Cardiogenesis and the Regulation of Cardiac-Specific Gene Expression

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Cardiogenesis and the Regulation of Cardiac-Specific

Gene Expression

Jau-Nian Chen, PhDa , Douglas B. Cowan, PhD b, John D. Mably, PhDc,*

a University of California Los Angeles, Los Angeles, CA, USA b

Children’s Hospital Boston, Boston, MA, USAc Massachusetts General Hospital, Boston, MA, USA

Development and maturation of the embryonic

vertebrate heart is an exquisitely conducted ensemble

of cell movements, interactions, and morphologic

transformations. The genetic orchestration of these

events is precise and can be reduced to dissonance by

the loss of a single component in this process. Proper

heart morphogenesis is important to normal embry-

onic development because the heart is the first organ

formed. All subsequent events depend on the ability

of the heart to supply oxygen and nutrients to fulfill

the metabolic requirements of the organism. Abnor-

malities in the formation of the heart often lead to

abnormal function and embryonic lethality or may

manifest later in life, causing severe health issues.

Cardiac defects are among the most common birth

defects, estimated at an incidence of 6 in 1000 live

births, with an even higher frequency in spontane-

ously aborted pregnancies [1]. Much of our under-

standing of the mechanisms and pathways regulating

cardiogenesis evolved from studies in model systems,

notably the mouse, chick, fly, frog, and zebrafish.

This article outlines the molecular events and mecha-nisms regulating heart formation, focusing on

recently identified members of the cardiogenic reper-

toire [2–5].

Early morphogenesis

Adult vertebrate hearts may vary greatly in their

overall structures, but the morphogenic processes that

shape the embryonic hearts of vertebrate species are

shared. In brief, vertebrate heart formation begins

when a bilaterally symmetric population of meso-

dermal cells in the anterior lateral plate becomes

committed to a cardiac fate in response to inductive

signals from the adjacent endoderm [6]. Migration of

these cardiogenic cells to the midline of the embryo

results in the formation of a linear heart tube (the

primitive heart tube). Cardiomyocytes adopt an atrial

or a ventricular cell fate during differentiation of the

primitive heart tube along the anterior-posterior axis,

although studies in zebrafish suggest this specifica-

tion occurs earlier, prior even to heart tube formation

[7]. The atrium and ventricle then undergo a right-

ward looping (cardiac looping) that is essential for

alignment of the inflow and outflow tracts and for

orienting the atrial and ventricular chambers [8]. Data

accumulated from studies over the last decadesuggest that in addition to these morphogenic events,

the genetic circuits critical for heart development also

are conserved across species [9].

This complex crosstalk between tissues is essen-

tial for cardiogenesis, in part contingent on the ex-

pression of an extensive assemblage of transcription

factors. Through precise temporal and spatial regu-

lation, the expression of these factors leads uncom-

mitted cells to enter the cardiac lineage. In the fruit

fly, Drosophila melanogaster , the activity of the

homeobox gene tinman is required for the formation

1551-7136/05/$ – see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.hfc.2005.03.002 heartfailure.theclinics.com

* Corresponding author. Cardiovascular Research Cen-

ter, Massachusetts General Hospital and the Department

of Medicine, Harvard Medical School, 149 13th Street,

Boston, MA 02129.

E-mail address: [email protected] (J.D. Mably).

Heart Failure Clin 1 (2005) 157 – 170

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of the primitive heart. Loss of function of tinman

blocks the formation of cardiac tissues in developing

fly embryos [10,11]. To confine tinman expression in

the cardiac lineage and to maintain its expression at

critical level requires signals from adjacent tissues.

Endoderm-derived dpp expression (the Drosophilaortholog of the bone morphogenic protein [bmp]

family) promotes formation of the cardiac lineage

[12], whereas wingless proteins (orthologs of the

vertebrate wnt proteins) have positive and negative

roles in this process [13,14]. In vertebrates, this

pathway is conserved, as shown in the chick, where

BMP2 can induce Nkx2.5 (the vertebrate ortholog of

tinman) in the anterior mesoderm. Studies in chick

and frog have revealed further that wnt3a and wnt8c,

signaling through the canonical wnt pathway, sup-

press the cardiac lineage (and promote the differen-tiation of hematopoeitic lineage) [15–17], whereas

wnt11 promotes cardiac differentiation through the

noncanonical wnt -signaling pathway [18].

