotonin is a recently identified candidate

(Fukumoto et al., 2005)–via unidirectional movement through

gap junctions, which have been shown to be necessary for

normal left–right patterning in Xenopus (Levin and Mercola,

1998). One possible role for motor proteins such as lrd, inv, and

Kif3-B is that they function as cytoplasmic transporters to

localize proteins and mRNAs involved in establishing left–

right asymmetry, e.g., the 14-3-3E protein and the H+/K+-

ATPase (Levin, 2004). Implicit in this model is that the

cytoskeleton is oriented in alignment with the future left–right

axis—a phenomenon that is possible given that left–right organ

reversals have been linked with disturbances in the microtubule-

es and genes that function upstream of left-side nodal expression in Xenopus.

th the dorsoventral and left – right axes. During subsequent cleavages, various

to the left or right side of the embryo. Nodal expression in the left lateral plate

MP signaling pathway represses right-side nodal expression. Arrows denote

rks indicate possible functional interactions. See text and references cited within

nodal expression in chick. Asymmetric depolarization of cells to the left of the

tivates left-side notch signaling. Notch signaling, in addition to left-side sonic

ship between these two pathways, if any, is not defined. A cascade of both left-

xpression in the lateral plate mesoderm. Arrows denote functional relationships

n for details.

A.F. Ramsdell / Developmental Biology 288 (2005) 1–2010

dependent, cortical rotation of the first cell cycle in Xenopus

(Yost, 1991). Although much remains to be tested, it is clear that

molecular asymmetry in Xenopus is established at least within

the first few cell cycles, well before the formation of the

Organizer and the appearance of ‘‘nodal’’ cilia. Thus, if cilia do

play a role in left–right development in Xenopus, it is very

likely that they participate in relaying left–right patterning cues,

rather than initiating the left–right axis.

As in Xenopus, studies in chick do not indicate a role for

nodal flow in establishing left–right asymmetry beyond the

circumstantial identification of cilia present in Hensen’s node.

In fact, a previous study implicated an earlier source of left–

right asymmetry signals in the embryo by showing that tissues

lateral to the node inductively interact with the node to specify

its left–right identity (Pagan-Westphal and Tabin, 1998).

Although the nature of the signal(s) that induce node left–

right identity is not known, they probably are involved in

regulating pH and voltage gradients, which as in Xenopus, are

required to set up early asymmetry in chick (Fig. 5B). In the

chick, there are distinct boundaries of gap junctional commu-

nication surrounding the node as well as asymmetric activities

in the H+/K+-ATPase pump that result in left-side depolariza-

tion of cells to the left of the node (Levin et al., 2002). The H+/

K+-ATPase driven depolarization causes a transient spike in

left-side extracellular Ca2+ levels (as opposed to intracellular

Ca2+ levels that are regulated by nodal flow), which

preferentially activates left-side notch signaling (Raya et al.,

2004). The latter occurs through modulating the affinity of

notch for its ligands, Dll1 and Srr1, an interaction that is

temporally influenced by locally expressed waves of lunatic

fringe (Przemeck et al., 2003; Raya et al., 2004). Notch

signaling is required for asymmetric nodal expression (Raya et

al., 2004); whether this regulation is direct or through

activation of other factors that are clearly involved in mediating

nodal expression (e.g., activin, sonic hedgehog) remains to be

determined. It is alternatively possible that notch acts in a

parallel pathway to these other factors in controlling nodal

expression. In mouse, notch-mediated induction of nodal

expression is direct, although how this interaction relates to

other processes of left–right axis determination in this

species, particularly nodal flow, is not known (Krebs et al.,

2003; Raya et al., 2003). In zebrafish, both notch signaling

and H+/K+-ATPase activity function in left–right development

prior to the formation of Kuppfer’s vesicle (and hence,

asymmetric nodal expression), suggesting that the role of nodal

cilia in this species is to relay previously established asymmetric

pattern (Kawakami et al., 2005).

