Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac Development, Degeneration and Regeneration

Abstract

Cardiovascular diseases are the most common cause of human death in the developing world. Extensive evidence indicates that various toxic environmental factors and unhealthy lifestyle choices contribute to the risk, incidence and severity of cardiovascular diseases. Alterations in the genetic level of myocardium affects normal heart development and initiates pathological processes leading to various types of cardiac diseases. Homeobox genes are a large and highly specialized family of closely related genes that direct the formation of body structure, including cardiac development. Homeobox genes encode homeodomain proteins that function as transcription factors with characteristic structures that allow them to bind to DNA, regulate gene expression and subsequently control the proper physiological function of cells, tissues and organs. Mutations in homeobox genes are rare and usually lethal with evident alterations in cardiac function at or soon after the birth. Our understanding of homeobox gene family expression and function has expanded significantly during the recent years. However, the involvement of homeobox genes in the development of human and animal cardiac tissue requires further investigation. The phenotype of human congenital heart defects unveils only some aspects of human heart development. Therefore, mouse models are often used to gain a better understanding of human heart function, pathology and regeneration. In this review, we have focused on the role of homeobox genes in the development and pathology of human heart as potential tools for the future development of targeted regenerative strategies for various heart malfunctions.

Full text

Adv Exp Med Biol –Cell Biology and Translational Medicine
https://doi.org/10.1007/5584_2019_349
#Springer Nature Switzerland AG 2019
Homeobox Genes and Homeodomain
Proteins: New Insights into Cardiac
Development, Degeneration
and Regeneration
Rokas Miksiunas, Ali Mobasheri, and Daiva Bironaite
Abstract
Cardiovascular diseases are the most common
cause of human death in the developing world.
Extensive evidence indicates that various toxic
environmental factors and unhealthy lifestyle
choices contribute to the risk, incidence and
severity of cardiovascular diseases. Alterations
in the genetic level of myocardium affects
normal heart development and initiates patho-
logical processes leading to various types of
cardiac diseases. Homeobox genes are a large
and highly specialized family of closely related
genes that direct the formation of body struc-
ture, including cardiac development. Homeo-
box genes encode homeodomain proteins that
function as transcription factors with character-
istic structures that allow them to bind to DNA,
regulate gene expression and subsequently con-
trol the proper physiological function of cells,
tissues and organs. Mutations in homeobox
genes are rare and usually lethal with evident
alterations in cardiac function at or soon after
the birth. Our understanding of homeobox gene
family expression and function has expanded
significantly during the recent years. However,
the involvement of homeobox genes in the
development of human and animal cardiac tis-
sue requires further investigation. The pheno-
type of human congenital heart defects unveils
only some aspects of human heart develop-
ment. Therefore, mouse models are often used
to gain a better understanding of human heart
function, pathology and regeneration. In this
review, we have focused on the role of homeo-
box genes in the development and pathology of
human heart as potential tools for the future
development of targeted regenerative strategies
for various heart malfunctions.
Keywords
Cardiac development · Cardiac regeneration ·
Heart disease · Homeobox genes
Abbreviations
AMHC1 atrial myosin heavy chain-1
ANTP Antennapedia
BMP bone morphogenetic protein
Cdh2 cadherin 2
CDK cyclin-dependent kinases
Cited2 Cbp/P300 interacting
transactivator with Glu/Asp Rich
Carboxy-Terminal Domain 2
CNS central nerve system
R. Miksiunas, A. Mobasheri, and D. Bironaite (*)
Department of Regenerative Medicine, State Research
Institute Centre for Innovative Medicine, Vilnius,
Lithuania
e-mail: daibironai@gmail.com;
daiva.bironaite@imcentras.lt

ESC embryonic stem cells
FGF fibroblast growth factor
FHF first heart field
Flk1 fetal liver kinase 1
GJA5 gap junction protein alpha 5
GSC goosecoid
H3K27me3 histone H3 methylation on the
amino (N) terminal tail
Hcn4 hyperpolarization-activated cyclic
nucleotide-gated channel 4 gene
HOXL homeobox transcription factor
Hox-like
Irx Iroquois family of homeobox
genes
ISL1 LIM-homeodomain transcription
factor islet 1/insulin gene enhancer
protein ISL-1
JMJD3 JmjC domain-containing protein 3
MEF2C myocyte-specific enhancer factor
2C
MESP1 mesoderm posterior BHLH tran-
scription factor 1
MSCs mesenchymal stem cells
Myocd myocardin
NKL NK-like
Nkx2-5 homeobox protein NK-2 homolog
E
Nodal nodal growth differentiation factor
Nppa natriuretic peptide A
OFT outflow tract
PCBP2 poly(rC)-binding protein 2
Pitx2 paired like homeodomain 2
Pitx2c paired-like homeodomain tran-
scription factor 2
PROS prospero
RA retinoic acid
SAN sinoatrial node
SHF second heart field
Shox2 short stature homeobox 2
SMAD main signal transducers for
receptors of the transforming
growth factor beta (TGF-β)
superfamily;
TALE three-amino-acid loop extension
Tbx5 T-box transcription factor 5
TF transcription factors
TGF-βtransforming growth factor beta;
VCS ventricular conduction system
ZEB2 zinc finger E-box binding homeo-
box 2
ZF zinc finger
Ziro zebrafish iroquois homeobox
genes
ZO-3 tight junction protein 3
1 Introduction
Homeobox genes are a large family of genes that
direct the formation of body structures along the
head-tail axis in multicellular animal species
(Innis 1997; Shashikant et al. 1991). It is also
known that homeobox genes (Hox genes), as an
ancient class of transcription factors, are impor-
tant for the body patterning during embryo devel-
opment (Innis 1997; Shashikant et al. 1991).
Many of the homeobox genes play very important
part in the spatiotemporal development of human
heart (Lage et al. 2010). Likewise, some of these
genes shape the human heart and control its mul-
tistep developmental process from simple cres-
cent cells to a fully functional organ. For
example, homeobox genes like homeobox protein
NK-2 homolog E (Nkx2-5), LIM-homeodomain
transcription factor islet 1 (Isl1), paired like
homeodomain 2 (Pitx2) are widely known to be
important for the proper development of human
heart (Akazawa and Komuro 2005; Luo et al.
2014; Franco et al. 2017). However, there are
many more homeobox genes that play substantial
roles in cardiac function but thus far, there are less
known and/or less investigated.
Specific inherited gene mutations cause con-
genital heart defects such as atrial or ventricle
septal defects, abnormalities of outflow tract and
etc. (Bao et al. 1999). Similarly, various patho-
logical lifestyle factors like smoking, low physi-
cal activity, toxic and noxious agents and other
environmental factors might also negatively
affect cardiovascular function and promote heart
failure (O’Toole et al. 2008; Nayor and Vasan
R. Miksiunas et al.