In vertebrates, Nkx2.5 is expressed in cardiac

muscle cells from the onset of embryonic heart

formation until adulthood. Although overexpression

of Nkx2.5 induces an expansion of cardiac tissues in

the frog and zebrafish [19,20], the role of Nkx2.5 in

establishment of cardiac lineage in vertebrates is not

as clear as the role of tinman in the fly. The fact that

mice lacking Nkx2.5 develop a heart suggests that

Nkx2.5 is dispensable for establishment of the cardiaclineage [21]. Although it is possible that Nkx2.5

serves a different function than tinman in the mouse,

it is more likely that other genes have redundant

functions in the specification of cardiac cell fate. This

notion is supported by the observations that other

Nkx genes, such as Nkx2.3, Nkx2.6 , and Nkx2.7 , are

expressed in cardiac tissues and that overexpression

of dominant negative forms of either Nkx2.5 and

Nkx2.3 in the frog blocks cardiogenesis [22,23].

In contrast to skeletal muscle, where a single

transcription factor, MyoD, is sufficient to activate thefull program of muscle differentiation, tinman/ Nkx2.5

cooperates with other transcription factors to com-

plete the program of cardiac gene expression. For

example, Nkx2.5 and GATA4 have synergistic effects

on the activation of downstream cardiac genes in fly

embryos and in cultured mammalian cells [24–26].

In addition, myocardin and serum response factor

(SRF) cooperate with Nkx2.5 in controlling the

expression of muscle structure genes [27].

Migration of the bilateral cardiac precursors

In fly and vertebrates, the cardiac precursors

initially are positioned bilaterally in the embryo. As

development proceeds, the bilaterally positioned car-

diac mesoderm migrates to and fuses at the midline to

form the primitive heart tube. In the fly, fgf signaling

has an instrumental role in guiding cardiac precursors

to the midline [28–30]. Although the role of fgf

in the migration of precardiac cells to the midlineneeds to be explored further in the vertebrate, genetic

studies in the zebrafish demonstrate that Fgf8 is

expressed in and required for development of the

zebrafish heart precursors, especially at the onset of

cardiac gene expression [31]. Because studies in

zebrafish have been instrumental in defining many

features of early cardiogenesis, this article discusses

zebrafish heart development and several of the

mutants identified from the large-scale ethyl nitro-

sourea mutagenesis screens.

The zebrafish heart is a primitive vertebrate heart consisting of two major cell types (myocardium and

endocardium) and two chambers (atrium and ven-

tricle), and its development is fast compared with

other vertebrate model organisms. The bilateral

cardiac primordia visualized at 14 somites (approx-

imately 16 hours postfertilization [hpf]) by Nkx2.5 in

situ staining (Fig. 1) fuse at the midline in the

zebrafish by 20 somites (19 hpf). The fused heart

soon grows into a long tubular structure, known as

the primitive heart tube, and can ensure circulation

throughout the body by 24 hpf. Within 2 days of de-

velopment, cardiomyocytes assume chamber-specificfates and cardiac looping is completed, placing the

ventricle to the right of the atrium [32].

The mechanisms underlying the fusion of the

cardiac mesoderm have been well described through

analysis of zebrafish mutations that block this pro-

cess and lead to the development of two hearts in

the affected embryos, a phenotype known as cardia

bifida [33–35]. Positional cloning of the natter

(nat ) and miles apart (mil ) mutations demonstrated

that fibronectin (the gene defective in nat ) [36] and

sphingosine-1-phosphate receptor (mil ) [37] areindispensable for the fusion of cardiac mesoderm

and revealed a significant role for the extracellular

matrix (ECM) in precardiac cell migration. The

essential role of endodermal signals in the migration

of the cardiac precursors was revealed through the

identification of the gene defects associated with the

bonnie and clyde (bon), casanova (cas), hands off

(han), and faust ( fau) mutations. bon encodes a Mix

family homeobox gene that regulates the generation

of endoderm precursors [38], whereas cas encodes a

sox -related protein that is sufficient to induce

endoderm differentiation [39]. The fau gene encodesthe zebrafish homolog of gata5 [40] and han encodes

dHAND ( Hand2) [41], each of which is expressed in

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endoderm and cardiomyocytes. Loss of function of bon, cas, han, and fau not only prevents cardiac

precursors from fusing at the midline, but also affects

the number and the differentiation of cardiac pre-

cursors, indicating an instrumental role of endoderm

in early cardiac patterning [40,42]. Similarly, mice

lacking GATA4 exhibit cardia bifida in addition to a

reduction of cardiomyocytes [43,44].

