1 Cardiovascular Research Institute, University of California, San Francisco, CA
94143-0130, USA
2 Department of Biochemistry and Biophysics, University of California, San
Francisco, CA 94143-0130, USA
* Author for correspondence (e-mail: bblack{at}itsa.ucsf.edu)
Accepted 6 May 2004
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SUMMARY |
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Key words: Mef2c, transgenic mouse, cardiac development, transcription, secondary heart field, anterior heart field, ISL1 (ISL-1), GATA4
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Introduction |
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Embryological studies in chick and mouse suggested that the conotruncal
region of the heart was likely to arise from a separate population of myogenic
precursor cells (Kelly and Buckingham,
2002; Yutzey and Kirby,
2002
). Indeed, very recent studies have identified that the
outflow tract and outlet region of the right ventricle are derived from a
precursor population that is distinct from the cells of the primary heart
field (Kelly et al., 2001
;
Mjaatvedt et al., 2001
;
Waldo et al., 2001
). This
population of cells, referred to as the anterior, or secondary, heart field,
is derived from mesoderm anterior to the primary heart field and appears to be
added to the heart at the time of cardiac looping
(Kelly et al., 2001
;
Mjaatvedt et al., 2001
;
Waldo et al., 2001
). The
identification of at least two distinct populations of cells contributing to
the heart provides a likely explanation for the spatial restriction of gene
expression and function that has often been observed in the heart
(Kelly and Buckingham, 2002
;
Schwartz and Olson, 1999
). For
example, the cardiac restricted T box gene Tbx5 plays an important
role in the formation of the atria and left ventricle but is not required for
proper formation of the right ventricle or outflow tract
(Bruneau et al., 2001
),
suggesting that it may not play a prominent role in the anterior heart field.
It is also clear that a number of cardiac genes are controlled by multiple,
separate enhancers that direct spatially restricted expression within the
developing heart (Schwartz and Olson,
1999
).
The observations that the amniote heart forms from multiple progenitor
populations and that a number of genes and transgenes exhibit spatially
restricted expression within the heart suggest that there are likely to be
important differences in the transcriptional pathways governing gene
expression between the two heart fields. Interestingly, many of the key
transcriptional regulators required for cardiac development are expressed in
both the anterior and primary heart fields, and thus it remains unclear how
spatial restriction within the heart is controlled. The homeodomain
transcription factor NKX2.5 and the zinc finger transcription factor GATA4 are
the first cardiac restricted transcription factors to be expressed during
development, and both are expressed throughout the precardiac mesoderm,
including the anterior heart field (Arceci
et al., 1993; Chen and Fishman,
2000
; Harvey,
1996
; Lints et al.,
1993
; Parmacek and Leiden,
1999
). The functions of GATA4 and NKX2.5 are essential for cardiac
development (Kuo et al., 1997
;
Lyons et al., 1995b
;
Molkentin et al., 1997
), and
the two factors function as part of a combinatorial transcriptional complex to
cooperatively activate cardiac gene expression
(Durocher et al., 1997
;
Lee et al., 1998
;
Sepulveda et al., 1998
;
Sepulveda et al., 2002
).
However, the role of these transcription factors in gene expression within the
anterior heart field, and how these factors fit together into transcriptional
pathways with other key transcriptional regulators, remains to be
determined.