In addition to asymmetric H+/K+-ATPase activity in

Xenopus, there is abundant evidence that left–right develop-

ment in the frog also requires TGFh-related signaling mediated

by a pathway comprised of Vg1, ALK4 (a type I Vg1 receptor),

and syndecan-2 (a co-factor that facilitates Vg1-ALK4 signal-

ing) (Chen et al., 2004; Hyatt and Yost, 1998; Kramer and Yost,

2002) (Fig. 5A). Ectopic activation of this pathway, either

through overexpression of mature Vg1 ligand or constitutively

active ALK4, results in a population of embryos exhibiting

predominately right-side or bilateral nodal expression and situs

inversus. Interruption of this pathway, either through interfer-

ence with Vg1-ALK4 signaling or syndecan expression or

function, causes abnormal nodal expression and situs ambiguus

(Chen et al., 2004; Kramer and Yost, 2002; Kramer et al., 2002;

Ramsdell and Yost, 1999). Elegant studies performed with

temporally regulated dominant-negative forms of syndecan-2

pinpointed the necessity of this pathway during gastrulation

(Kramer and Yost, 2002), which is the same developmental

window in which ALK4 expression is detected in the organizer

of the frog embryo (Chen et al., 2005). Consistent with these

findings, syndecan-2 is asymmetrically expressed to the right

side of Hensen’s node (Fukumoto and Levin, 2005), suggesting

conservation of TGFh signaling function downstream of the

initial symmetry-breaking events. Since the discovery of a role

for Vg1 and components of its signaling pathway in mediating

left–right axis determination in Xenopus, it has been unclear

though, how this pathway might be mechanistically linked to

the other processes upstream of nodal expression. The recent

discovery that ALK4 signaling in Xenopus can induce

expression of notch and its ligands, delta-1 and delta-2 (Abe

et al., 2004), raises the possibility that the Vg1-ALK4 pathway

could function to link asymmetric pH and voltage gradients

with notch pathway activation. Because null mutations in the

mouse Vg1 orthologue, Gdf1, illustrate that this pathway also is

necessary for left–right axis determination in this species (Wall

et al., 2000), studies aimed at coupling the Vg1/Gdf1 pathway

with activation of notch signaling are logical and potentially

very interesting directions for future investigation.

Maintenance of asymmetric nodal expression and relay of

left–right positional information to developing tissues

Once established, nodal expression in the perinodal area is

believed to activate left-side nodal expression in the lateral

plate mesoderm, and the latter domain of nodal asymmetry is

maintained by complex interactions among a number of

positive- and negative-acting regulators (Fig. 5B). This

regulatory network is best characterized in chick, where sonic

hedgehog induction of nodal expression initiates an autoregu-

latory loop that also requires the action of BMPs and caronte, a

member of the Cerberus/DAN family of BMP antagonists

(Monsoro-Burq and Le Douarin, 2000, 2001; Piedra and Ros,

2002; Rodriguez Esteban et al., 1999; Yokouchi et al., 1999).

