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First published online 18 October 2006
doi: 10.1242/dev.02625
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1 Molecular Medicine Unit, UCL Institute of Child Health, London WC1N 1EH,
UK.
2 Developmental Biology Division, National Institute for Medical Research, The
Ridgeway, Mill Hill, London NW7 1AA, UK.
* Author for correspondence (e-mail: p.riley{at}ich.ucl.ac.uk)
Accepted 12 September 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Heart, Hand1, Mouse, Tet-Off, Outflow tract, Cardiomyocyte, Proliferation, Differentiation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). These cells
comprise the so-called primary heart field that later contributes mainly to
the left ventricle (Cai et al.,
2003). Subsequent rapid elongation coincident with cardiac looping
then occurs following the recruitment of cells to the growing poles of the
heart (Kelly and Buckingham,
2002). At the arterial pole, the heart tube is extended by the
addition of extracardiac cells that originate from progenitors in the
pharyngeal mesoderm, dubbed the anterior (or secondary) heart field (AHF)
(Kelly et al., 2001;
Mjaatvedt et al., 2001;
Waldo et al., 2001).
Cardiomyocytes arising from the AHF take on right ventricular and outflow
tract (OFT) identities (Zaffran et al.,
2004). More recently, the AHF itself has been further redefined as
a subpopulation of a broader second lineage that not only contributes to the
right ventricle and OFT at the arterial pole of the heart, but also to the
atria and inflow regions at the venous pole
(Meilhac et al., 2004).
Inroads into deciphering transcriptional regulation in the cardiac lineages
suggest a requirement for combinatorial activities of multiple transcription
factors that have an impact on differentiation and deployment of myocardial
precursor cells. However, factors that maintain a balance between
proliferation and differentiation of different populations of myocardial
precursors have not been identified.
Hand1 (previously known as eHAND, Hxt and Thing1), a member of the bHLH
transcription factor family, is a candidate regulator of cardiac precursor
cell fate. Hand1 is expressed in distinct regions of the linear heart
tube and, post-looping, becomes localised to both primary heart field and
second lineage derivatives, specifically in the outer curvature of the
presumptive left ventricle and the developing OFT, and also at lower levels in
the outer curvature of the right ventricle. The role of Hand1 in heart
development has previously been explored in mice lacking Hand1, but
detailed analysis is confounded by early embryonic lethality resulting from
extra-embryonic defects (Firulli et al.,
1998; Riley et al.,
1998). Rescue of the extra-embryonic defects by tetraploid
aggregation experiments yielded limited insight into the precise cellular role
of Hand1 in the heart because embryos were only partially rescued, with failed
looping morphogenesis, defective chamber septation and impaired ventricular
development (Riley et al.,
1998).
Controversy surrounds the specific function of Hand1 in the heart in terms
of whether it is involved in regulating looping morphogenesis as a primary
role that feeds back directly onto ventricular development, or whether it is
restricted to ventricular specification and maturation coincident with
looping. Conditional targeting of Hand1 has not resolved this issue,
as the defects observed following heart-specific knockout of Hand1
(McFadden et al., 2005) are
less severe than would be predicted based on the reported phenotype of
tetraploid-rescued conventional Hand1-null embryos
(Riley et al., 1998). In the
conditional targeting study by McFadden and colleagues
(McFadden et al., 2005),
Hand1 cardiac knockouts under the control of a Nkx2.5 Cre driver
formed hearts that clearly looped and had a degree of left-ventricular
expansion. These observations are significantly milder than previously
described for the tetraploid-rescued mutants, which are characterised by a
complete failure in looping morphogenesis and aberrant ventricular
differentiation (Riley et al.,
1998). Putative limitations with respect to the Cre approach taken
are probably caused by mosaicism of the Cre expression under the described
Nkx2.5 promoter (McFadden et al.,
2005) and/or a temporal delay in efficient Cre excision resulting
in Hand1 being inactivated at the heart tube stage, excluding an effect on
earlier cells expressing Hand1
(Buckingham et al., 2005).