The association of cardia bifida with defects in

cardiac patterning and cardiomyocyte differentiation

had led to a model in which the fusion of the bilateral

cardiac primordia at the midline is essential for subsequent cardiac development and morphogenesis.

This notion has been challenged recently by a study

on mice lacking Foxp4, however [45]. Foxp4 is a

member of the Fox gene family whose expression is

abundant in the anterior foregut endoderm but cannot

be detected in the cardiomyoctyes. Foxp4 mutants

exhibit cell death–mediated loss of anterior foregut

mesoderm and cardia bifida, but each cardiac

primordium successfully differentiates into a four-

chambered heart with proper alignment according to

the left-right axis. Thus, although many factors that

regulate fusion of the cardiac primordia also playessential roles in the maturation of the heart, the

processes are not inseparable. These results hint at a

greater level of preprogramming in the bilateral precursors than previously appreciated.

Heart tube formation

In contrast to the extensive studies on early

cardiac patterning, molecular and cellular mecha-

nisms involved in the morphogenesis of the primi-

tive heart tube, which occurs immediately after the

bilateral cardiac primordia fuse at the midline, are

largely unknown. The survival of mammalian em-

bryos depends on proper cardiac function for circu-lation. Therefore, mutations affecting early cardiac

patterning decisions such as primitive heart tube

morphogenesis often cause embryonic lethality at

early developmental stages, making such studies

difficult in traditional models such as the mouse.

Another attribute of the zebrafish that has facilitated

such studies is the ability of embryos to survive

through early development without an active circu-

lation. In particular, two zebrafish mutations affect-

ing primitive heart formation, heart and soul (has)

[46,47] and heart and mind (had ) [48], provide

points of entry to understanding the molecular andcellular mechanisms guiding primitive heart tube

morphogenesis. In has and had mutants, the bi-

Fig. 1. Expression pattern of Nkx2.5 defines the heart field. ( A) Wholemount in situ analysis of Nkx2.5 expression in zebrafish

embryos stains the two bilateral heart fields at 14 somites (approximately 16 hpf ), indicated by the two arrowheads. Arrow

indicates midline expression of no tail (ntl , zebrafish T brachyury homolog) [149]. ( B) By 20 somites (approximately 19 hpf)

these two populations of cells fuse at the midline (arrowhead ). Arrow indicates staining by midline marker ntl . (C ) The fused

heart field develops into the primitive heart tube by 24 hpf, highlighted by Nkx2.5 in situ staining, and the embryonic onset of

circulation begins with contraction of the heart at this time. ( D) By 2 days of development, cardiomyocytes have assumed

chamber-specific fates and cardiac looping is complet ed, placing the ventricle to the right of the atrium [32]. Both chambers are

visible by staining with the myosin antibody MF20 [7].

cardiogenesis & gene expression 159



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lateral myocardial precursors migrate to the midline

normally, but myocardial precursors of both mutants

remain at the midline as a clump of cells. As a

result, the had mutants develop a small heart with

severe defects in the differentiation and contractility

of cardiomyocytes [48], and the has mutants have amispatterned heart with the ventricular cells encom-

passed by a single layer of atrial cells [46,47].

Identification of the genetic lesions responsible

for these defects has revealed roles for the genes

encoding atypical protein kinase C l (aPKC l, has)

[46,47] and the a1B1 isoform of Na,K-ATPase (had )

[48]. The identification of these genes in the

regulation of primitive heart tube formation suggests

that changes in myocardial cellular architecture

during heart development can have striking effects

on this process. aPKC l and Na,K-ATPase are po-larized proteins. In the chick, Na,K-ATPase a1 as-

sumes a lateral position in precardiomyocytes [49],

and mislocalization of Na,K-ATPase has been asso-

ciated with Type 4 Long-QT cardiac arrhythmia in

humans [50], indicating the importance of Na,K-

ATPase polarity in maintaining cardiac function. On

the other hand, aPKC is part of the evolutionarily

conserved Par3/Par6/aPKC protein complex, a key

player in the establishment of cell polarity. In the

zebrafish, the medial myocardial precursors transform

from a cuboidal to columnar shape upon the fusion of

the bilateral primordia, and this change in cell shapeis accompanied by the transition of aPKC cellular

localization from cell-cell contacts to the apicolateral

domain [36]. Overexpression of a dominant negative

form of aPKC in MDCK cells abolishes polarized

distribution of Na,K-ATPase [51] and prevents the

initial spotlike adherens junction complexes from

maturing into beltlike adherens junctions [52,53].