Very recent studies by Evans and colleagues demonstrated a critical role
for the LIM-homeodomain transcription factor ISL1 (also known as ISL-1) in
anterior heart field development (Cai et
al., 2003). ISL1 was first identified as a key regulator of the
rat insulin I gene enhancer and expression was observed in endocrine
cells of the pancreas (Karlsson et al.,
1990
). During embryonic development, however, Isl1 is
also expressed in the crescent-shaped pattern of the anterior heart field,
medial and dorsal to the MLC2a (My17 Mouse Genome
Informatics) expressing cells in the primary heart field
(Cai et al., 2003
). By
mid-gestation, Isl1 is expressed broadly outside the anterior heart
field, but expression in the cardiogenic region is restricted to the
pharyngeal mesoderm of the anterior heart field, and fate mapping studies
demonstrated that the Isl1 expressing cells from the anterior heart
field contribute to the vast majority of cells in the outflow tract and right
ventricle in the embryo (Cai et al.,
2003
). Targeted disruption of Isl1 results in embryonic
lethality between 9.5 and 10.5 dpc (Pfaff
et al., 1996
). Mice lacking Isl1 have severely misshapen,
unlooped hearts at 9.5 dpc and the right ventricle and outflow tract fail to
form in these embryos, demonstrating an essential role for ISL1 in anterior
heart field development (Cai et al.,
2003
). While these studies demonstrated a critical and previously
unappreciated role for ISL1 in cardiac development, no direct targets of ISL1
have been identified in the anterior heart field.
Members of the myocyte enhancer factor 2 (MEF2) transcription factor family
also play key roles in cardiac myogenesis. There are four mef2 genes
in vertebrates and a single mef2 gene in Drosophila
(Black and Olson, 1998). In
mice, Mef2c is among the earliest markers of the cardiac lineage with
transcripts evident in the developing heart beginning at about 7.5 dpc,
shortly after the expression of the Nkx2.5 and Gata genes
(Arceci et al., 1993
;
Edmondson et al., 1994
;
Harvey, 1996
;
Lin et al., 1997
;
Lints et al., 1993
;
Morrisey et al., 1996
;
Morrisey et al., 1997
;
Parmacek and Leiden, 1999
).
Despite the early expression of Mef2c in the heart, MEF2 activity is
not required for muscle specification; instead, it appears to play a key role
in differentiation. This notion is strongly supported by work in
Drosophila in which inactivation of the single Mef2 gene
results in a complete loss of muscle differentiation in all three muscle
lineages with no apparent defect in myoblast specification
(Bour et al., 1995
;
Lilly et al., 1995
;
Ranganayakulu et al., 1995
).
The role of MEF2 factors in vertebrate muscle is supported by the observation
that a dominant negative form of MEF2 is sufficient to block differentiation
in cultured mouse myoblasts (Ornatsky et
al., 1997
). The essential role for MEF2 activity in vertebrate
cardiac development is apparent from genetic studies in mice, in which
disruption of the Mef2c gene results in embryonic lethality at 9.5
dpc due to cardiac and vascular defects (Bi
et al., 1999
; Lin et al.,
1997
; Lin et al.,
1998
). The cardiac phenotype in Mef2c null embryos is
nearly identical to the heart phenotype observed in Isl1 null embryos
(Cai et al., 2003
;
Lin et al., 1997
). The hearts
of Mef2c null embryos fail to undergo looping, they have gross
abnormalities in the outflow tract, and the right ventricle fails to form
(Lin et al., 1997
), suggesting
a key role for Mef2c in the transcriptional pathways regulating
anterior heart field development. However, despite the importance of
Mef2c in cardiac development, nothing has been elucidated regarding
its transcriptional regulation in the heart.
In the present study, we used in vivo bacterial artificial chromosome (BAC) recombination to generate a Mef2c-lacZ BAC that recapitulates the endogenous pattern of Mef2c expression in transgenic embryos. By comparing the human and mouse genomic sequence in the region encompassed by the mouse Mef2c-lacZ BAC, we identified multiple regions of conservation in Mef2c noncoding regions. One of these regions of conservation represents an enhancer that is sufficient to direct transcription to the anterior heart field, but not to other regions where endogenous Mef2c is expressed. We show that this Mef2c enhancer begins to function by 7.5 dpc in the precardiac mesoderm and continues to direct expression to the anterior heart field and its derivatives in the outflow tract and right ventricle throughout embryonic and fetal development. This enhancer contains two evolutionarily conserved GATA binding sites that are bound by GATA4 with high affinity and are completely required for enhancer function in vivo. We also show that the Mef2c anterior heart field enhancer is a direct transcriptional target of ISL1 via two evolutionarily conserved, perfect consensus ISL1 binding sites, which are essential for enhancer function in vivo. Thus, these studies identify Mef2c as the first direct target of ISL1 in the anterior heart field and support a model in which ISL1 and the GATA family of transcription factors serve as the earliest regulators of the transcriptional program controlling the development of the outflow tract and right ventricle.