Nevertheless, the conditional, heart-specific Hand1 mutant embryos
presented with left-ventricular hypoplasia, leading to the postulation that
this specific anomaly could be the result of a proliferation defect in the
primary heart field lineage (Buckingham et
al., 2005). However, because of the early lethality of mouse
embryos lacking Hand1 and the limited insight gained from the
conditional knockout approach, it was necessary to develop a model that would
enable us to express Hand1 in an appropriate spatially and temporally
controlled manner, circumventing complications associated with extra-embryonic
defects, which could then be used to assess its precise role during heart
development. To achieve this, we engineered the tetracycline system into the
endogenous Hand1 locus. The Tet-Off transactivator (tTA) was targeted
specifically by homologous recombination and thus placed under the control of
the endogenous Hand1 promoter and regulatory regions. The resulting
mouse tTA knock-in strain (driver) was then crossed to multiple transgenic
lines (responders) to regulate expression of a tet-responsive Hand1
transgene. In light of the limitations of pre-existing loss-of-function mouse
models, we sought to use the tet-system knock-in approach to induce
overexpression of Hand1, restricted exclusively to
Hand1-expressing regions, and to facilitate the analyses of early-
and late-onset functions of Hand1 in the developing heart. At E9.5,
overexpression of Hand1 disrupted cardiac morphogenesis and brought
about an extension of the heart tube and extraneous looping caused by the
elevated proliferation of cardiac precursors in the distal OFT. In
complimentary studies employing in vitro differentiated embryonic stem (ES)
cells, the complete loss of Hand1 resulted in elevated cardiomyocyte
differentiation, whereas Hand1 gain of function maintained
cardiomyocyte proliferation, via G1 progression and delayed cell-cycle exit.
The combined in vivo and in vitro studies, therefore, indicate a
cell-autonomous role for Hand1 in regulating the balance between cardiomyocyte
differentiation and proliferation during cardiac development.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and neomycin-resistance cassette from pPNT
(Tybulewicz et al., 1991)
flanked by loxP sites were cloned downstream of the tTA. This construct was
then subcloned into a SalI-BglII digest of a
Hand1-targeting vector described previously
(Riley et al., 2000). For the
responder transgenic construct, a full-length Hand1 cDNA was cloned
downstream of the tetracycline response element (TRE) in pTRE2 (Clontech) and
a neomycin-resistance cassette was inserted downstream in the opposing
orientation. The driver and responder mouse lines were generated by genOway
(Lyon, France). The driver targeted line was established by homologous
recombination in 129sv ES cells. Correctly targeted ES-cell clones were
identified by Southern blot analysis, using a BglII digest of genomic
DNA and a 5' probe that is a 2 kb EcoRI/KpnI fragment
that hybridises to a 13 kb wild-type band and a 9 kb targeted mutant band. The
responder transgenic strain was generated by `Safe DNA
Transgenesis'
(http://www.genoway.com/).
Positive ES-cell clones were again identified by Southern blot (see
Fig. 1A-D). Transgene copy
number was estimated by scanning densitometry using the Scion Image program
(Scion Corporation;
http://www.scioncorp.com/frames/fr_scion_products.htm).
Positive clones were subsequently used for injection into C57BL/6-derived
blastocysts for generation of chimeras using standard procedures, for both the
targeted driver and transgenic responder lines.
Mouse maintenance and genotyping
Mice were maintained on a mixed background. Embryos were generated by
mating heterozygous driver and responder mice. A total of 654 embryos were
analysed and all were stage-matched to controls by somite count. The targeted
driver allele was identified by a 690 bp product
(S-5'-GGGGTGGGGTGGGATTAGAT-3' and
AS-5'-AGAAGGGCCCAGGGAAGACT-3'). The presence of the responder
transgene was detected by a 2.2 kb product
(S-5'-GCGCCTGGCTACCAGTTACA-3' and
AS-5'-GGGGTGGGGTGGGATTAGAT-3').
ES cells and embryoid bodies
ES cells containing the transgenic responder construct were co-transfected
with pBIG3r (Strathdee et al.,
1999) containing the tTA under the control of a TK promoter and
pTK-Hyg (Clontech). Positive clones stably expressing the tTA were isolated
following hygromycin selection and presence of the tTA was determined by a 150
bp PCR product (S-5'-CAGCGCATTAGAGCTGCTTA-3' and
AS-5'-ATCTCAATGGCTAAGGCGTC-3'). RT-PCR was performed to confirm
that the positive clones expressed both the tTA (primers as above) and the
Hand1 transgene, as detected by a 400 bp PCR product
(S-5'-GATGGGACTGGAGAAGACCA-3' and
AS-5'-GAAGTCAGATGCTCAAGGGG-3').
ES cells were cultured and differentiated in vitro to form embryoid bodies
(EBs) derived from wild-type, control (transgenic responder construct alone),
Hand1-overexpressing and Hand1 heterozygous or null lines,
as previously described (Riley et al.,
2000; Smart et al.,
2002). Single cardiomyocytes were isolated from adherent EB
cultures at days 10 and 14, according to the protocol described previously
(Maltsev et al., 1993).
RT-PCR
mRNA was isolated from whole embryos, cells and EBs using the Micro
FastTrack 2.0 kit (Invitrogen), according to the manufacturer's instructions.