These data suggest that aPKC and Na,K-ATPase may

function in a common pathway for the establishment

or maintenance of cell polarity.

Chamber maturation

Growth of the myocardium

The regulation of normal myocardial development

is exquisitely sensitive and can be disrupted by

mutations within many different genes [2]. The

growth and thickening of the ventricular myocardium

is essential to vertebrate development because of the

vigorous contraction required to maintain blood

circulation and pressure. The control of myocardialdevelopment continues to be elucidated through

analyses of gene disruption in model systems such

as fly, zebrafish, and mouse. The serum response

factor (SRF) is one protein that has been implicated

as a controller of multiple signaling pathways essen-

tial for cardiac development, including the regulation

of immediate-early response genes and muscle-

specific genes [54]. Generation of a conditionalmouse transgenic mutant that deleted the cardiac-

specific expression of SRF resulted in embryonic

lethality associated with an abnormally thin myocar-

dium and dilated chambers, as well as a loss of nor-

mal trabeculation. In addition, several genes essential

for normal heart development also were downregu-

lated, suggesting a role for SRF as a global regulator

essential for cardiac maturation. Similarly, inactiva-

tion of the homeodomain Hop in mice and zebrafish,

which physically interacts with SRF and inhibits its

activity, results in a thin ventricular myocardium withfewer cells than normal [55,56].

The role of the ligand neuregulin and its co-

receptors ErbB2/ErbB4 in the trabeculation of the

ventricle was revealed through knockout studies in

mice [57–59]. Loss of any of these three pathway

members resulted in embryonic lethality between E10

and E12. All were associated with a failure of the

ventricle to develop normally, although atrial devel-

opment was not affected severely [57–59]. The

endocardial cushions also were reduced, with fewer

mesenchymal cells forming these valvular prescursor

structures [57–59]. Although ErbB2 and ErbB4 areexpressed on the myocardial surface, their ligand,

neuregulin, is expressed in the endocardium, impli-

cating crosstalk between these cell types for myo-

cardial development. In addition, the clustering of

endocardial-derived mesenchymal cells at the sites of

valve formation was disrupted by loss of the two myo-

cardial receptors ErbB2/4. These results established

the paradigm for signaling between the adjacent

endocardial and myocardial layers of the heart.

Another aspect of ventricular development that

has received less attention is the orientation of cellgrowth in a concentric direction to produce a thick,

multicell-layered myocardium. This type of oriented

cell growth may contribute to normal chamber

formation by thickening at appropriate positions

along the axis of the early heart tube. Studies in

zebrafish [60] and mouse [61] have revealed roles for

this process in normal heart morphogenesis. Charac-

terization of a zebrafish mutant with a single-cell–

layered myocardium has revealed a novel endocardial

transmembrane protein, heart of glass (heg ), which is

essential for the concentric thickening of the ven-

tricle. Although the hearts of these mutants developwith the normal complement of myocardial cells, they

are not added along the axis from the endocardium

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to myocardium, but instead spread over the circum-

ference of the hear t, resulting in two massive cham-

bers (Fig. 2) [60]. Reminiscent of the neuregulin/

ErbB2/ErbB4 mice knockouts, loss of the endocardial

gene heg also results in a failure to develop endo-

cardial cushions, further implicating this molecule inthe communication between the two adjacent cell

layers of the heart.

The analysis of patterns of clonal cell organization

in the mouse heart supports the importance of

directionality of cell growth in proper heart forma-

tion. Using a construct containing the lacZ reporter

gene targeted to an allele of the endogenous a-cardiac

actin gene [62], Meilhac and colleagues [61] dem-

onstrate expansion in clonal cells in a concentric

direction within the ventricle (along the axis from

endocardium to myocardium). This proliferation of cells correlated with the thickening within regions of

the ventricle during development of this chamber.

This process of oriented cell growth may facilitate

regional increases in the thickness of the myocardium

along the axis of the heart tube, thereby playing a

prominent role in chamber morphogenesis.