2015). Since it is impossible to exactly pinpoint
how certain gene mutations influence develop-
ment of human heart at the earliest stages, differ-
ent mouse models have been created to better
understand regulation of human heart develop-
ment and its relation to various diseases
(Camacho et al. 2016). Many of the genes studies
in mouse models have similar vital roles in the
development and function of human heart
(Xu and Baldini 2007). Therefore, investigation
of human disease and cues from mouse heart
development models have revealed an important
role of homeobox genes, including those that
encode transcription factors.
Aside from already known homeobox genes,
there are more homeobox genes that are essential
for the formation of human and/or mouse myo-
cardium. Some of these homeobox genes code
transcription factors (TF), whereas other form a
tight network regulating heart development and
fate of heart progenitors. Several review articles
have explored individual families of homeobox
gene and their roles in embryo development.
However, knowledge concerning the involve-
ment of homeobox genes and homeodomain TF
in the development of human heart referring
mouse models are still lacking. Therefore, in this
review we describe the role of more than
20 homeobox genes that are mainly involved in
heart development and around 15 homeobox
genes that are known to play minor or less
investigated, but nonetheless important roles in
cardiac development. Data summarized in this
review will help to broaden the possible future
applications of homeobox genes and their coded
TF in targeted therapeutic strategies for cardiac
regeneration and therapy.
2 Development of the Human
Heart
Starting from the day first of fertilization, the
zygote undergoes multiple cell divisions leading
to the formation of third germ layer, known as the
mesoderm (Moorman et al. 2003). Later
mesodermal cells migrate towards anterior part
of embryo to form a distinct crescent-shaped epi-
thelium, named the cardiac crescent
(Buckingham et al. 2005). Cells situated in the
distinct anterior-lateral territory within the cardiac
crescent contribute to the formation of first heart
field (FHF), distinguished by the expression of
hyperpolarization-activated cyclic nucleotide-
gated channel 4 gene (Hcn4) (Liang et al. 2013).
Cardiac progenitor cells also develop into second
heart field (SHF), which is located medially to the
cardiac crescent and extend posteriorly (Cai et al.
2003). Formation of SHF is marked by the
expression of LIM-homeodomain transcription
factor islet 1 (ISL1) (Cai et al. 2003). Sometimes
progenitors of FHF and SHF are called cardio-
genic or cardiac mesoderm (Dupays et al. 2015;
Liu et al. 2014; Kitajima et al. 2000). These
distinct heart fields fuse to form heart tube,
which eventually develops into functional heart
(Fig. 1) (Moorman et al. 2003; Nemer 2008).
During this time, the primitive cardiac conduction
system, including sinoatrial node (SAN), ventric-
ular conduction system and other, starts to form
(van Weerd and Christoffels 2016). The FHF
cells develop into the left ventricle, as well as
into the atrioventricular canal and part of the
atria, whereas SHF cells develop into the right
ventricle and outflow tract, with contribution to
the formation of atria and inflow vessels
(Buckingham et al. 2005). Once the heart fields
are formed, they fuse into heart tube and undergo
process called heart looping. During this phase
the whole heart tube twists in the rightward direc-
tion eventually forming clearly visible, but still
primitive, heart chambers (Santini et al. 2016).
Later on, the heart undergoes septation to fully
separated left and right sides of the heart.
There are many factors regulating human and
mouse heart development, however only some of
them may be considered to be core regulators of
cardiogenesis. One of the most important TF is
GATA4 which orchestrates expression of multi-
ple transcriptions including other major
determinants of cardiomyogenesis like Nkx2-5,
T-box transcription factor 5 (Tbx5), heart- and
Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac...

neural crest derivatives-expressed protein
1 (HAND1/2) and others (Bruneau et al. 2001a;
Belaguli et al. 2000; Sepulveda et al. 1998).
GATA4 integrates bone morphogenetic proteins
(BMP) and SMAD, the main signal transducers
for receptors of the transforming growth factor
beta (TGF-β) superfamily, to ensure cardiac cell
survival and stable lineage during cardiac devel-
opment (Benchabane and Wrana 2003). Of
course, there are other factors that promote devel-
opment of various structures within the heart. For
example, it is know that Tbx5 controls atrial gene
expression, whereas myocyte-specific enhancer
factor 2C (MEF2C) promotes development of
ventricle and vasculogenesis (Bruneau et al.
2001a; Lin et al. 1997). Altogether, the develop-
ment of human heart is a carefully controlled
multistep process involving many genes, intracel-
lular and extracellular signalling factors leading
to proper cardiac function. The miss-controlled
heart development process leads to various
inherited or acquired cardiac disorders. This
review focuses mostly on the homeodomain
proteins, as one of the most important group of
transcription factors regulating heart develop-
ment, function and impairment.
3 Homeobox Genes
Homeodomain proteins are one of the most
important group of proteins/transcription factors
regulating plan of body structure and
organogenesis in eukaryotes including heart
development and disorders. DNA binding
proteins have been extensively studied, but even
today there are no established rules for predicting
the specificity of DNA sequence based upon the
amino acid sequence of the proteins.
Homeodomain proteins are characterized by spe-
cific 60 amino acid long helix-turn-helix DNA
binding homeodomain motif (Seifert et al.
2015). The homeodomain is a very highly
conserved structure and consists of three helical
regions folded into a tight globular structure that
binds a 50-TAAT-30core motif. The high degree
of conservation of homeodomain proteins is an
ideal model to study specific protein-DNA
interactions. The DNA sequence that encodes
the homeodomain is called the “homeobox”and
homeobox-containing genes are known as “hox”
genes.
Most of the transcription factors belonging to
this group are not only structurally but also evolu-
tionary conserved and play crucial roles in embry-
onic patterning and differentiation (Pearson et al.
2005). The main role of homeodomain proteins
in vivo is to control the genetic determination of
development and implementation of the genetic
body plan. There are 102 homeobox gene families
that represent 235 active human homeodomain
proteins, but only some homeodomain classes
have close association with cardiac development
and/or diseases (Bürglin and Affolter 2016). This
review covers description of around 20 homeobox
genes that up today are known to have a major
Fig. 1 Schematic representation of heart development in human and mouse. FGF first heart field, SHF second heart
field, RV right ventricle, LV left ventricle, RA right atrium, LA left atrium. (Scheme adapted from (Nemer, 2008))
R. Miksiunas et al.