BMP2 expression in the lateral plate mesoderm is symmetric

(Rodriguez Esteban et al., 1999; Yokouchi et al., 1999), and its

role in regulating nodal expression is to induce CFC-cripto, a

co-factor that is necessary for cellular responsiveness to nodal

(Fischer et al., 2002; Fujiwara et al., 2002; Schlange et al.,

2002). Caronte also is induced by BMP2; while the function of

caronte in this cascade is not certain, it might be to limit

(indirectly) the extent of nodal autoinduction (Piedra and Ros,

2002). Two important targets of nodal expression include

Pitx2, a bicoid-related transcription factor that is discussed in

detail below, and the homeobox gene NKX 3.2 (also termed

BapX1. In chick, NKX 3.2 is detected in the left, but not right,

lateral plate mesoderm, where its expression is positively

regulated by nodal BMP2 (Schlange et al., 2002; Schneider et

A.F. Ramsdell / Developmental Biology 288 (2005) 1–20 11

al., 1999). Despite its inverted expression pattern in mouse

(Schneider et al., 1999), null mutations nevertheless are

associated with defects in laterality of the spleen and pancreas

(Hecksher-Sorensen et al., 2004). In addition to nodal, lefty-1

and lefty-2 are two other TGFh family members which are

involved in relay of left–right pattern and which are expressed

in the left half of the prospective floor plate, and the left lateral

plate mesoderm, respectively (Bisgrove et al., 1999; Meno et

al., 1996, 1997, 1998). Both lefty proteins function as nodal

antagonists to prevent the spread of left-side signals to the

opposite side of the embryo (Branford and Yost, 2004; Cheng

et al., 2000; Meno et al., 1998; Thisse and Thisse, 1999). As

observed for nodal, lefty-2 expression is similarly altered in iv/

iv and inv/inv mice, and a number of experimental perturba-

tions of nodal activity also cause abnormal lefty-1 and lefty-2

expression patterns (Meno et al., 1996, 1997). On the right side

of the node, a series of signaling pathways is necessary for

repression of right-side nodal expression and for induction of

cSnr, a transcription factor that is normally restricted to the

right lateral plate mesoderm (Isaac et al., 1997). Beginning

with right-side activin signaling, which is necessary for BMP4

expression, BMP4 in turn induces FGF8 and PCL2 expression,

the latter which functions to represses right-side sonic

hedgehog (Boettger et al., 1999; Wang et al., 2004). BMP2

signaling also is active on the right side of the embryo, where it

cooperatively functions with the FGF8 pathway to induce cSnr

expression (Boettger et al., 1999).

The importance of BMP, FGF, and nodal antagonists in

maintaining left-side specific nodal expression is suggested by

functionality of these pathways in other vertebrate species. In

mouse, BMP4 is required for left-side nodal expression

(Fujiwara et al., 2002) and null mutations in Cerl-2, which

encodes a novel nodal antagonist, result in situs ambiguus

(Marques et al., 2004). In zebrafish, functional inhibition of the

nodal antagonist charon leads to bilateral nodal-related gene

expression and reversed heart looping (Hashimoto et al., 2004).

Null mutations in mouse ACRVRI (also termed ALK2), which

encodes a type I BMP receptor, result in situs ambiguus and

bilateral nodal expression, the latter probably due to loss of

lefty-1 expression in the midline (Kishigami et al., 2004). Gain-

and loss-of-function studies of ALK2 in Xenopus also indicate

that this pathway is necessary for normal left–right develop-

ment of the heart and visceral organs (Ramsdell and Yost,

1999). It should be emphasized that even though many of the

same signaling pathways that are active in chick, frog, and fish

are employed in mouse, this species differs from other

vertebrates in that some of its left–right signaling molecules

either act on the contralateral side of the embryo (e.g., FGF8 is

a left-side determinant in mouse), or are expressed in a

symmetric fashion while nevertheless being involved in left–

right patterning (e.g., sonic hedgehog) (Meyers and Martin,

1999). Other genes that affect nodal expression and heart and

visceral organ asymmetries in mouse include rotatin (Faisst et

al., 2002), cited2 (Bamforth et al., 2004), PKD2 (Pennekamp

et al., 2002), and BPI/PLUNC (Hou et al., 2004).

In conjunction with left- and right-side regulatory networks,

gene expression in the midline itself is important in regulating

asymmetric nodal expression. In addition to lefty-1, another

important midline gene is Zic3, a member of the GLI

transcription factor family that is normally present in midline

mesoderm and that is induced by activin/Vg1 signaling in

Xenopus (Kitaguchi et al., 2000, 2002; Nagai et al., 1997;

Purandare et al., 2002). Misexpression of either wild type or

deletion mutant forms of Zic3 in Xenopus embryos causes situs

ambiguus and predominantly bilateral or right-side nodal

expression (Kitaguchi et al., 2000). In mouse, Zic3 null

mutations result in a failure to maintain asymmetric nodal

expression in the node and situs ambiguus (Purandare et al.,

2002). Zic3 is thought to function in left–right development by

controlling formation of midline tissues such as the notochord.

One of the main consequences of maintaining a midline

barrier, via midline gene expression such as Zic3 or lefty-1, is

that cell populations positioned on opposite sides of the

midline propagate left–right patterning information indepen-

dently of one another. Clues to how the midline operates on a

cellular level to prevent left–right signals from reaching the

contralateral side come from notable studies in which cell death

in the primitive streak (Kelly et al., 2002) and a novel

population of ventral foregut cells derived from Hensen’s node

(Kirby et al., 2003) are shown to contribute to left–right

patterning activity. Further investigation of these two newly

identified midline cell populations and how they interface with

left–right signaling cascades should greatly increase our

understanding of the specific mechanisms employed at the

embryonic midline.

It should be noted that concomitant with the development of

left–right asymmetry, there are paired tissues located to either

side of the midline (e.g., presomitic mesoderm) that must

undergo symmetric morphogenesis. Exactly how this coordina-

tion occurs, and particularly how these tissues remain refractory

in response to opposing left- and right-side lateralizing

influences, has been a longstanding question. A series of very

recent studies demonstrate that, in chick, mouse, and zebrafish,

somite formation is labile to left–right signaling pathways, but

that under normal conditions, retinoic acid mediates synchro-

nized left and right side somitogenesis by preventing presomitic

mesoderm from responding to left–right asymmetric signaling

(Kawakami et al., 2005; Vermot et al., 2005; Vermot and

Pourquie, 2005).