The secondary heart field

The contribution by the secondary (or anterior)

heart field to development of the myocardium and

outflow tract of the heart has emerged as an important

and potentially clinically significant aspect of cardio-

genesis [63–65]. (Although these field names often

are used interchangeably, there are distinctions

between them as a result of the experimental ap-

proaches taken for their definition [66].) The con-tribution of the heart-forming fields at the venous

Fig. 2. Endocardial-myocardial signaling is necessary for normal myocardial development. ( A) During normal heart development

in vertebrates, the myocardium thickens by proliferation of cells in a concentric direction, along the axis from the lumen

outwards. This process results in a thickened myocardial wall by 48 hpf of zebrafish development. ( B) In a class of zebrafish

mutants, including the mutant heart of glass (heg ), this process is interrupted. The result is a single-cell–layered myocardium

and a failure of the myocardium to thicken. The chambers dilate resulting in a massively enlarged heart. The endocardial

cushions, which normally develop as swellings at the atrial-ventricular junction, also fail to develop in this mutant.




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pole of the heart to normal myocardial growth is well

established, but the secondary heart field at the

arterial pole of the heart is less well characterized.

Many cardiac defects are associated with a failure

of the outflow tract to develop normally, including

atrioventricular and ventricular septal defects andtetralogy of Fallot, and together may account for as

much as one third of all congenital cardiovascular

disease [67]. Therefore, understanding the mecha-

nisms regulating the proper formation and migration

of the secondary heart field may provide insights into

the pathogenesis of these conditions.

DiGeorge syndrome (DGS) is one relatively com-

mon disorder associated with severe outflow tract

defects and anomalies in the glands and facial struc-

tures. Mice with a null mutation in the gene Tbx1,

which is also within the deleted interval associatedwith the human disease, exhibit cardiac outflow tract

defects, consistent with a role for this transcription

factor in outflow tract formation and the pathology of

DGS [68,69]. Further analysis of the requirement for

Tbx1 in outflow tract formation has implicated it in

the maintenance of cell proliferation within the

secondary heart field, consistent with the outflow

tract defects associated with the mouse knockout

and DGS [70]. Expression of Tbx1 within Nkx2.5 –

positive cells is essential for septation of the heart,

and in part may be mediated through the action of

Fgf10 [70]. These results provide a firm link betweenthe secondary heart field and a congenital disorder of

the heart.

Neural crest cells are also essential for outflow

tract formation, and experiments in which neural crest

cells were ablated in chick embryos led to a shortened

outflow tract and abnormal heart looping [71]. The

molecular interactions between these migrating neu-

ral crest cells and those within the secondary heart

field seem essential to the normal formation of the

outflow tract myocardium. Loss of the transcription

factors Hand1 (dHand ) [72], Nkx2.5 [73], and Mef2c[74], expressed in these cells, exhibit outflow tract

abnormalities. Also, mice lacking the transcription

factor Foxh1 can form a primitive heart tube but fail

to form normal outflow tract and right ventricular

myocardium [75]. The similarity of the phenotype of

the Foxh1/ and Mef2c/ mice is consistent

with the ability of Foxh1, through interaction with

Nkx2.5, to directly target the regulation of Mef2c

[75]. Through a single transforming growth factor– b

(TGF-b) response element in Mef2c, von Both and

colleagues [75] demonstrate that Foxh1 can mediate

Smad-dependent activation of Mef2c, and targets expression of a transgene to the anterior heart field, and

subsequently to the outflow tract and right ventricle.

Islet-1 (Isl1), a LIM-homeodomain protein, is best

known for its important role in the embryogenesis of

the pancreatic islets of Langerhans [76]. Examination

of the hearts of mice lacking Isl1, however, reveals

a complete loss of the outf low tract, right ventricle,

and much of the atria [77]. Expression analysis andlineage tracing of Isl1-positive progenitors have con-

firmed that this gene is a marker for a set of undiffer-

entiated cardiac progenitors, loss of which correlates

with t he loss of cardiac structures in the Isl1 mutant

mice [77]. An enhancer within Mef2c contains two

consensus binding sites for Isl1 that are essential to

its transcriptional regulation [78], further evidence of

the interplay between the genes expressed within this

heart field.

Valve formation

The endocardial cushions are the precursors to the

valves that eventually will complete the segmentation

of the chambers comprising the mature heart. They

develop as swellings of the cardiac jelly, the ECM

layered between the endocardium and the myocar-

dium at the chamber junctions. These regions then are

populated by a specific subset of endocardial cells

that undergo an epithelial-mesenchymal transition

(EMT) and delaminate from the surface of the endo-

cardial lining to migrate into the cardiac jelly [79].Once localized to the site of the future valves, these

cells proliferate to form the swellings that partition

the chambers. These structures then undergo exten-

sive remodeling to form the mature, tapered leaflet

structures characteristic of mature valves. The devel-

opment of these structures is perturbed easily, and not

only are defects in valve formation associated with

many congenital heart defects, but adult valvular

disease is a significant cause of morbidity and

mortality [67]. The coordinated expression of many

genes is essential for proper valve formation, and wellestablished roles for the connexins and members of

the notch, wnt , and TGF-b signaling pathways have

been defined [80,81].