impact in the regulation of heart development and
functioning (Fig. 2).
4 Homeobox Genes in Mouse
Heart Development
and Human Disease
Humans have more than 67 genes that are impor-
tant for cardiac hypertrophy and over 92 genes
that control cardiovascular system, therefore it is
reasonable to assume that some of the genes
might be responsible for cardiac development
and disease (van der Harst et al. 2016; Smith
and Newton-Cheh 2015). Congenital heart dis-
ease (CHD) have structural heart anomaly,
including atrial or ventricle septal defects,
overriding aorta, right atrium isomerism and
other structural changes in new born heart.
Patients do usually display multiple symptoms,
like, rapid breathing, bluish skin, poor weight
gain and feeling tired in general (Sun et al.
2015). The main cause of these abnormalities is
reduced blood oxygen levels in whole organism,
which is a result of improper heart septation lead-
ing to the mixture of oxygenated and deoxygen-
ated blood in systemic blood circulation.
Additionally, CHD could be caused by genetical
or environmental factors like infections during
pregnancy such as Rubella, drugs and maternal
illness (Sun et al. 2015). Since homeobox genes
are important for the heart development, some
mutation of the homeobox genes including
MEIS2,Nkx2-5 and others can potentially cause
CHD (Zakariyah et al. 2017; Johansson et al.
2014). Of course, homeobox gene mutations is
not a sole cause of heart defect present at human
birth. For example, mutations in GATA4 and Tbx5
can also affect integrity of heart tissue (McCulley
and Black 2012). In addition, impaired gene func-
tioning might be also related to the other heart
diseases like cardiomyopathy, hypertrophy,
Fig. 2 Hierarchy of homeobox genes involved in heart
development. Pink boxes indicate homeobox gene classes,
yellow box indicates TALE gene superclass. Red boxes
indicate genes with major involvement in heart develop-
ment and diseases. Blue boxes indicate genes having less
important role in heart development and diseases
Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac...

defects of the heart rhythm and other
abnormalities (Kathiresan and Srivastava 2012).
However, the impact of homeobox genes in heart
disorders is still not clear and needs further inves-
tigation to clarify their role not only in the cardiac
development but also in the physiological and
pathophysiological conditions.
5 ANTP Class of Homeobox
Genes
The Antennapedia (ANTP)-class of homeobox
genes are involved in the determination of pattern
formation along the anterior-posterior axis of the
animal embryo. NK-like (NKL) and homeobox
transcription factor Hox-like (HOXL) are an
ancient subclasses of homeobox genes that
belong to an ANTP homeobox gene class (Hol-
land et al. 2007). It is likely that HOXL gene
clusters originate from NKL, since NKL genes
are widespread throughout the genome as tight
clustered Hox genes (Bürglin and Affolter 2016).
Both subclasses of the genes (NKL and HOXL)
are evolutionarily conserved and play
predetermined roles in heart patterning and
disease.
6 Hox Gene Families of HOXL
Subclass
Hox gene families belong to the HOXL subclass of
ANTP class of homeobox. Hox genes code tran-
scription factors which are important for whole
body patterning and development (Pearson et al.
2005). It total, there are 39 human Hox genes
grouped in HOXA, HOXB, HOXC and HOXD
gene clusters. Hox genes are highly conserved,
because they play a vital role in anterior-posterior
formation of body axis (Pearson et al. 2005). The
precise function of these genes is achieved by their
specific temporal and spatial expression over the
life course. During early mouse cardiac develop-
ment, the retinoic acid (RA) might be responsible
for the anterior-posterior patterning in SHF
(Bertrand et al. 2011). Hoxb1, Hoxa1, and Hoxa3
act as downstream targets of RA and participate in
forming outflow tract (OFT) and normal SHF
development (Bertrand et al. 2011). Hoxb1/
or Hoxa1/,Hoxb1+/mouse embryos
develop shortened OFT and display abnormal pro-
liferation and premature differentiation of cardiac
progenitors (Bertrand et al. 2011). This is probably
related to the altered fibroblast growth factor
(FGF) and BMP signaling pathways in developing
mouse embryo (Roux et al. 2015). Clinical studies
have revealed that Hoxa1 mutations might cause
congenital human heart defects and other
abnormalities like, mental retardation, deafness,
horizontal gaze restriction and etc. (Bosley et al.
2008). Several other studies have shown that Hox
genes might be also related to the human heart
diseases, however more research is needed to
unveil exact functions of these genes in
human heart development (Gong et al. 2005;
Haas et al. 2013).
7 Nk4 Gene Family of NKL
Subclass
There are multiple NKL genes in mouse and
humans regulating various developmental pro-
cesses, however only some of them contribute to
the development of the heart (Larroux et al.
2007). The Nkx2-5 and Nkx2-6 genes are the
members of the NK4 homeobox gene family of
NKL subclass and are closely related to the Dro-
sophila tinman gene (Bürglin and Affolter 2016;
Harvey 1996). To our knowledge, only Nkx2-6
and Nkx2-5 relate to the mouse and human heart
development and disease, whereas Nkx2-3,Nkx2-
7,Nkx2-8 and Nkx2-10 might be important for the
heart development of zebrafish, frog or chicken
(Newman and Krieg 1998; Wang et al. 2014;Tu
et al. 2009; Allen et al. 2006; Brand et al. 1997).
During the early stages of embryogenesis,
Nkx2-5 is expressed in myocardium and pharyn-
geal endoderm, whereas Nkx2-6 can be found in
sinus venosus, pharyngeal endoderm and myo-
cardium of the outflow tract (Lints et al. 1993).
R. Miksiunas et al.