‘‘Translating’’ left–right axis information into anatomical

asymmetry

The nodal Y Pitx2c pathway

The connection between asymmetric nodal expression in

the lateral plate mesoderm and cardiac asymmetry is the

regulation of downstream genes that have direct roles in heart

development. One such gene that is induced to be expressed in

the left lateral plate mesoderm by nodal and that is required for

normal heart development is Pitx2c (Fig. 6). Pitx2c is one of

three Pitx2 isoforms (Pitx2a, Pitx2b, and Pitx2c) that belong to

the bicoid group of paired homeobox transcription factors.

Pitx2 is a homolog of the human RIEG gene, whose mutation

Fig. 6. Conserved, left-side Pitx2c expression in the lateral plate mesoderm. Whole-mount in situ hybridization reveals left-side Pitx2c expression (arrowhead) in the

lateral plate mesoderm in mouse (A, ventral view), chick (B), tadpole (anterior to left and dorsal to top), and zebrafish embryo (D). Panel A courtesy of P. Tam

(Davidson et al., 1999), panels B and C courtesy of M. Blum (Campione et al., 1999).

A.F. Ramsdell / Developmental Biology 288 (2005) 1–2012

results in Rieger syndrome, a haploinsufficiency condition

characterized by dental hypoplasia, craniofacial dysmorphism,

umbilical stump protrusions, and ocular anomalies (Amendt et

al., 2000). Less frequently, defects in cardiac, limb, and

pituitary development are observed (Amendt et al., 2000).

Once induced by nodal (Logan et al., 1998; Long et al.,

2003; Piedra et al., 1998; Shiratori et al., 2001; Yoshioka et al.,

1998), Pitx2c expression in the left lateral plate mesoderm is

maintained by nkx2.5 (Shiratori et al., 2001). Similar to nodal,

a combination of right side only, bilateral, and absent

expression Pitx2c patterns is observed in experimentally

induced cases of situs ambiguus in mouse, chick, and frog

(Bamforth et al., 2004; Bisgrove et al., 2000; Boettger et al.,

1999; Branford et al., 2000; Chen et al., 2004; Piedra et al.,

1998; Schlange et al., 2001; St Amand et al., 1998; Yoshioka et

al., 1998). The observation that null mutations in many

different left–right axis genes result in a similar defective

left–right phenotype can be attributed to the downstream

effects of these genes on the nodal Y Pitx2c pathway. For

example, null mutations that result in absent Pitx2c expression

include cryptic, FGF8, gdf1, and nodal and the predominant

phenotype in these mice is right isomerism. Null mutations that

cause bilateral Pitx2c expression include lefty-2 and ACRIa/

ALK2 and these mice predominantly exhibit left isomerism.

Midline genes that are necessary for left–right development

also converge at the nodal Y Pitx2c pathway; shh and lefty-1

are two examples that cause abnormal nodal (and hence Pitx2c)

expression in null mutants. Mice that are null for all three Pitx2

isoforms (Pitx2abc) exhibit defective body wall closure, gut

malrotation, right pulmonary isomerism, and the spectrum of

cardiac defects that are usually associated with heterotaxy,

including DORV, TGA, common AV canal, ASD/VSD, and

anomalous venous return (Gage et al., 1999; Kitamura et al.,

1999; Liu et al., 2001, 2002). Right atrial isomerism, PTA, and

aortic arch anomalies such as right aortic arch or double aortic

arch are also sometimes present. Interestingly, despite the

numerous cardiac defects caused by Pitx2abc null mutation, all

of these can be rescued with hypomorphic Pitx2c alleles except

right atrial isomerism, which requires a higher dosage of Pitx2c

expression/activity (Liu et al., 2001). Consistent with these

findings, analysis of Pitx2c function in Xenopus has shown that

Pitx2c antisense oligonucleotides cause OFT, AVC, and atrial

septation defects (Dagle et al., 2003).