Proper valve formation depends on the precise

communication between the myocardial and endo-

cardial layers of the heart through the ECM or cardiac

jelly that separates these layers. Vascular endothelial

growth factor (VEGF) is a key regulator of endothe-

lial cell proliferation and is essential for the EMT

required for valve development [82]: increases and

decreases in VEGF levels adversely affect valve

development. Loss of a single VEGF allele in mice issufficient to impair endocardial cushion development

[83], but defects in EMT and valve formation are

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observed when myocardial VEGF levels are stimu-

lated artificially at E9.5 [84,85]. Normally, upon

completion of EMT by E10.5, VEGF levels increase

dramatically, suggesting that high VEGF levels are

required to terminate this process as well, whereas

lower levels at earlier stages are essential for itsinitiation [84,85].

Recently, the role of VEGF in valve formation has

been linked with nuclear factor of activated T-cells

(NFATc1), long known to be required for this pro-

cess [86]. NFATc1 is a transcription factor ex-

pressed only in the endothelial cells of the heart,

but it is not essential for the EMT associated with the

early stages of endocardial cushion formation [87].

Inactivation of this gene in mice results in embryonic

lethality associated with the selective absence of the

aortic and pulmonary valves [87,88]. The normaldevelopment of the tricuspid and mitral valve is of

note, however, because these two structures are

derived entirely from the endocardial cushion tissue

[89], whereas the final stages of development of the

aortic and pulmonary valves are less well defined and

include contributions from neural crest cells [90]. At

E9.0 to E9.5 in mice hearts, calcineurin stimulates

expression of the other NFATc isoforms, NFATc2, c3,

and c4 in the myocardium and dampens VEGF

expression in the endocardium, facilitating EMT [86].

Subsequently, by E10.5, endocardial NFATc1 is

activated in the endocardium, which is essential for valve maturation [86].

These results are especially interesting when

examined in the context of the endocardial cushion-

derived defects associated with Down syndrome

because the gene DSCR1 (also known as ‘‘regulator

of calcineurin 1’’) is located in the minimal candidate

region for the Down syndrome phenotype and in-

hibits calcineurin activity [91]. The gene is expressed

in heart tissue and overexpressed in the brain of

Down syndrome fetuses [91]. Further linking these

molecules in a single regulatory network is theobservation that DSCR1 is induced by VEGF and

that expression of DSCR1 in endothelial cells can

block dephosphorylation, nuclear translocation, and

activity of NFATc1, presumably through loss of

calcineurin signaling [92]. In mice, DSCR1 is

expressed in the endocardium of the developing

atrioventricular and semilunar valves, as well as in

the interventricular septum and the ventricular myo-

cardium [93]. In the outflow tract of the Nfatc1/

mice, DSCR1 also is decreased, consistent with the

transactivation by NFATc1 of an intragenic element

within DSCR1 [93]. Thus, as previously described for VEGF, precise regulation of DSCR1 and associated

calcineurin activity is essential for valve formation.

VEGF also exerts its influence on valve formation

through other signaling conduits, including the

regulation of b-catenin activity. Phosphorylation of

b-catenin and subsequent association with PECAM-1

(CD31) is increased by VEGF signaling, modulating

b-catenin localization and signaling [94]. The wnt / b-catenin signaling pathway also is activated con-

stitutively by truncation of the tumor suppr essor

protein adenomatous polyposis coli (Apc) [95].

Although mice embryos with truncated Apc do not

complete gastrulation, a zebrafish mutant expressing

a truncated form of Apc completes gastrulation but

develops defects in heart morphology, including

failure of the hearts to loop and excessive endocardial

cushion formation [96]. Although b-catenin is upre-

gulated normally only in valve-forming cells, there is

accumulation of nuclear b-catenin in all cardiac cellsif the zebrafish mutant, correlating with the expansion

in cushion formation and concomitant increase in

valve marker expression [96]. Thus, cell proliferation

and EMT, normally restricted to the site of the endo-

cardial cushions, occur throughout the endocardium.