Moreover, during normal heart development,
Nkx2-5 expression is essential for the looping of
vertebrate embryonic heart, heart septation and
formation of cardiac conduction system, whereas
most of the Nkx2-5 mutations are related to
human congenital heart disease and conduction
defects (Tanaka et al. 1999). Inactivation of Nkx2-
5arrested heart formation at the looping stage
revealing its critical role in cardiac development
(Lyons et al. 1995). However, targeted disruption
of Nkx-2.6 did not cause any abnormalities in the
heart suggesting a possible compensatory func-
tion of Nkx-2.5 (Tanaka et al. 2000).
It is important to note that Nkx2-5 mutations
lead to an altered spatiotemporal development of
human heart, improper heart septation and forma-
tion of cardiac conduction system (Dupays et al.
2015; McCulley and Black 2012; McElhinney
et al. 2003). Analysis of human Nkx2-5 mutants
and gene truncations showed that most of the
mutations affected Nkx2-5 binding to DNA or
its localization but not protein-protein
interactions (McCulley and Black 2012;
Reamon-Buettner et al. 2004). Several different
studies of mice Nkx2-5 knockout and human
embryonic stem cells (ESC) revealed that Nkx2-
5mutations might alter gene expression of spe-
cific transcription factors like SP1, SRY, JUND,
STAT6, MYCN,PRDM16,HEY2 and others
(Anderson et al. 2018; Li et al. 2015). Also
some studies support an idea that, Nkx2-5 mutant
proteins might alter space and time specific
human cardiac development by dysregulating
BMP, Notch and Wnt signalling pathways
(Anderson et al. 2018; Wang et al. 2011; Luxán
et al. 2016; Cambier et al. 2014). There is a
possibility that Nkx2-5 modulates these pathways
by interacting with multiple transcription factors
in time-dependent mode. For example, in mouse
heart Nkx2-5 interacts with Hand2 transcriptions
factor to activate Irx4, which is necessary for the
ventricular identity (Yamagishi et al. 2001). Con-
versely, Nkx2-5 expression is also timely
regulated since Nkx2-5 overexpression leads to
an improper SAN formation in early mouse
development (Roux et al. 2015). Mammalian
heart development is also regulated by the com-
bination of cardiac transcription factors having
specific DNA motifs in their centrally located
DNA binding domains. It was also shown that
Nkx2-5, GATA4 and Tbx5 can physically inter-
act and synergistically regulate targeted genes
(Hiroi et al. 2001; Pradhan et al. 2016). Since
these genes are the master regulators of heart
development, functional mutations in these
genes are linked to various types of congenital
heart diseases (Benson 2002; Hatcher et al. 2003).
Taken together, Nkx2-5 and other transcription
factors like Isl1, GATA4, Tbx5, Hand2, MEF2C,
Irx4 form a core of transcription factors essential
for the heart development and congenital heart
disease (Fig. 3) (McCulley and Black 2012).
8 HHEX Gene Family of NKL
Subclass
Proline rich homeodomain protein or homeobox
protein (PRH/HHEX) expressed by
hematopoietic system is a transcription factor
belonging to the family of NKL subclass gene
(Bedford et al. 1993). As the name implies, it is
important for the development of hematopoietic
cell, but not less is essential for the development
of other systems, including heart (Bedford et al.
1993). Mouse double HHex mutants have multi-
ple developmental issues, including defective
vasculogenesis, hypoplasia of the right ventricle,
aberrant development of the compact myocar-
dium and other complications related to forebrain,
thyroid and liver developmental disorders (Hallaq
et al. 1998). Additional studies have revealed that
HHex plays distinct role in mouse cardiac meso-
derm specification and development. HHex
expression is controlled by Sox17 transcription
factor, which is known to be essential for the
formation of mouse cardiac mesoderm (Liu
et al. 2014). Several studies of human population
have shown that common variants of HHex gene
(rs7923837 and rs1111875) may also be
associated with diabetes (Karns et al. 2013;
Kelliny et al. 2009; Pechlivanis et al. 2010).
Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac...

9 PRD and PRD-Like Homeobox
Class
The PRD class is the second largest of the homeo-
box gene classes in animal genomes and, like the
ANTP class, these genes have been found only in
animals. The PRD class derives its name from the
Paired (Prd) gene of Drosophila. Multiple gene
families belong to the PRD homeobox class,
including Shox2,Hopx,GSC,Pitx2 and others,
which are important for the heart development
and disease.
10 Shox Gene Family of PRD Class
Short stature homeobox 2 (Shox2) is a homeobox
gene belonging to the PRD class of homeobox.
Shox2 is an essential for the development of limb
and cardiac conduction systems, including forma-
tion of sinoatrial node (SAN) in mice and humans
(Gu et al. 2008; Blaschke et al. 2007; Liu et al.
2011). Studies of Shox2 function during the
mouse development revealed several cues how
this homeodomain transcription factor in particu-
lar controls formation of SAN (Blaschke et al.
2007). Shox2 mice null mutants displayed severe
cardiac conduction defects, such as low heart
rhythm rate and drastically reduced cell prolifera-
tion (Espinoza-Lewis et al. 2009). This phenotype
is probably related to the downregulation of
HCN4,Tbx3 and the upregulation of natriuretic
peptide A (Nppa), gap junction protein alpha
5(GJA5) and Nkx2-5 gene expressions
(Espinoza-Lewis et al. 2009). It is also known
that HCN channels play a vital role in autonomic
control of heart rate, so it is no surprise why
Shox2 null mutants do not develop SAN (Alig
et al. 2009). During the normal development of
mouse cardiac expression of Shox2 is also tightly
controlled by several transcription factors. For
example, transcription factor Tbx5 activate
Shox2 expression, however transcription factors
like Pitx2c and NKX2-5 potentially silence
Shox2 expression (Espinoza-Lewis et al. 2011;
Puskaric et al. 2010). Paired-like homeodomain
transcription factor 2 (Pitx2c) also can potentially
inhibit left-sided pacemaker specification by
suppressing Shox2 expression in left atrium,
therefore SAN develops only in the region of
right atrium (Wang et al. 2010). All these results
indicate that Shox2 is essential for the maintaining
pacemaker cell program during the heart develop-
ment. On the other hand, in adulthood Nkx2-5
Fig. 3 Core transcription factors important for the heart development and congenital heart disease. (Scheme adapted
from (McCulley and Black, 2012))
R. Miksiunas et al.

antagonizes Shox2 and promotes cardiomyocyte
formation (Fig. 4) (Liang et al. 2017).
Taken together, these studies indicate that
Shox2 might be a good candidate to develop
biological pacemaker. Results from mouse ESCs
and canine mesenchymal stem cells (MSCs) have
shown that cells overexpressing Shox2 induce
expression of SAN markers such as HCN4,
Cx45 and Tbx3 (Ionta et al. 2015; Feng et al.
2016). In addition, mouse embryonic bodies
overexpressing Shox2 showed better contractile
phenotype compared to the control group of
embryonic bodies (Ionta et al. 2015). Moreover,
human patients with an early-onset atrial fibrilla-
tion had significantly downregulated expression
of Shox2 gene (Hoffmann et al. 2016). These
results are promising for patients suffering from
heart rhythm defects, however, more studies are
needed to test functions of biological pacemaker
in order to treat human arrhythmias.
11 HOPX Gene Family of PRD
Class
HOPX is another PRD-class homeobox gene
family important for the multiple organ
development and tissue homeostasis in adults
(Chen et al. 2015; Schneider et al. 2015; Mariotto
et al. 2016). Since it lacks a DNA binding
domain, HOPX can only modulate gene expres-
sion by forming complexes with other regulatory
proteins (Kook et al. 2006). In general HOPX acts
as a cell proliferation inhibitor in humans cancer
cells, however its function in mouse cardiac cell
differentiation is not entirely clear (Chen et al.
2015; Waraya et al. 2012; Yap et al. 2016). Stud-
ies of mouse development have shown that
HOPX plays a critical function in early formation
of cardiomyocyte progenitors. HOPX integrates
BMP and WNT signalling in developing mouse
heart by interacting with SMAD proteins and
inhibiting WNT signalling pathway leading to
the formation and differentiation of
cardiomyocyte progenitors (Jain et al. 2015).
On the other hand, there are some cues that
HOPX might act as a negative regulator of car-
diac differentiation in mice. HOPX interacts with
HDAC2, thus reducing GATA4 transcriptional
activity by deacetylation (Trivedi et al. 2010).
These findings are consistent with previous
reports that overexpression of HDAC2 inhibits
the development of cardiomyocytes by down-
regulating the expression of GATA4 and Nkx2-5
Fig. 4 Signaling networks governing pacemaker and atrial differentiation of cardiomyocyte in developing mouse heart.
(Scheme adapted from (Liang et al. 2017))
Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac...