Pitx2c is implicated in regulating multiple aspects of cardiac

left–right development

Unlike nodal, Pitx2c is expressed during stages of heart

tube formation when it is detected exclusively in the left, but

not right, side of the developing heart (Campione et al., 1999;

Logan et al., 1998; Ryan et al., 1998; St Amand et al., 1998;

Yoshioka et al., 1998) (Fig. 7). Cardiac Pitx2c expression

persists through looping stages, where it is present in the entire

left side of the heart, spanning from the left atrium to the most

distal part of the outflow tract, reaching into the left side of the

SANF and the left pharyngeal arch mesenchyme (Liu et al.,

2002) (Fig. 7). In addition, Pitx2c plus two other Pitx2

isoforms (Pitx2a and Pitx2b) are expressed in migrating

cardiac neural crest (Hamblet et al., 2002; Kioussi et al.,

2002). During later stages, Pitx2c expression is observed in the

left atrioventricular canal, left atrium, interatrial septum, and

the left caval vein (Franco and Campione, 2003).

Initial studies showed that right side ectopic Pitx2c expres-

sion in chick and Xenopus causes reversed heart looping

(Campione et al., 1999; Logan et al., 1998; Piedra et al., 1998;

Ryan et al., 1998; Yoshioka et al., 1998). These findings,

coupled with the observations that Pitx2c is expressed by

components of other organs that undergo looping, suggested that

Pitx2c mediates looping morphogenesis (Campione et al., 1999;

Logan et al., 1998; Ryan et al., 1998). However, Pitx2abc and

Pitx2c null mutant mice do not exhibit reversed cardiac looping

(Gage et al., 1999; Kitamura et al., 1999), suggesting that Pitx2c

is dispensable for this aspect of the looping process. In Xenopus,

Pitx2c loss-of-function causes abnormal (though not necessarily

reversed) shifting of the OFT, confirming that although Pitx2c

can mediate later aspects of looping, it is not involved in

controlling initial directionality of looping. Errors in the leftward

shifting and/or rotation of the OFT are causatively linked with

DORV, TGA, and VSDs; therefore, the abnormal OFT looping

that is associated with impaired Pitx2c function could account

for these types of CHDs.

The mechanism by which Pitx2c regulates OFT looping is

undefined. During looping, the outlet region of the heart

undergoes lengthwise growth due to the addition of myocar-

dium derived from the SANF (Brand, 2003; Mjaatvedt et al.,

2001; Waldo et al., 2001). Thus, one possible function of

Pitx2c in OFT looping is that its expression in the SANF may

Fig. 7. Conserved, left-side Pitx2c expression during early stages of heart

development. Whole-mount in situ hybridization reveals left-side Pitx2c

expression (arrows) during initial formation of the heart tube in chick (A)

and in mouse (B). As the heart tube continues to elongate, Pitx2c expression

(arrows) is maintained in the left half of the chick (C) and mouse (D) heart.

During looping, Pitx2c expression is detected in the entire left half of the chick

heart, including the aortic sac (AS, arrow) (E) and in the entire left half of the

mouse heart (F), as seen along the inner curvature (asterisk). All panels ventral

views, with left (L) and right (R) indicated. Figures courtesy of M. Campione

and M. Blum (Campione et al., 2001).

A.F. Ramsdell / Developmental Biology 288 (2005) 1–20 13

somehow be involved in the recruitment of cells from the

SANF to the conotruncus. Alternatively, Pitx2c might mediate

OFT looping by affecting the cardiac neural crest. In Pitx2abc

null mice, the population of neural crest cells that contributes to

the heart appears decreased (Kioussi et al., 2002; Liu et al.,

2002). Although Pitx2 does not directly regulate migration of

neural crest cells, null mutations in Pitx2abc do reduce the

number of neural crest cells that accumulate in the OFT by

inhibiting proliferative expansion of this cell population

(Kioussi et al., 2002). This inhibition requires the upstream

activation a wnt/Dvl/h-catenin pathway that is responsible for

inducing Pitx2 expression (Kioussi et al., 2002). In other

mouse mutants (e.g., FGF8�/�, BMP4�/�), decreased

numbers of neural crest cells in the OFT have been associated

with defective OFT looping (Abu-Issa et al., 2002; Liu et al.,

2004). It has been proposed that the link between neural crest

cells and altered OFT looping is that neural crest cells

somehow regulate the factors that mediate the addition of

SANF cells to the outflow myocardium to the heart (Farrell et

al., 1999, 2001; Yelbuz et al., 2002, 2003). In turn, it has been

proposed that when cardiac neural crest cells are ablated, the

impaired recruitment of SANF cells to the OFT causes the OFT

to develop a shortened stature that impedes its ability to

normally loop (Farrell et al., 2001; Yelbuz et al., 2002, 2003).