In contrast, endocardial cushion formation is inhib-

ited by overexpression of full-length Apc or Dickkopf

1 ( Dkk1), a secreted wnt inhibitor [96].

The signaling required for normal valve formation

between and within endocardial and myocardial cell

layers is facilitated by the cardiac jelly, the ECM of

the heart. The role of one component of this complex,hyaluronic acid (HA), has been characterized particu-

larly well for its role in mediating signaling, such

as ErbB2/4 activity [81]. HA is a glycosaminogly-

can comprised of alternating glucoronic acid and

N-acetylglucosamine (NAG) residues, with no pro-

tein backbone [97]. HA is synthesized by HA

synthases (HAS) at the plasma membrane, and is

released to the outside of the cell where it forms a gel

that occupies the extracellular space. Through inter-

actions with other matrix components, such as

versican [98], HA can regulate ligand activity withinthe ECM. In mammals, three HAS genes exist: has1,

has2, and has3. Loss of has2 in mice results in

embryonic lethality by E9.5, and is characterized by a

complete loss of cardiac jelly, severe pericardial

edema, and abnormal vessel development. The im-

portance of adequate matrix formation also has been

revealed by analysis of the zebrafish valve mutant

jekyll , characterized by a lack of endocardial cushions

with toggling of blood back and forth between

chambers and pericardial edema [99]. Mutations were

defined within the gene 50-diphosphate (UDP) –

glucose dehydrogenase (Ugdh), an enzyme requiredfor the conversion of UDP-glucose into UDP-

glucoronic acid, and the subsequent production of


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the matrix com ponents HA, chondroitin sulfate, and

heparin sulfate [100]. Clearly, alterations in the com-

position of the ECM can have profound effects on

valve development, and may affect further other attri-

butes of cardiac growth, such as myocardial growth

and trabeculation.

Development of the conduction system

There has also been substantial progress in

understanding early development of the cardiac con-

duction system. Central to this understanding are

questions regarding the origin of the cells involved

in generating and conducting electrical impulses to

the atrial and ventricular chambers. The finding

that some cells of the conduction system expressneural markers, such as neurofilament [101], HNK-1

[102], and EAP-300 [103,104], and more recent

experiments that show the contribution of neural

crest-derived cells to the maturation of conductive

phenotypes and the development of autonomic

innervation [105,106], suggest that cardiac neural

crest-derived cell populations are recruited to form

the conduction system. Several recent studies, how-

ever, have demonstrated that cells of the central and

peripheral conduction tissues emanate from a multi-

potent cardiomyogenic lineage, rather than from

migratory neurogenic populations or outgrowth froma specified pool of myogenic precursors [107,108].

The recruitment of these cells seems continuous

throughout the early stages of heart development

[109]. Moreover, the ability to record coordinated

depolarization of distinct cardiac compartments

before the arrival of neural crest-derived or epithe-

lial-derived cells lends credibility to the argument that

even before different components of the conduction

system can be identified morphologically, the basic

electrical configuration of the heart already has been

established [110].Although the entire conduction system shares

some common transcriptional mechanisms that drive

specification and differentiation of recruited cells, the

functional differences between the central and

peripheral conductive regions point toward a complex

genetic program [111]. For example, loss of Nkx2.5

function causes hypoplastic development throughout

the conduction system, which contributes to func-

tional defects in the postnatal heart [112]. This in-

dicates that Nkx2.5 is either important in the

recruitment of cells to the conduction system in early

cardiac development or in the retention of those cellsthrough the prevention of apoptosis [113,114]. The

T-box transcription factor Tbx-3 similarly delineates

most of the conduction system, including what seems

to be a remnant of the embryonic slow-conducting

regions that contribute to the ventricular conduction

tissue [115,116]. In this instance, Tbx-3 (and possibly

Tbx-2, which is also expressed transiently in the

conduction system) may govern development of the conduction system by preventing expression of

chamber-specification genes such as atrial natriuretic

factor [116,117]. In contrast, the Tbx-5 protein seems

to function in specification and patterning of the

central conduction system [118]. Demonstration of

the interaction of Nkx2.5 and Tbx proteins indicates

cooperation of these transcription factors in the

development of specific cardiac compartments. Other

transcription factors such as GATA6 , HF-1b, MyoD,

and Msx-2 have been implied to be associated with

the development of sections of the conduction sys-tem, but the precise role of these proteins in trig-

gering a conduction specific genetic program remains

enigmatic [109,119,120].