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Materials and methods |
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Cloning and mutagenesis
A 6142 bp fragment of the mouse Mef2c gene and eight additional
deletion fragments were generated by PCR with primers designed to correspond
to the nucleotide numbers given for each enhancer construct in this study (see
Fig. 3A). Each Mef2c
enhancer fragment was cloned into the transgenic reporter plasmid
HSP68-lacZ (Kothary et al.,
1989) except for Mef2c-F6/Frag3, which uses an endogenous
promoter from Mef2c and was cloned into the promoterless
lacZ reporter plasmid AUG-ß-gal
(McFadden et al., 2000
).
Mutations were introduced into the wild-type Mef2c-F6/Frag2 cardiac
enhancer fragment (nucleotides 1-3970) by PCR as described
(Dodou et al., 2003
) to create
the following mutant sequences: mGATA-p,
5'-AAGTCACCCGCTTGCTAGCGGTCAGGGGAGC-3'; mGATA-d,
5'-CTAAGAGTTCTGGCCAGTGTCTGCTC-3'; mISL-p,
5'-GGTTTACTTGCTAGGTACCTGGATAAAG-3'; mISL-d,
5'-GGTCAGGGGAGCCTAGGTCATTTGGG-3'. For the double GATA mutant,
mGATA-p/d, the GATA-p mutation was introduced into the GATA-d mutant
background to create the double mutant enhancer. For the double ISL mutant,
mISL-p/d, the ISL-d mutation was introduced into the ISL-p mutant background
to create the double mutation. The entire sequence of each mutant fragment was
confirmed by sequencing on both strands. The GenBank accession number for the
sequence of the mouse Mef2c anterior heart field enhancer described
in these studies is AY324098.
|
Electrophoretic mobility shift assay (EMSA)
DNA-binding reactions were performed as described previously
(Dodou et al., 2003). Briefly,
double-stranded oligonucleotides were labeled with 32P-dCTP using
Klenow to fill in overhanging 5' ends and purified on a nondenaturing
polyacrylamide-TBE gel. Binding reactions were pre-incubated at room
temperature in 1xbinding buffer (40 mM KCl, 15 mM HEPES pH 7.9, 1 mM
EDTA, 0.5 mM DTT, 5% glycerol) containing 2 µg of either recombinant GATA4,
recombinant ISL1, or unprogrammed reticulocyte lysate, 1 µg of poly dI-dC,
and competitor DNA (100-fold excess where indicated) for 10 minutes prior to
probe addition. Reactions were incubated an additional 20 minutes at room
temperature after probe addition and electrophoresed on a 6% nondenaturing
polyacrylamide gel. Recombinant GATA4 and a truncated version of the ISL1 cDNA
containing the homeodomain were generated from plasmids pCITE-GATA4 and
pCITE-ISL1, respectively, using the TNT Quick Coupled
Transcription/Translation System as described in the manufacturer's directions
(Promega). pCITE-GATA4 was generated by cloning the complete Rattus
norvegicus GATA4 coding region into the translational enhancement vector
pCITE-2A (Novagen). pCITE-ISL1 was generated by cloning an EcoRI
fragment from the hamster Mesocricetus auratus Isl1 cDNA
(Wang and Drucker, 1994
)
encoding the C-terminal 231 amino acids of ISL1 as an in-frame fusion into the
translational enhancement vector pCITE-2A (Novagen). This fragment of the ISL1
cDNA has had the N-terminal 118 amino acids removed. These residues include
the ISL1 LIM domains, which have been shown previously to inhibit DNA binding
in vitro (Sanchez-Garcia and Rabbitts,
1993
). The Nkx2.5 gs1 control GATA4 binding site and the
mutant gs1 (M1) oligonucleotides have been described
(Lien et al., 1999
). The sense
strand sequence of the wild-type and mutant ISL1 site control oligonucleotides
from the Insulin I promoter were generated based on previously
published studies (Karlsson et al.,
1987
) and were: In,
5'-GCCCTTGTTAATAATCTAATTACCCTAG-3'; mIn,
5'-GCCCTTGTCGACGATCCGGTTACCCTAG-3'. The sense strand sequences of
the Mef2c oligonucleotides used for EMSA were: GATA-p,
5'-GGTCACCCGCTATCTATCGGTCAGGCC-3'; GATA-d,
5'-GGTAAGAGTTCTTATCAGTGTCC-3'; mGATA-p,
5'-GGTCACCCGCTTGCTAGCGGTCAGGCC-3'; mGATA-d,
5'-GGTAAGAGTTCTGGCCAGTGTCC-3'; ISL-p,
5'-GGTTTACTTGCTAATGACCTGGATAAC-3'; ISL-d,
5'-GTCAGGGGAGCCTAATGCATTTGGGAAC-3'; mISL-p,
5'-GGTTTACTTGCTAGGTACCTGGATAAC-3'; mISL-d,
5'-GTCAGGGGAGCCTAGCTCATTTGGGAAC-3'.