The RNA was DNase treated before reverse transcription was performed using
Superscript II RT (Invitrogen), according to the manufacturer's instructions.
PCR was performed using standard conditions.
Optical projection tomography
Optical projection tomography (OPT) was performed essentially as described
(Sharpe et al., 2002) on
whole-mount embryos. Analysis and visualisation of OPT data was performed with
Improvision Volocity 3.1.
Histology
Embryos were fixed, dehydrated and wax embedded as previously described
(Moorman et al., 2001). They
were then serially sectioned at 10-15 µm. In situ sections were
counterstained with 0.5% Eosin, as described previously
(Smart et al., 2002).
RNA in situ hybridisation
RNA in situ hybridisation on whole-mount embryos and sections was performed
as previously described (Moorman et al.,
2001; Smart et al.,
2002), using riboprobes specific for Hand1
(Cserjesi et al., 1995), atrial
natriuretic factor (Anf; Nppa - Mouse Genome Informatics) and
Gata4 (Kuo et al.,
1997), islet 1 (Isl1) and Wnt11
(Cai et al., 2003),
Nkx2.5 (Lints et al.,
1993), Hand2
(Srivastava et al., 1997), and
myocyte enhancer factor 2c (Mef2c)
(Edmondson et al., 1994).
Immunofluorescence
Sections were dewaxed and rehydrated
(Moorman et al., 2001), and
stained with antibodies against MF20 (1/10; Developmental Studies Hybridoma),
phospho-histone H3 (1/200; Upstate) and cleaved caspase 3 (1/100; Cell
Signalling Technology). Sections were then counterstained with the nuclear
marker bis-benzamide, as previously described
(Hill and Riley, 2004;
Riley et al., 2000).
Real-time PCR
Real-time qRT-PCR was carried out on RNA extracted from embryos at E9.5 and
EBs following 14 days of differentiation. This was performed on an ABI 7000
Sequence Detector (Applied Biosystems) using SYBR Green (Absolute QPCR SYBR
Green Mix, ABgene). Complementary DNA PCR primers were obtained from published
sequences (Fijnvandraat et al.,
2003a; Fijnvandraat et al.,
2003b; Lekanne Deprez et al.,
2002) or designed using Primer Express software (version 2.0,
Applied Biosystems).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
To determine whether the tTA was expressed in Hand1-expressing
lineages in the driver strain, we performed RNA in situ hybridisation analysis
to detect tTA transcripts in whole-mount embryos and in sections at E9.5. The
results confirmed that tTA expression from a gross whole-mount perspective
colocalises with Hand1-expressing regions, notably in the branchial
arches, lateral plate mesoderm, distal OFT and left ventricular (LV)
myocardium at E9.5 (Fig. 2A,B).
In histological sections, there were some differences between Hand1 and tTA
expression, reflecting a degree of mosaicism in the tTA driver, notably
ectopic expression in the first branchial arch
(Fig. 2E,F). As we have taken
the approach of knocking-in the tTA driver into the endogenous Hand1
locus and we know from previous studies that Hand1 is not required for its own
expression (Riley et al.,
2000), it is reasonable to assume that the tTA is subject to all
of the appropriate temporal and spatial control as endogenous Hand1.
Any variation, therefore, between Hand1 and tTA expression should be
minimal and inexplicable in light of our current understanding of the knock-in
approach.
The fact that tTA expression, on the whole, recapitulated that of endogenous Hand1 confirmed the knock-in approach to be suitable for driving Hand1 overexpression exclusively in Hand1-expressing cells. In order to assess levels of Hand1 expression, compound heterozygote embryos were generated by crossing the driver and responder strains. Compound heterozygotes expressed the transgenic Hand1 cDNA by RT-PCR and northern blot (not shown), and, furthermore, had approximately twice as much Hand1 expression, as measured by real-time quantitative PCR, than control embryos (Fig. 1F). The single founder animal (Fig. 1F) and subsequent F1 and F2 generations were used throughout the study. The transgene copy number for the original founder was four and no correlation was observed between copy number and level of Hand1 expression (data not shown). The Hand1 responder transgene was not expressed in the absence of the tTA driver (data not shown), confirming that the system was not `leaky'.
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) and the
fact that tetraploid-rescued Hand1-null embryos have hypoplastic
hearts with downregulation of differentiation markers such as Mlc2v
(Myl2 - Mouse Genome Informatics)
(Riley et al., 1998), suggest
that Hand1 overexpression in the developing heart may promote
cardiomyocyte differentiation in vivo. In compound driver/responder
heterozygotes we observed, as early as E8.0, expansion of the primary heart
tube and extension at the earliest stages of looping compared to littermate
controls (embryos containing either only the driver targeted allele or the
responder transgene), suggesting hyperplasia and a putative expansion of cell
number (Fig. 3). Extension of
the primary heart tube at E8.0-E8.5 (Fig.