genes (Kawamura et al. 2005; Karamboulas et al.
2006). All these results highlight the complex
nature of HOPX and its partners in heart develop-
ment. HOPX undeniably plays a critical role in
early heart development, because some mouse
mutants cannot develop a functional myocardium
and display cardiac conduction defects (Chen
et al. 2002; Ismat et al. 2005). HOPX also might
be related to the human heart failure, since HOPX
is downregulated in patients having cardiac
hypertrophy (Güleç et al. 2014; Trivedi et al.
2011).
12 Goosecoid Gene Family of PRD
Class
Goosecoid (GSC) is another protein that belongs
to the bicoid related paired (PRD) homeobox
class of genes. Goosecoid is often associated
with limb, skeletal and craniofacial development,
although, it might be important for cardiac meso-
derm formation, since its expression is controlled
by Mesp1 (Zhu et al. 1998). Mesoderm posterior
BHLH transcription factor 1 (MESP1) preferen-
tially binds to two variations of E-box sequences
and activates critical mesoderm modulators,
including Gata4, mix paired-like homeobox
(Mixl1) and GSC homeobox (Soibam et al.
2015). In addition, mesoderm formation can be
induced with l-proline and trans-4-hydroxy-l-pro-
line resulting in increased expression of Mixl1
and GSC (Date et al. 2013). GSC also is impor-
tant for the cell migration in early embryonic
development, therefore the overexpression of
goosecoid enhances oncogenic cell growth and
metastasis (Kang et al. 2014).
13 Pitx Gene Family of PRD-Like
Class
Paired like homeodomain 2 (Pitx2) is a PRD-like
homeobox class gene which is important for the
establishment of the left-right axis and for the
asymmetrical development of the mouse and
probably human heart, lung, and spleen, twisting
of the gut and stomach, as well as the develop-
ment of the eyes (Campione et al. 1999; Shiratori
et al. 2006; Evans and Gage, 2005). There are
several alternative Pitx2 transcripts, however only
Pitx2c isoform plays determined role in the asym-
metric development of mouse heart (Liu et al.
2002). Higher vertebrates, at an early heart devel-
opment stage and after the heart tube formation,
undergo embryonic heart looping, which is the
first visual evidence of embryo asymmetry
(Harvey 2002). Transcription factors like nodal
growth differentiation factor (Nodal) and Cbp/P300
interacting transactivator with Glu/Asp Rich
Carboxy-Terminal Domain 2 (Cited2) activate
Pitx2 transcription leading to the rightward twist
of the heart tube and forming prospective embry-
onic atrial and ventricular chambers. Deletion of
Pitx2c in mouse caused drastic alteration of
looping process leading to various heart defects
including the isomerism of right atrium and ven-
tricle (Lin et al. 1999; Yu et al. 2001).
Humans with Pitx2c mutations develop vari-
ous heart abnormalities, including an improper
formation of ventricle and atrial chambers septa,
atrial fibrillation and others. It is quite likely that
septation defects are caused by the
downregulation of transcription factors down-
stream of Pitx2, since certain Pitx2 mutants
displayed reduced cardiac transcriptional activity
in human patients (Wang et al. 2013; Wei et al.
2014). Surprisingly, the overexpression of Pitx2c
in mouse R1-embryonic stem cells results in ele-
vated gene expression of essential cardiac tran-
scription factors like GATA4, MEF2C, Nkx2-5
and others (Lozano-Velasco et al. 2011). Conse-
quently, Pitx2c might be a good candidate for
heart regeneration, since it positively regulates
multiple transcription factors important for car-
diac development. Recently it was shown that
mouse embryonic stem cells overexpressing
Pitx2c could restore mouse heart function after a
myocardium infarct through the multiple
mechanisms including efficient terminal
R. Miksiunas et al.

differentiation, regulation of action potentials of
cardiomyocytes and positive paracrine effects
(Guddati et al. 2009). However, more studies
need to be done to determine the utility of
Pitx2c in human heart regeneration strategies.
14 TALE Homeobox Superclass
Three-amino-acid loop extension (TALE) is
another superclass of homeobox genes, which
codes for highly conserved transcription
regulators essential for various developmental
programs. These genes encode proteins with
atypical homeodomain structure, defined by hav-
ing three additional amino acids in
homeodomain. TALE homeobox gene superclass
includes the main zinc finger (ZF), PBC and Meis
homeobox 1 (Meis) classes. Out of 20 human
homeodomains only Meis1, Meis2 and Iroquois
homeobox proteins 1-6 (Irx1-6) have their clearly
defined function in heart development and
disease.
15 Meis Genes Family of Meis
Class
Meis1 encodes the TALE superclass homeobox
transcription factor implicated in cardiac,
hematopoietic and neural development (Mariotto
et al. 2013; Azcoitia et al. 2005; Hisa et al. 2004).
Meis1 deficient mice have malformed cardiac
outflow tracts with overriding aorta and ventricu-
lar septal defect (Stankunas et al. 2008).
Downregulation of Meis1 leads to cardiac hyper-
trophy in humans and mice. Meis1 binds poly
(rC)-binding protein 2 (PCBP2) gene promoter
and activates its expression in order to suppress
human or mouse heart hypertrophy (Zhang et al.
2016). In turn, PCBP2 represses angiotensin II,
which enhances hypertrophic human or mouse
cardiac growth (Zhang et al. 2015). There are
around 79 cardiac specific genes that have
Meis1 and NKX2-5 binding sites in developing
mouse heart, some of them are associated with
cell signaling and cardiac progenitor differentia-
tion, like Tbx20, myocardin, cadherin 2 (Cdh2),
Wnt11, and Wnt2 (Dupays et al. 2015). Adult
mouse cardiomyocytes with mutant MEIS1
exhibit increased proliferation and progression
of the cell cycle (Mariotto et al. 2013). This
function is emphasized in adult mouse hearts
since Meis1 activates inhibitors of cyclin-
dependent kinases (CDK) like p15, p16 and p21
(Mariotto et al. 2013). In humans
non-synonymous Meis1 gene variants might be
associated with congenital heart defects, whereas
patients carrying 2p14 microdeletions show
symptoms of deafness and cardiomyopathy
(Mathieu et al. 2017; Arrington et al. 2012). It is
likely that Meis1 is required for the control of
spatiotemporal cell proliferation in early develop-
ing heart to prevent hypertrophy, however more
studies need to be done to fully understand the
role of Meis1 in cardiac development and disease.
Meis2 encodes TALE homeobox superclass
transcription factor essential for the development
of mouse cranial and cardiac neural crest (Machon
et al. 2015). Recent findings indicate that Meis2
might be an important factor for the proliferation of
fetal human cardiomyocyte cells (Wu et al. 2015).
Reduction of Meis2 gene expression by miR-134
results in slowed progression of human
cardiomyocyte progenitor cell cycle (Wu et al.
2015). A clinical and genetic study also revealed
that small Meis2 deletion can negatively affect
several developmental processes: human patients
with small Meis2 non-frame shift deletion
(c.998_1000del:p.Arg333del) had serious cleft
palate and cardiac septal defects (Louw et al.
2015). It is known that Meis2 interacts with
DNA and forms multimeric complexes with Hox
and Pbx proteins (Louw et al. 2015). Single dele-
tion of arginine residue affects the ability of Meis2
to bind DNA leading to serious developmental
problems of human heart (Louw et al. 2015). Clin-
ical studies have shown that patients having only
one functioning Meis2 gene copy survive, how-
ever they have similar phenotype such as clefting
and ventricular septal defects leading to delayed
Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac...