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Results |
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To identify transcriptional enhancers within the Mef2c locus, we
isolated a 120 kb bacterial artificial chromosome (BAC) encompassing the
5' end of the mouse Mef2c gene and introduced lacZ
using in vivo BAC recombination (Yang et
al., 1997). The lacZ gene was introduced into the first
coding exon such that the Mef2c start codon was deleted and replaced
with the lacZ gene, but the 5' and 3' boundaries of the
exon were preserved (Fig. 1A).
This BAC recombinant (Mef2c-L8-lacZ) was used to generate
transgenic embryos to determine if the BAC sequence contained transcriptional
enhancers sufficient to direct expression of lacZ in vivo. Expression
directed by the BAC transgene could be observed first at 7.5 dpc in the
anterior heart field (Fig. 1C),
a crescent-shaped region of splanchnic mesoderm adjacent and medial to the
primary heart field (Cai et al.,
2003
; Kelly et al.,
2001
). Similarly, expression at 8.0 dpc was readily apparent in
the pharyngeal mesoderm at the anterior end of the linear heart tube
(Fig. 1D). By 8.5 dpc,
expression was still readily apparent in the anterior heart field, but other
regions known to express endogenous Mef2c, including neural crest and
vascular endothelium, began to be marked by lacZ expression
(Fig. 1E). By 9.5 dpc, all
developing tissues known to express endogenous Mef2c
(Edmondson et al., 1994
),
including pharyngeal mesoderm, heart, somites, branchial arches, neural crest
and vasculature expressed the Mef2c-L8-lacZ BAC transgene
(Fig. 1F). These results
indicated that the transcriptional enhancers responsible for directing
Mef2c expression in vivo were contained within the sequence of the
120 kb Mef2c BAC. In particular, the early expression of the BAC
transgene in the anterior heart field (Fig.
1C,D) indicated that elements responsible for expression in this
domain were present within the Mef2c BAC sequence. Therefore, to
identify discrete enhancer elements residing within the BAC sequence, we
compared the mouse and human Mef2c genes for homology by
visualization tools for alignments (VISTA) analysis
(Mayor et al., 2000
), which
revealed 11 regions of strong conservation in Mef2c noncoding
sequences.
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The Mef2c anterior heart field regulatory region contains an endogenous promoter and is controlled by a small, evolutionarily conserved enhancer module
Our screen of all the conserved sequences from the Mef2c locus
utilized a strategy in which each conserved region was fused to the HSP68
heterologous promoter to determine if a given region conferred specific
enhancer activity. Because the anterior heart field regulatory region from the
Mef2c gene encompasses the third untranslated exon from the
Mef2c gene (depicted in Fig.