Whether this model is correct is somewhat controversial

because it is difficult to reconcile that a shortened OFT can

simultaneously occur with PTA, a defect that is unequivocally

and causally linked to neural crest cell ablation. Thus, if the

function of Pitx2 in the neural crest is required for normal OFT

looping, it is alternatively possible that the neural crest plays a

role unrelated to SANF cell recruitment. Besides Pitx2, other

left–right asymmetry genes–ALK2, ALK4, and FGF8 –are

expressed in the cardiac neural crest, suggesting that neural

crest might be subject to left–right specification prior to or

during its migration (Abu-Issa et al., 2002; Chen et al., 2005;

Kaartinen et al., 2004; Kishigami et al., 2004). However,

whether expression of Pitx2 or other laterality genes by cardiac

neural crest cells is induced as part of the left–right signaling

cascade that specifies the midline remains to be determined.

With the availability of neural crest mutants in mouse and

zebrafish, as well as the ease of experimentally ablating neural

crest in chick and Xenopus, the different potential roles for

neural crest cells in cardiac looping could be tested using a

variety of molecular, embryological, and genetic approaches.

In addition to OFT looping, Pitx2c might play a role in

regulating septation in the IFT and OFT (Fig. 8). The

myocardium in the AVC exhibits left, but not right, side Pitx2c

expression (Campione et al., 2001), which is required for

migration of cells into cardiac cushions (Liu et al., 2002). The

origin of the Pitx2c expressing cells within the cushions is

probably the AV myocardium, which together with epicardial-

derived cells, is required to complete septation and valve

formation in the heart (Lamers and Moorman, 2002). Pitx2 also

appears to be required for migration of a small subset of

myocardial cells from the left to the right side of the OFT (Liu

et al., 2002); although the significance of this migratory

process is unknown, it does suggest that Pitx2 may be involved

in conferring cellular and molecular asymmetry to this portion

of the heart. With respect to the OFT, Pitx2abc null mice

exhibit PTA, a condition in which the conotruncus and AS are

not divided properly into the base of the aorta and pulmonary

artery by the APS (Kitamura et al., 1999; Lu et al., 1999; Liu et

al., 2001). This suggests that Pitx2 is necessary for septation of

the OFT. Finally, the expression of Pitx2c in cells that will

form the IAS suggests it too plays a direct role in atrial

septation, perhaps by affecting competency of cells to

transform to mesenchyme, as appears to occur in the AVC

region. Consistent with this idea, IAS defects are frequently

present in Pitx2c null mice (Liu et al., 2001).

Pitx2c and aortic arch anomalies

Aortic arch anomalies are frequently present in heterotaxy

patients, indicating that patterned regression and persistence of

vessels are governed by the left–right axis (Bowers et al., 1996).

Fig. 8. Conserved, left-side Pitx2c expression during later stages of heart

development. Whole-mount in situ hybridization reveals left-side Pitx2c

expression (arrows) during looping and septation stages of the heart in chick

and mouse. Ventral view of a HH stage 16 chick heart (A) and left lateral view

of an E10.0 mouse heart (B) showing expression in the left atrium (LA),

atrioventricular canal (AVC), ventricle (VEN), outflow tract (OFT), and aortic

sac (AS). Dorsal view of a HH stage 23 chick heart (C) and ventral view of an

E10.0 mouse heart (D) showing lack of Pitx2c expression in the right atrium

(RA), the right side of the OFT, and the original right side of the ventricular

chambers (positioned dorsally at this stage due to looping). Pitx2c is expressed

in the LA and the interatrial septum (IAS) as shown in the chick heart (E) and in

a cross-section of an E10.0 mouse heart (F). Figures courtesy of M. Campione

and M. Blum (Campione et al., 2001).

A.F. Ramsdell / Developmental Biology 288 (2005) 1–2014

However, virtually nothing is known about how this occurs, or

for that matter, even if vessels (including the endocardium of the

heart) truly possess any type of cellular or molecular asymmetry.

Despite their mesodermal origin, it is possible that blood vessels

themselves are not lateralized, but rather that they develop

asymmetric pattern in response to biomechanical feedback from

blood flow. An alternative, though not necessarily exclusive

possibility, is that vessels develop asymmetric pattern based on

signals derived from the tissues that induce their formation.