3A-L) was associated with a corresponding temporal and spatial
overexpression of Hand1, as confirmed by in situ analysis
(Fig. 3M-P). At E9.5,
Hand1-overexpressing embryos revealed a distinct cardiac phenotype
(Figs 4,
5). Hearts in which
overexpression occurred clearly looped to the right, suggesting no apparent
defect in the initiation and onset of looping. However, looping was abnormal,
with a significant overextension of the heart tube. Most notably, the OFT was
elongated distally and physically displaced from the inflow region
(Fig. 4D). Coincident with the
abnormal looping was defective LV development (Figs
4,
5). The Hand1
overexpression embryos appeared morphologically normal in other lineages, and
the phenotype could be reversed with the addition of the tet derivative
doxycycline to pregnant mothers (data not shown), indicating that the
embryonic defects observed were specific and indeed caused by overexpression
of Hand1.
The outflow tract is elongated in Hand1-overexpressing embryos
In Hand1-overexpressing embryos, the distal OFT region was
significantly extended and the heart tube displaced both ventrally away from
the body wall and laterally to reveal extraneous rightward looping (Figs
4,
5). The abnormal looping of the
heart is most probably a direct consequence of the OFT extension, as the heart
tube might be expected to undergo additional turns/folds to become
accommodated within a restricted pericardial region. In order to confirm the
observation of OFT elongation, distal OFT length was measured in moderately
(n=11) and severely (n=5) affected overexpression embryos
and compared with transgene positive (n=9) controls
(Fig. 5A-D). The distal OFT was
defined as the region of the heart tube extending from the body wall to the
apex of the so-called `dog leg bend'
(Anderson et al., 2003). In
severely affected embryos, the OFT was found to be approximately twice as long
as controls. In the moderately affected embryos, OFT length was found to be
approximately 50% longer than in controls. Variation in the cardiac phenotype
between Hand1-overexpressing embryos at mid-gestation stages (compare
Fig. 4D-F with 4G-H) is most
likely to be attributable to a degree of mosaicism at the level of tTA
expression despite the knock-in approach (data not shown). However, the
mosaicism fortuitously enabled us to assess a range of hypermorphic cardiac
phenotypes, with the most severe case illustrated in
Fig. 4G-I and
Fig. 5E-H. Furthermore,
hypermorphic embryos provided a valuable insight into changes in gene
expression profiles in the heart, even when the anatomical defects observed
were mild to modest (see below).
Abnormal ventricular development in severely affected Hand1 overexpression embryos
In severely affected embryos, extraneous looping was accompanied by
impaired LV differentiation and expansion leading to severe necrosis
(Fig. 4G-I,M,
Fig. 5H). In moderately
affected embryos, the presumptive left ventricle was reduced in size but had
otherwise undergone a degree of expansion and appeared to be developing
normally (Fig. 4D-F,K).
Sections through the hearts of the severely affected
Hand1-overexpressing embryos revealed that the failure in ventricular
expansion was associated with an almost complete absence of chamber lumen
(Fig. 4M) caused by a
combination of elevated myocyte density (control, 110±15
(n=6); overexpression, 185±12 (n=6);
P<0.05) and cellular hypertrophy. The LV phenotype in the severely
affected embryos may be a primary effect of Hand1 overexpression in
the LV myocardium itself, or could be a secondary effect caused by the
impaired haemodynamics of the expanded OFT and extraneously looped heart
tube.
In severely affected embryos, the extended OFT and impaired ventricular expansion was not compatible with survival beyond E10.5 (data not shown). Moderately and mildly affected embryos recover such that, at E12.5-14.5, heart development appears entirely normal (see Fig. S1 in the supplementary material) and these embryos survive to birth. Neither the mild nor moderate class of Hand1-overexpressing embryos revealed overtly abnormal cardiac morphogenesis. There appears to be a tolerance of variation in outflow-tract length, even amongst control somite-matched embryos (see Fig. 5D), which ensures normal development. The expansion of the OFT observed in the mildly and moderately affected mutants is presumably within this variance threshold such that the OFT is remodelled appropriately and LV maturation remains comparable with controls.