1A), we reasoned that this regulatory module might contain its
own, endogenous promoter. To determine if promoter sequences were present
within the Mef2c anterior heart field regulatory module, we cloned
nucleotides 1-3970, which includes the upstream region of the module and the
first 76 nucleotides of untranslated exon 3, into the promoterless transgenic
reporter plasmid AUG-ß-gal (McFadden
et al., 2000) and tested for activity in transgenic embryos
(Fig. 3A, Frag #3). This region
of the enhancer directed robust expression in transgenic embryos when fused to
the HSP68 promoter (Fig. 3A, Frag #2; Fig. 3B). Similarly,
this region of the Mef2c gene also directed strong expression to the
anterior heart field in the absence of a heterologous promoter
(Fig. 3C), indicating the
presence of an endogenous promoter immediately upstream of the third
untranslated exon (Fig.
3A).
Next we wanted to define the minimal region of the enhancer that was required for expression in vivo as a first step toward defining a transcriptional pathway upstream of Mef2c in the anterior heart field. Toward this goal, we tested a series of enhancer deletion fragments for the ability to direct cardiac specific expression in transgenic embryos (Fig. 3A). These analyses identified a 1.4 kb fragment (Fig. 3A, Frag #6) that was sufficient to direct robust expression of lacZ exclusively to the anterior heart field (Fig. 3D). By contrast, a 1.1 kb fragment (Fig. 3A, Frag #7) was completely incapable of directing any expression in vivo (Fig. 3E). Thus, these results indicate that the 286 bp region between nucleotides 2584 and 2869 is essential for cardiac expression and that one or more critical cis-acting elements must reside in that region. Based on these observations, we tested the 286 bp region for enhancer function in vivo to determine if this region of the Mef2c gene was sufficient for enhancer activity (Fig. 3A, Frag #9). In five independent transgenic events, this region of the enhancer was never sufficient to direct lacZ expression (Fig. 3F), indicating that additional sequences were required for enhancer function in vivo. Importantly, addition of 163 nucleotides (Fig. 3A, Frag #10) conferred strong anterior heart field specific expression (Fig. 3G). These results indicated that additional cis-acting elements required for anterior heart field expression must also reside within the 163 bp between nucleotides 2869 and 3032. Taken together, these deletional analyses identified a 449 bp minimal enhancer element (nucleotides 2584-2869) sufficient for anterior heart field specific expression in vivo, which contains two smaller regions that are each required for enhancer function.
The Mef2c anterior heart field enhancer is dependent on essential GATA and ISL1 sites
To identify specific transcription factor binding sites within the minimal
sufficiency region of the enhancer, we analyzed it for evolutionarily
conserved sequences that might serve as potential cis-regulatory
elements. These analyses identified several putative candidate binding sites
for cardiac gene expression. Among these were three conserved candidate
binding sites for the GATA family of transcription factors that resided
between nucleotides 2584 and 2869. The proximal GATA site (GATA-p) consisted
of a conserved, perfect consensus element; the medial GATA site (GATA-m)
consisted of a conserved, imperfect GATA element; and the distal GATA site
(GATA-d) represented a conserved, perfect consensus GATA sequence
(Fig. 4). These analyses also
identified two conserved perfect consensus elements for the LIM-homeodomain
transcription factor ISL1 that were present between nucleotides 2869 and 3032
(Fig. 4). In addition, we
identified two candidate E boxes, representing potential binding sites for
HAND factors, and a putative NKE binding site for NK class factors such as
NKX2.5 (Fig. 4). As a first
test to determine if these candidate sites might represent bona fide binding
sites, we tested each of these elements for the ability to bind to their
respective candidate transcription factors by EMSA.
|
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Taken together, the results of the EMSA and mutational analyses demonstrate that the Mef2c anterior heart field enhancer is bound by GATA factors and by ISL1 and that the evolutionarily conserved binding sites for these factors are completely required for enhancer function in vivo. These data also suggest that Mef2c expression in the anterior heart field is not dependent on direct activation by NKX2.5 or HAND proteins. Overall, the results presented in this study support a model for anterior heart field expression of Mef2c that is dependent on cardiac restricted GATA transcription factors and ISL1 through direct binding and activation of a discrete anterior heart field enhancer module.