motor development and learning disability
(Johansson et al. 2014).
16 Irx Gene Family of IRX Class
Iroquois homeobox genes and their coded
homeodomain proteins are another class of tran-
scription factors belonging to TALE superclass of
homeobox genes. Iroquois-class homeodomain TF
(Irx) defining feature is atypical homeodomain
structure and specificIroquois(IRO)
homeodomain family sequence motif, which is
important for the recognition of DNA sequence
(Gómez-Skarmeta and Modolell 2002; Cavodeassi
et al. 2001). Humans and mice have six Irx
proteins, which are important for the development
of lung, nervous system, eye, pancreas, female
gonad, early limb and, of course, heart patterning
(Cavodeassi et al. 2001; Cheng et al. 2005;
Schwab et al. 2006; van Tuyl et al. 2006; Ragvin
et al. 2010; Jorgensen and Gao 2005;McDonald
et al. 2010). Irx1 and Irx2 are expressed in inter-
ventricular septum from E14.5 onward, however,
mouse Irx2 mutants are viable and display no
notable phenotype defects in the developing heart
(Christoffels et al. 2000; Lebel et al. 2003). Irx1
gene variants might be related to the congenital
heart disease in humans (Guo et al. 2017).
Irx3 gene in mice seems to be very important
for the ventricular conduction system (VCS)
(Christoffels et al. 2000). Various studies suggest
that Irx3 is required to maintain rapid electric
conduction through the VCS for proper ventricu-
lar activation, via antithetical regulation of Cx40
and Cx43 expression (Zhang et al. 2011;
Kasahara et al. 2003). Clinical studies have
revealed that defects of Irx3 gene can cause lethal
cardiac arrhythmias in human patients (Koizumi
et al. 2016). Irx3 function appears to be evolu-
tionary conserved, since expression of Ziro3a, a
Irx3 homologue in zebrafish, is detected in devel-
oping fish heart (Zhang et al. 2011).
Irx4 is associated with the formation of ven-
tricular myocardium in mouse and humans
(Christoffels et al. 2000; Cheng et al. 2011).
Data from mouse and chicken indicate that Irx4
suppresses atrial gene expression by down
regulating atrial myosin heavy chain-1
(AMHC1) (Bao et al. 1999; Bruneau et al.
2001b). Several Irx4 mutations have been
identified that might be associated with human
congenital heart disease, particularly ventricular
septal defect (Cheng et al. 2011).
Irx5 is expressed in adult mouse heart and
maintains proper action potentials, particularly
regulates T-wave seen in ECG (Costantini et al.
2005). Mice lacking Irx5 develop properly with-
out any structural abnormalities in the heart
(Costantini et al. 2005). This indicates that Irx5
is not required for cardiac development or that
other Irx genes can compensate for the loss
of Irx5.
Irx6 is detectable in mouse developing heart,
however its expression is relatively weak com-
pared to other Irx genes (Christoffels et al. 2000).
17 Zeb Gene Family of ZF Class
ZEB2 or zinc finger E-box binding homeobox 2 is
a gene coding transcription factor belonging to
class of ZF homeobox gene and homeodomain
class of ZN proteins (Bürglin and Affolter, 2016).
It has multiple functional domains (E-box, Zinc
finger, homeobox), so naturally it can control
gene expression with a variety of transcription
factors (Gheldof et al. 2012). The complex nature
of Zeb2 shows that it drives multiple processes
including the development of heart and neural
systems, however, it usually acts as a transcrip-
tion repressor rather than activator (Hegarty et al.
2015). Systematic study of mouse and human
ESC transcriptome differentiation profiles
revealed that Zeb2 might play important role in
cardiac specialization. Human ESC with silenced
Zeb2 gene proliferate more slowly and fail to
differentiate into mature cardiomyocytes com-
pared to the wild cells (Busser et al. 2015). In
addition, cardiomyocytes with silenced Zeb2 do
not show any contractile properties, although car-
diac differentiation program is activated. More
R. Miksiunas et al.