Consistent with this possibility, the assortment of pulmonary and

systemic venous anomalies that are frequently found in cases of

isomerism suggest that vascular endothelial cells are competent

to respond to the same set of left–right patterning signals,

regardless of which side of the midline they are located (Bowers

et al., 1996; Ruscazio et al., 1998). Moreover, vessel persistence

during vascular remodeling involves recruitment of cells that

interact with the endothelium to promote its survival and

differentiation. Failure to recruit support cells or disrupted

interaction between support cells and the endothelium would

result in vessel regression. Therefore, with respect to asymmetric

AA remodeling, laterality gene expression in pharyngeal arch

mesenchyme may be important. Pitx2c is expressed in the left,

but not right, pharyngeal arch mesenchyme (Liu et al., 2002),

and it has been suggested that Pitx2c null mutations could impair

recruitment, maintenance, or cross-talk between supporting cells

and endothelial cells in this region. Because Pitx2c is expressed

only by left-side AAs, the absence of Pitx2c expression would

be predicted to result in abnormal patterning on this side,

consistent with the right-side aortic arch, right-side ductus

arteriosus, and left inominate arteries that frequently occur in

Pitx2c null mice.

Genes in addition to Pitx2 that regulate cardiac left–right

morphogenesis

In addition to Pitx2c, BMP4 and flectin are two other genes

that play important roles in cardiac left–right morphogenesis. In

zebrafish and Xenopus, BMP4 is asymmetrically expressed in

the nascent heart tube (Chen et al., 1997), and transgenic studies

in Xenopus using cardiac-targeted inhibitors of BMP signaling

demonstrate that BMP4 is required for looping morphogenesis,

independent of Pitx2c function (Breckenridge et al., 2001).

Flectin is an extracellular matrix protein that is asymmetrically

expressed in chick, with predominant expression observed in the

left heart fields (primary and anterior), the left dorsal mesocar-

dium, and the left side of the heart tube during looping stages

(Linask et al., 2003; Tsuda et al., 1996, 1998). Ectopic Pitx2c

expression causes a rightward shift in flectin expression in the

heart fields and the dorsal mesocardium, coincident with

reversed or absent heart looping (Linask et al., 2002). Treatment

of embryos with CFC antisense oligonucleotides, which

likewise cause reversed or absent looping, also inverts the

asymmetry of flectin expression (Linask et al., 2003). However,

this treatment does not alter normal Pitx2c expression, indicat-

ing either that flectin is a downstream target of Pitx2c that is

directly mediating looping, or that flectin acts in a pathway

parallel to the nodal Y Pitx2 pathway. In addition to the role of

flectin in looping, these studies also showed that Pitx2c could

alter the position of the developing foregut relative to the

embryonic midline. As shown in these studies and in the

previously mentioned study of node-derived ventral midline

cells (Kirby et al., 2003), this finding is significant because the

orientation of heart looping is somehow regulated by cells of the

ventral floor of the foregut. Given that CFC antisense treatment

also causes a rightward shift of the ventral foregut relative to the

midline, this suggests that the ventral foregut may be a primary

tissue target in the relay of left–right axis signals.

Some ‘‘isolated’’ CHDs might actually be subtle laterality

defects

Although the majority of human laterality defects are

thought to be sporadic, it is clear that left–right defects also

A.F. Ramsdell / Developmental Biology 288 (2005) 1–20 15

can arise from environmental perturbations and from heritable

modes of transmission (Kuehl and Loffredo, 2002). Clinical

analyses of familial cases indicate three modes of inheritance

for laterality defects: X-linked, autosomal dominant (usually

with incomplete penetrance), and autosomal recessive (Bel-

mont et al., 2004; Kosaki and Casey, 1998). In many cases,

specific mutations have been mapped and include ZIC3,

ACVR2B, CRYPTIC/CFC1, LEFTYA, and NKX2.5 (Bamford

et al., 2000; Belmont et al., 2004; Gebbia et al., 1997; Kosaki

et al., 1999a,b). Because these genes also have been identified

or implicated in animal models of heterotaxy, this confirms that

our current models are (and presumably will continue to be) of

great value in understanding the etiology of human laterality

disease.

Observations made in animal models of heterotaxy on the

role of the midline in left–right development (reviewed by

Yost, 1998) have prompted investigators to look for clinical

correlations between human midline defects and laterality

disturbances (Goldstein et al., 1998; Morelli et al., 2001).