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Discussion |
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A GATA- and ISL1-dependent model for transcriptional activation in the anterior heart field
The Gata4/5/6 genes are among the earliest markers of the cardiac
lineage in the developing mouse, with expression appearing throughout the
precardiac mesoderm as early as the expression of Nkx2.5
(Arceci et al., 1993;
Parmacek and Leiden, 1999
).
This very early expression suggests that GATA transcription factors are likely
to be involved in the initial specification of cardiomyocytes, which is
supported by studies in embryonal carcinoma stem cells that showed that
expression of GATA4 dominantly induced precocious cardiac differentiation
(Grepin et al., 1995
).
Furthermore, expression of a dominant negative form of GATA4 in those cells
blocked cardiac differentiation (Grepin et
al., 1997
). Additional evidence for a role for GATA factors in
cardiac specification comes from studies in zebrafish, which demonstrated that
GATA5 could dominantly induce cardiogenesis and the expression of
Nkx2.5 (Reiter et al.,
1999
). Likewise, misexpression of murine GATA4 in
Drosophila results in expansion of cardiac cells in the dorsal
mesoderm (Gajewski et al.,
1999
).
The expression directed by the Mef2c enhancer described here is
restricted to the anterior heart field throughout development. This enhancer
contains two conserved GATA sites, which are bound by GATA4 with high affinity
and are required for enhancer activity. These results indicate that
Mef2c is a direct target of GATA factors and support a model in which
GATA factors play a crucial role in the specification of myocytes in the
anterior heart field. Additional support for this model comes from studies
showing that Nkx2.5 and dHAND also appear to be direct
transcriptional targets of GATA factors in the anterior heart field
(Lien et al., 1999;
McFadden et al., 2000
;
Searcy et al., 1998
).
Importantly, the cardiac restricted GATA transcription factors, GATA4/5/6, are
expressed more broadly than the anterior heart field restricted pattern of the
Mef2c-lacZ transgene described in this study
(Arceci et al., 1993
;
Morrisey et al., 1996
;
Morrisey et al., 1997
;
Parmacek and Leiden, 1999
).
The broader expression pattern of the GATA factors suggests that additional
regulators must be required to restrict enhancer function to the anterior
heart field. Indeed, in this study, we show that the Mef2c anterior
heart field enhancer is also a direct target of ISL1. As is the case for the
GATA factors, ISL1 expression within the embryo is broader than the pattern
directed by the Mef2c enhancer described here, but expression of ISL1
within cardiogenic lineages appears to be limited to the anterior heart field
(Cai et al., 2003
). Thus, these
data suggest a model for anterior heart field-specific transcriptional
activation dependent on the combined activities of ISL1 and GATA transcription
factors.
The Mef2c enhancer described here is the first identified direct
transcriptional target of ISL1 in the anterior heart field. It will be
interesting to determine if other anterior heart field restricted genes or
enhancers are also dependent on ISL1 for activation. Notably, the
GATA-dependent cardiac enhancer from the Nkx2.5 gene, which appears
to direct expression to the anterior heart field, also contains a perfect
consensus site for ISL1 in close proximity to the essential GATA binding site,
although the function of the ISL1 site in that enhancer was not investigated
(Lien et al., 1999). GATA
factors have been shown to function cooperatively with other transcription
factors expressed in the heart, including SRF, NKX2.5, dHAND and MEF2C
(Dai et al., 2002
;
Durocher et al., 1997
;
Lee et al., 1998
;
Morin et al., 2000
;
Sepulveda et al., 2002
), and
it will be interesting to determine if GATA factors function as part of a
cooperative transcriptional complex with ISL1 as well.