The proximity of the Purkinje fiber network with

branching coronary arteries led to speculation that

progressive conscription of cardiomyogenic cells to-

ward a conductive fate was the result of autocrine and

paracrine interactions. In fact, induction of cells in the

peripheral conduction system seems dependent on

vascular-derived endothelin-1 (ET-1), Neuregulin-1

(NG-1), and their receptors, despite some spatial and

temporal inconsistencies likely caused by variationacross species [121–123]. Exposure to ET-1 can

confer pacemaker activity upon cultured embryonic

stem cells [124] and the regulated expression of

endothelin-converting enzyme may provide an addi-

tional layer of control over the specification of con-

duction tissues in early development [101,125]. In

addition, treatment of embryonic cardiomyocytes

isolated from looped, tubular hearts with ET-1 re-

sulted in increased expression of wnt7a and wnt11,

suggesting a role for these proteins in patterning

the conduction system [126]. Because ET-1 is respon-sive to hemodynamic forces such as shear stress

and stretch, blood flow and pressure in the develop-

ing heart may serve as a fundamental inductive

signal for the formation of the conduction system

[123,127,128].

Aberrant development of the conduction system

has been implicated in arrhythmias and other con-

genital abnormalities of cardiac activation [109,129].

Perturbations in cellular excitability, intercellular

communication, and tissue architecture are associated

with a slowing of conduction velocity and, as a result,

increase the likelihood for the development of life-threatening arrhythmias [130,131]. In particular,

inadequate electrical coupling of cardiomyocytes

chen et al164

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through gap junctions seems central to arrhythmo-

genesis. Null mutations have been made in genes

encoding each of t he principal cardiac connexin

proteins [132–138]. Although Cx43- and Cx45-

deficient mice die prematurely from congenital

deformations of the cardiovascular system, mice that completely lack Cx40 are viable. The Cx40-knockout

mice show partial atrioventricular (AV)– bundle

branch block that results in a greater susceptibility

to arrhythmia formation. In addition, the Cx40-null

mice exhibit some characteristics reminiscent of

human congenital heart defects. These include myo-

cardial hypertrophy, an AV canal defect, incomplete

formation of the mesenchymal cap of the atrial sep-

tum, and various ventricular septal defects [139].

Heterozygous Cx45-deficient mice have conduction

blockade through the AV canal and contractions inthe outflow track that are not coordinated with those

of the ventricle. The Cx45-null embryos also exhibit

an endocardial cushion defect believed to arise from a

failure of cells in the endocardium to undergo the

requisite epithelial-mesenchymal transformation. The

combination of conduction block and congenital

abnormalities results in Cx45-knockout mice dying

of heart failure at embryonic day 10. Cx43-knockout

mice die from a pulmonary outflow tract obstruction

shortly after birth and conditionally deleted mice

have slow ventricular conduction velocities and

propensity for ventricular fibrillation [140,141]. Micedeficient for Cx40 and Cx43 have validated the idea

that loss of either isoform causes reduction in

propagation velocity and leads to congenital defor-

mations. Similarly, experiments that involve ablation

or swapping of connexin gene combinations show

defects in conduction and deformations in other

cardiovascular structures [142–144].

Summary

This article summarizes some recent contributions

to our understanding of cardiogenesis, although these

new findings build on the framework of earlier

studies. One paradigm that has emerged is the im-

portance of signaling between the endocardium and

myocardium for normal heart morphology. A require-

ment for endothelial cells in organogenesis has been

shown in mice, zebrafish, and frog, which have

demonstrated that kidney [145] and pancreas for-

mation [146] are contingent on the presence of these

cells. Similarly, studies in mouse and zebrafish

describe the loss of genes expressed in either cardiaccell type that have profound effects on the develop-

ment of the adjacent cell layer. The endocardium of

the heart seems to be a vertebrate-specific develop-

ment because the primitive chordate heart has no such

structure [2]. Consistent with this observation, much

of normal vertebrate cardiogenesis is predicated on

the sophisticated interweaving of signals between the

endocardial and myocardial layers, as well as signal-ing within these layers. As our understanding of the

genetic control of cardiogenesis evolves, new regu-

latory models will emerge and others will progress,

including the role of the endocardium in mediating

signals from the circulation. In fact, a role for flow

itself already has been defined for formation of the

zebrafish kidney [147] and heart [148]. Inevitably,

the principles derived from these studies will frame

the foundation upon which the heart is built.

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