detailed analysis has revealed that silencing of
Zeb2 gene negatively affects human striated mus-
cle contraction program, including genes related
to calmodulin pathway, HCN and potassium
channels (Busser et al. 2015). Targeted regulation
of Zeb2 gene expression improves
cardiomyogenic processes and heart regeneration.
Zeb2 mutation is also often associated with
Mowat-Wilson syndrome (Garavelli and
Mainardi 2007). Major signs of this disorder fre-
quently include distinctive facial features, intel-
lectual disability, delayed development, an
intestinal disorder called Hirschsprung disease,
Congenital Heart Disease and other types of
birth defects (Garavelli and Mainardi 2007). All
mentioned disorders are related to the improper
heart development caused by the Zeb2 defective
heart cells. In addition, Zeb2 repress epithelial
genes (claudins, tight junction protein 3 (ZO-3),
connexins, E-cadherin, plakophilin
2, desmoplakin, and crumbs3) in order to induce
epithelial to mesenchymal transition (EMT),
which is crucial for the developmental processes
such as gastrulation, neural crest formation, heart
morphogenesis, formation of the musculoskeletal
system, and craniofacial structures (Vandewalle
et al. 2009; Garavelli et al. 2017).
18 LIM Homeobox Class
LIM homeobox class genes encode two Lim
domains and one homeodomain. Lim domain is
a50–60 amino acid length zinc finger motif,
which is primarily involved in protein-protein
interactions, so naturally LIM transcription
factors can interact with multiple proteins in
cell, thus regulating its phenotype.
19 Isl Gene Family of LIM
Homeobox Class
Isl1 is a LIM homeobox class member that
encodes a homeodomain transcription factor
important for cell differentiation, fate determina-
tion and generation of cell diversity in multiple
mouse and human tissues including central nerve
system (CNS), pancreas and heart (Zhuang et al.
2013). During early cardiac development, Isl1,
Nkx2-5 and fetal liver kinase 1 (Flk1) support
the formation of SHF, which gives rise to the
right ventricle, outflow tract and part of the atria
(Dyer and Kirby, 2009). Isl1 promotes expansion,
migration and proliferation of SHF progenitor
cells during the development of the mouse heart
(Witzel et al. 2012). Additionally, Isl1+ mouse
heart cells have potential to differentiate into
multiple cell types within the heart, including
cardiomyocytes, smooth muscle, pacemaker and
endothelial cells (Laugwitz et al. 2007).
There are multiple mechanisms explaining
how Isl1 can promote expression of target
genes, which suggests the expression of Isl1 is
tightly controlled during the mouse heart devel-
opment. For example, Nkx2-5 homeodomain
transcription factor downregulates Isl1 expression
in order to promote ventricular development in
mouse heart (Witzel et al. 2012; Prall et al. 2007).
The newest studies indicate, that Isl1 may repress
development of mouse heart ventricle in order to
promote the development of SAN (Dorn et al.
2015). Mouse embryos overexpressing Isl1
develop SAN-like cells instead of ventricle myo-
cardium (Dorn et al. 2015). It is likely that the
expression of Isl1 activates Nkx2-5 expression in
SHF progenitor cells, however, in later staged of
heart development Nkx2-5 shuts down ISL1
expression to promote ventricular development
(Dorn et al. 2015). Isl1 orchestrates the expres-
sion of hundreds of potential genes implicated in
cardiac differentiation, mainly through epigenetic
mechanisms (Wang et al. 2016). Isl1 in mouse
ESCs acts together with JmjC domain-containing
protein 3 (JMJD3) histone demethylase to pro-
mote the demethylation or tri-methylation of core
histone H3 on the amino (N) terminal tail
(H3K27me3) at the enhancer’s place of key
downstream target genes, such as myocardin
(Myocd), MEF2C and others (Wang et al. 2016).
In addition, Isl1 may reduce histone methylation
near GATA4 and Nkx2-5 genes after the expres-
sion of Isl1 lentiviral gene, and can also recruit
p300 histone acetyltransferase to the promoter of
Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac...

MEF2C gene in order to promote Mef2c expres-
sion in developing mouse embryo (Yu et al.
2013). Other data suggest that lentiviral-induced
overexpression of Isl1 gene promotes not only
MEF2C gene acetylation, but also GATA4 and
Nkx2-5 in C3H10T1/2 mouse cell line (Xu et al.
2016). The tight control of Ils1 gene expression is
required, since it acts as a positive
cardiomyogenic gene regulator reducing methyl-
ation and increasing acetylation levels of genes
and histones by direct and indirect methods. Most
of the published data concerning Isl1 function
have come from the studies of mouse develop-
ment, however there are some studies that link
Isl1 gene expression with the susceptibility to
human congenital heart disease (Luo et al. 2014;
Stevens et al. 2010). Development of mechanisms
that could control expression of Isl1 might be
important target in further regulation of heart
regeneration.
20 PROS Homeobox Class
Homeobox prospero (PROS) genes code atypical
C terminal prospero domain and belongs to a
distinctive class of Prospero homeodomain
proteins (Yousef and Matthews, 2005). The
PROS domain is a DNA binding domain of
approximately 100 amino acids. In addition,
PROS homeobox genes code additional three
amino acids in their HD domain (Yousef and
Matthews 2005).
21 PROX Gene Family of PROS
Homeobox Class
Prospero homeobox 1 or Prox1 is a gene coding a
transcription factor that plays important role in
the development of mouse heart, CNS, eye, liver
and lymphatic system (Elsir et al. 2012). Firstly, it
was discovered in Drosophila as an important
player in the development of central nervous sys-
tem in insects. However, later Prox1 homologues
were found in vertebrates and mammals (Elsir
et al. 2012). In mouse heart development of
Prox1 is important for the sarcomere formation
and muscle contraction (Risebro et al. 2009).
Mouse Prox1 conditional mutants show increased
number of fast twitch fibers compared to slow
twitch fibers. Prox1 mutant mice develop fatal
dilated cardiomyopathy and die around 7–14th
week (Petchey et al. 2014). It was shown that in
mice Prox1 acts as a transcriptional repressor of
genes like Tnnt3,Tnni2 and Myl1 that are essen-
tial for the formation of fast twitch fibbers
(Petchey et al. 2014). Prox1 might also be impor-
tant for the maintenance of cardiac conduction
system in adult mice. It was also shown that
uncontrolled Nkx2-5 expression led to cardiac
conduction defects, surprisingly suggesting that
Prox1 might act as a direct upstream modifier of
Nkx2-5 gene expression (Risebro et al. 2012). In
humans dysregulation of Prox1 gene expression
might also lead to congenital heart disease, like,
hypoplastic left heart (Gill et al. 2009). Thus, the
close connection of Prox1 with Nkx2-5 and other
heart development and diseases regulating genes
makes it an attractive target in cardiac regenera-
tion field.
22 Role of Homeobox Genes
in Cardiomyogenesis
The summarized and reviewed data of estimated
involvement of homeobox genes in the heart
development, diseases and/or regeneration pro-
cesses suggest that some homeobox genes play
more important role than the other. Data
summarized in Table 1show the homeobox
genes that have been most commonly
investigated with important roles in
cardiomyogenesis.
It is quite evident that dozens of homeobox
genes are required for early cardiomyogenesis,
heart septation, formation of pacemaker cell,
cardiomyocyte and etc. Some of the homeobox
genes are directly related to the development of
CHD, atrial fibrillations and other cardiac
pathologies. However, there are much more
R. Miksiunas et al.