Intriguingly, in one of these studies, a significant association

was observed not only between laterality defects and midline

defects, but also between laterality defects and ‘‘isolated’’

cardiac defects (Morelli et al., 2001). The latter association

has been observed in both animal and human studies, leading

to the suggestion that some instances of isolated cardiac

defects may actually represent subtle, ‘‘forme fruste’’ laterality

defects (Goldmuntz et al., 2002). For example, in the cited-2

null mouse, which lacks Pitx2c expression in the left lateral

plate mesoderm and in the heart, embryos develop the cohort

of CHDs that is typical of heterotaxy (i.e., defects in OFT,

septal, and AA remodeling) in addition to abnormal body

turning, right atrial and pulmonary isomerism, and hypoplastic

spleen (Bamforth et al., 2004; Weninger et al., 2005).

Comparison of these mice with cited-2 null mutations made

on a mixed genetic background gave surprising results

(Bamforth et al., 2004). In the latter group, Pitx2c expression

was present in the left lateral plate mesoderm but absent or

reduced specifically in the OFT of the heart. These mice

exhibited DORV and VSD but laterality of other organs was

not affected. These results suggest that there are genetic

modifiers that can influence the cited-2 null phenotype by

directly or indirectly regulating Pitx2c expression. Moreover,

the occurrence of CHDs in the mixed background mutant

embryos suggests that some CHDs actually may result from

perturbations of left–right axis genes, even in instances of

otherwise normal body situs. As a direct test of this

hypothesis in a clinical setting, a population of patients with

TGA (but no overt laterality defects) was examined for

mutations in CFC1, a gene that is expressed in the OFT of the

embryonic mouse heart (Dono et al., 1993) and that is

required for cellular responsiveness to nodal signaling in the

lateral plate mesoderm, probably by functioning as a co-

receptor for nodal (Fischer et al., 2002; Fujiwara et al., 2002;

Schlange et al., 2002). A subset of patients afflicted with TGA

(but not laterality defects) was found to indeed harbor CFC1

mutations, indicating for the first time, a common genetic

etiology between human TGA and laterality disease (Gold-

muntz et al., 2002). Because many laterality genes identified in

animal models are known also to participate in processes of

cardiac morphogenesis, it is predicted that many more

seemingly ‘‘isolated’’ cardiac defects will be found in situs

solitus individuals that represent a mutation or other perturba-

tion in left–right axis gene(s).

Prospects

The past decade has brought immense progress in decon-

structing the complexities of left–right axis determination and

its relationship to cardiac development while at the same time

illuminating areas in which further investigation is much needed.

Studies in mouse, chick, frog, and zebrafish reveal that

establishing the left–right axis involves a number of intriguing

physiological processes that extend beyond the conventionally

studied, growth factor-mediated signaling pathways. Whether

processes of ‘‘nodal’’ flow, gap junctional communication, and

membrane voltage potential are unique for axis determination in

each species is an important but unresolved issue. Relatively

more is known about the role of the midline in left–right

patterning, but despite unraveling many of the complicated

genetic interactions, the specifics of which cell types are

engaged in relay of left–right signals are less well defined.

With regard to asymmetric morphogenesis, work on Pitx2c has

accelerated our understanding of the types of cardiac defects that

can arise from impaired function of a single laterality gene. Yet,

how this one gene actually regulates the three different endpoints

of cardiac left–right patterning (looping, regression/persistence,

and lateralization) is by no means definitively answered, and is

limited, in part, by our current understanding of fundamental,

morphogenetic processes that drive normal cardiac develop-

ment. Finally, there is exciting interplay between clinical

observations and experimental observations in animal models

of laterality disease, indicating that some seemingly ‘‘isolated’’

congenital cardiac defects might actually arise from disturbances

in left–right signaling pathway(s). Future progress in this latter

area is anticipated to be especially significant, as elucidating

common genetic etiologies will greatly advance our understand-

ing of the causes of many types of deleterious and life-

threatening congenital heart malformations.

Acknowledgments

Thanks to colleagues near and far for enjoyable discussions

of cardiac left–right development and to R. Markwald, D.

Bernanke, and C. Drake for comments on the manuscript.

Special thanks to Sue Tjepkema-Burrows for drawing the

schematic figures. Cardiac asymmetry research in the author’s

lab is supported by HL73270 (to A.F.R.) and a SC COBRE for

Cardiovascular Disease P20-RR-1634 (to Roger R. Markwald).

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