Distinct transcriptional hierarchies in the primary and secondary heart fields
A GATA- and ISL1-dependent transcriptional cascade in the anterior heart
field contrasts with the hierarchy present in the primary heart field and in
the Drosophila heart. In Drosophila, the Nkx2.5
ortholog, tinman, is the earliest marker of the heart
(Frasch, 1999) and is thought
to be the initial transcriptional activator responsible for cardiac
specification (Cripps and Olson,
2002
; Frasch,
1999
). In the fly heart, the GATA transcription factor gene,
pannier, is a direct downstream target of Tinman, which also directly
activates the transcription of Mef2
(Gajewski et al., 1997
;
Gajewski et al., 2001
). A
similar NKX dependent hierarchy appears to exist in the primary heart field in
vertebrates as well. NKX2.5 is among the earliest known cardiac transcription
factors expressed in the primary heart field in vertebrates
(Brand, 2003
;
Harvey, 1996
;
Lints et al., 1993
), and some
evidence suggests that NKX2.5 may play a role in cardiomyocyte specification
(Yamagishi et al., 2001
).
Furthermore, recent studies of the Nkx2.5 gene have identified
several enhancers regulating expression in the heart
(Schwartz and Olson, 1999
).
One of these enhancers directs expression to the cardiac crescent and primary
heart tube and was shown to be dependent on SMAD proteins in vivo
(Liberatore et al., 2002
;
Lien et al., 2002
). These
observations suggest that Nkx2.5 may be a direct target of
BMP-inducing activity, and that this enhancer may serve as the initiator of
cardiac specification in the primary heart field
(Liberatore et al., 2002
;
Lien et al., 2002
). Taken
together, all these observations suggest that the transcriptional hierarchies
for myocyte development in the two vertebrate heart fields might be divergent,
with NKX2.5 as the critical factor in the primary heart field and GATA
factors, along with ISL1, as the key transcriptional regulators in the
anterior heart field.
Modular regulation of Mef2c expression
An important question that remains to be defined is how the cells derived
from the anterior and primary heart fields are integrated during cardiac
morphogenesis. Cells appear to be added progressively from the pharyngeal
mesoderm into the conotruncal region of the heart at the time of cardiac
looping, and evidence suggests that this addition ultimately contributes to
the outflow tract and much of the right ventricle
(Kelly et al., 2001;
Mjaatvedt et al., 2001
;
Waldo et al., 2001
). However,
the precise boundaries within the adult mouse heart derived from the anterior
heart field remain to be defined (Kelly
and Buckingham, 2002
). In this regard, the Mef2c enhancer
described here might be useful in fate mapping the contributions of the
anterior heart field to the adult heart. The results presented in
Fig. 2 show that the
Mef2c enhancer is sufficient to direct expression to the anterior
heart field but not to other regions where endogenous Mef2c is
expressed and that this enhancer does not function in the adult heart. Thus,
we are currently using this enhancer to drive the expression of Cre
recombinase in lacZ reporter mice
(Soriano, 1999
) to identify
regions of the adult heart that are derived from the Mef2c expressing
cells in the anterior heart field in the embryo.
The expression pattern of Mef2c during mouse development is
complex. Transcripts are abundant in heart, skeletal muscle, vasculature and
neural crest (Edmondson et al.,
1994; Leifer et al.,
1993
; Lyons et al.,
1995a
). The studies presented here have defined for the first time
a transcriptional enhancer from Mef2c sufficient to direct expression
only to the anterior heart field, and previous studies have identified a
transcriptional enhancer from Mef2c that functions only in skeletal
muscle (Dodou et al., 2003
;
Wang et al., 2001
). The
identification of these discrete enhancer modules strongly suggests that the
expression of Mef2c is controlled by separate transcriptional
enhancers in each lineage where it is expressed. This type of modular model
for transcriptional regulation dependent on multiple, separate enhancers has
been proposed previously as a mechanism for regulatory diversity
(Firulli and Olson, 1997
).
This seems to be the case for Mef2c, as we have recently identified
additional enhancers from the Mef2c BAC
(Fig. 1A) that are capable of
independently directing lacZ expression to vascular endothelium,
smooth muscle and neural crest (M.V., J.A. and B.B., unpublished
observations). It will be interesting to determine whether these enhancers
function as truly independent modules or whether they operate together in the
context of the entire Mef2c locus.
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ACKNOWLEDGMENTS |
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REFERENCES |
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