homeobox genes related to the heart develop-
ment, that so far have been less investigated or
in one or another model system showed less direct
involvement in cardiomyogenic processes
(Table 2). Data summarized in Table 2also high-
light the fact that many more studies are needed to
understand regulation of homeobox genes and
their role in cardiomyogenic processes.
Table 1 Homeobox genes with major involvement in heart development and diseases
Gene Development Disease Reference
Hox Hoxa1, Hoxb2 and Hoxb2 is
important for anterior-posterior
patterning in SHF. Integrating FGF
and BMP signalling.
Hoxa1 mutations might
cause CHD.
Pearson et al. (2005), Bertrand et al.
(2011), Bosley et al. (2008), Gong
et al. (2005) and Haas et al. (2013)
HOXB13, and HOXC5
mutations might be related
to heart disease.
Nkx2–5Heart looping, heart septation and
cardiac conduction system formation.
Integrates BMP, notch and WNT
signaling during development.
Multiple gene variants and
truncations are related to
CHD.
McCulley and Black (2012), Tanaka
et al. (1999), McElhinney et al.
(2003), Anderson et al. (2018),
Wang et al. (2011), Luxán et al.
(2016) and Cambier et al. (2014)
Hhex Cardiac mesoderm specification. HHex gene variants might
be associated with diabetes.
Liu et al. (2014), Karns et al. (2013),
Kelliny et al. (2009) and Pechlivanis
et al. (2010)
Shox2 Cardiac conduction system
development.
Downregulation during
early-onset atrial fibrillation.
Blaschke et al. (2007) and Hoffmann
et al. (2016)
Hopx Cardiomyocyte progenitor formation
in mouse early heart. Negative
regulator of GATA4 expression.
Downregulated in patients
having cardiac hypertrophy.
Jain et al. (2015), Trivedi et al.
(2010) and Trivedi et al. (2011)
GSC Cardiac mesoderm specification. Zhu et al. (1998)
Pitx2c Establishment of the left-right axis in
heart development. Heart looping and
chamber septation.
Mutations cause improper
ventricle and atrial
chambers septa formation,
atrial fibrillation.
Liu et al. (2002), Wang et al. (2013)
and Wei et al. (2014)
Meis1
and
Meis2
Meis1 and Meis2 control of cell cycle
progression during heart
development.
Meis1 and Meis2 gene
variants might be associated
with CHD.
Mariotto et al. (2013), Arrington
et al. (2012), Wu et al. (2015) and
Louw et al. (2015)
Irx1-6 Irx3 very important for ventricular
conduction system.
Irx1 gene variants might be
associated with CHD.
Christoffels et al. (2000), Guo et al.
(2017), Koizumi et al. (2016) and
Cheng et al. (2011)
Irx4 is associated with the formation
of ventricular myocardium in mouse
and humans.
Irx3 gene defects can cause
lethal cardiac arrhythmias
in human patients.
ZEB2 Controls striated muscle
development and contraction.
Gene variants cause Mowat-
Wilson syndrome. Patients
display CHD and other
defects.
Busser et al. (2015) and Garavelli
and Mainardi (2007)
Islet1 Cell expansion, migration and
proliferation. Marks formation of
SHF. Repress ventricular fate in order
to promote sinoatrial node
development. Positive gene
regulator, which reduces gene and
histone methylation levels and
increase acetylation.
Gene variants might be
related to CHD.
Witzel et al. (2012), Dorn et al.
(2015), Wang et al. (2016) and
Stevens et al. (2010)
Prox1 Important for sarcomere formation
and muscle contraction.
Dysregulation of Prox1
might lead to CHD.
Elsir et al. (2012) and Petchey et al.
(2014)
Homeobox Genes and Homeodomain Proteins: New Insights into Cardiac...

23 Concluding Remarks
Heart development is a complex process requir-
ing strict spatiotemporal development to form a
healthy organ providing properly functioning
organism. Multiple transcription factors, signal-
ling pathways, morphogens and other stimuli
govern the heart development process. However,
it is possible to state that multiple homeobox
genes and their coded transcription factors come
into the heart developmental stages when their
function is needed (Tables 1and 2). None of
these homeodomain transcription factors can be
separated from each other, since their ability to
bind DNA affects patterns of multiple gene thus
resulting in changed transcriptome level of multi-
ple cells.
Homeodomain proteins are not the only tran-
scription factors important for cardiac develop-
ment. The transcription factors of other gene
families also significantly contribute heart devel-
opment. For example, GATA, Tbx, HAND,
Mef2c and other accompany homeodomain
factors like Nkx2-5, Isl1, etc. (Hiroi et al. 2001;
Gao et al. 2011; Maves et al. 2009; Skerjanc et al.
1998). Most of these homeodomain transcription
factors are conserved and display coexistence and
codependence in heart development of human as
well as simple invertebrates like fruit flyor
ascidians (Jensen et al. 2013a; Olson 2006).
Only birds and mammals display fully separated
heart, however reptilians still have no septum
between right and left ventricles (Jensen et al.
2013b). Deeper further insights into septum for-
mation of lower vertebrates like snakes, lizards
and turtles could also help to understand signal-
ling networks of human congenital heart diseases.
Maybe in the future will be possible to engineer a
reptile with four chambered heart, thus leading to
better understanding of cardiac regeneration pro-
cess and allowing to develop new therapeutic
Table 2 Homeobox genes having less important role in heart development and diseases
Class Subclass Gene Development and disease Reference
ANTP HOXL MEOX1
and
MEOX2
Control of vascular endothelial cells proliferation in
mice. Dysregulation might be associated with heart
disease in mouse.
Lu et al. (2018), Douville
et al. (2011)
ANTP NKL Msx1 and
Msx2
Regulate survival of secondary heart field precursors
and post-migratory proliferation of cardiac neural crest
in the outflow tract
Chen et al. (2007)
ANTP NKL Lbx1 Specification of a subpopulation of cardiac neural crest
necessary for normal heart development.
Schäfer et al. (2003)
ANTP NKL Nkx2-6 NKX2-6 mutation predisposes to familial atrial
fibrillation.
Wang et al. (2014)
PRD Prrx1
and
Prrx2
Formation of cardiovascular system and connective
tissues of the heart and in the great arteries and veins.
Bergwerff et al. (2000)
PRD Pax3,
Pax7
Involved in neural crest and cardiac development JA (1996)
ZF
(TALE)
ZFHX3 Genetic polymorphisms in are associated with atrial
fibrillation in a Chinese Han population.
Liu et al. (2014)
PBC
(TALE)
Pbx1 Patterning of the great arteries and cardiac outflow
tract.
Stankunas et al. (2008)
and Arrington et al.
(2012), Chang et al.
(2008)
Pbx acts with Hand2 in early myocardial
differentiation in zebrafish. Non-synonymous variants
in PBX genes are associated with congenital heart
defects.
TGIF
(TALE)
Tgif1 and
Tgif2
Left-right asymmetry formation and embryonic heart
looping.
Powers et al. (2010)
SIX/SO Six2 Six2 marks a dynamic subset of second heart field
progenitors.
Zhou et al. (2017)
R. Miksiunas et al.

strategies for human cardiac congenital and other
types of diseases.
Acknowledgements The study is funded by the Lithua-
nian Research council, project No. S-MIP-17-13.
Ethics Approval and Consent to Participate Not
applicable.
Consent for Publication All authors agree to the publi-
cation of this manuscript.
Availability of Data and Material Not applicable.
Competing Interests The authors declare that they have
no competing interests.
Authors’Contributions RM wrote the manuscript draft.
DB revised the manuscript. AM read, corrected and
approved the final manuscript.
Funding The study is funded by the Lithuanian Research
council, project No. S-MIP-17-13.
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