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), we
deemed it unlikely that the OFT expansion would be attributed to elevated
migration, or elevated number, of extracardiac cells from the anterior heart
field. To confirm this hypothesis, we investigated the contribution of the AHF
in the Hand1-overexpressing embryos by examining the expression
levels of: the LIM-domain-containing transcription factor Isl1, which
is involved in the differentiation of AHF cells
(Cai et al., 2003);
Mef2c, which is believed to be a target of Isl1
(Dodou et al., 2004), and
changes in which are thought to reflect defects in the formation of the AHF
and its derivatives (Kelly and Buckingham,
2002); and Hand2 (downstream of Mef2c in the
same pathway) (Lin et al.,
1997). In the case of all three markers - Isl1
(Fig. 6A-F), Mef2c
(Fig. 6G-L) and Hand2
(data not shown) - there were no observable differences in expression between
control and Hand1-overexpressing embryos, suggesting that the distal
OFT expansion is not caused by an elevated deployment of AHF cells. This
suggests that the effect of Hand1 overexpression was autonomous to
the distal OFT and may have arisen as a result of an inherent expansion in
cardiac precursors in this region. To test this hypothesis, we looked at
markers of cardiomyocyte differentiation by in situ hybridisation and
quantitative real-time PCR. Atrial natriuretic factor (ANF) is a marker of
differentiated myocardium in the atrial and ventricular chambers, but has
previously been reported to be undetectable in the OFT
(Habets et al., 2002). Not
only was Anf (Nppa - Mouse Genome Informatics) downregulated
in the trabeculated myocardium of the LV in modestly affected embryos
(Fig. 7A-D), it was also
downregulated in the OFT and restricted exclusively to the proximal OFT,
suggesting alternate differentiation status along the proximal and distal axes
(Fig. 7E,F). To assess the
differentiation status specifically of the extended distal OFT in the
overexpression embryos, we examined the levels of Wnt11, a marker of
the myocardium of the OFT (Cai et al.,
2003) implicated in the induction of cardiogenesis and in the
cardiomyocyte differentiation program
(Koyanagi et al., 2005;
Pandur et al., 2002).
Wnt11 was expressed at high levels throughout the distal OFT in
control embryos (Fig. 7G,I) but
restricted to low-level expression in a subpopulation of cells in the
myocardial wall of the OFT in overexpression embryos
(Fig. 7H,J). This suggests a
reduction in the level of myocardial differentiation coincident with distal
OFT extension. Reduced myocardial differentiation in the primary heart tube
and, notably, the LV was confirmed in the overexpression embryos by in situ
hybridisation for the early cardiac markers Nkx2.5 and Gata4
(Fig. 8). Whereas robust
expression for both markers was observed in the primary heart tubes of control
embryos (Fig. 8A,C,E,G),
Nkx2.5 and Gata4 were significantly downregulated throughout
the heart, most notably in the left ventricle of the overexpression embryos
(Fig. 8B,D,F,H).
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Elevated proliferation of cardiomyocytes in the distal outflow tract
As there appears to be reduced cardiomyocyte differentiation in the
Hand1 overexpression embryos, we determined whether there were
altered levels of apoptosis or elevated cell proliferation by immunostaining
with antibodies against cleaved caspase 3 (CC3) and phospho-histone H3 (PH3),
respectively. We observed no difference in expression of CC3 between wild-type
and overexpressing embryos, with very few cells throughout the developing
heart undergoing apoptosis at this stage (data not shown). By contrast, there
were significantly more proliferating cells located in the distal OFT region
of overexpressing hearts compared with wild type, with large numbers of PH3
positive cells, as an indicator of cells undergoing mitosis, situated in the
wall and lumen of the OFT [control, 125±10 (n=6);
overexpression, 238±14 (n=6) P<0.01]
(Fig. 9A-H). The observation of
an over-proliferation of cardioblasts in the OFT of overexpression embryos is
further supported by the downregulation of Wnt11 described above
(Fig. 7G-J).
Taken together, a lack of change in markers of the AHF, the downregulation of key genes associated with promoting cardiomyocyte differentiation and elevated levels of proliferation of cells in the distal OFT suggest that Hand1 does not affect deployment of cells into the OFT from the AHF, but rather that, once the extracardiac cells arrive in the OFT, they respond to Hand1 gain of function by continuing to proliferate as opposed to committing to a terminally differentiated cell fate.
Hand1 dosage influences cardiomyocyte differentiation
To confirm the in vivo findings and enable us to compare directly
Hand1 gain of function with a loss-of-function model, we
characterised Hand1 activity in vitro using ES cells differentiated into EBs
over a period of 14 days in floating culture. EB-derived cardiomyocytes
represent a faithful model of the in vivo situation: not only do they exhibit
characteristics of early chamber myocardium but a substantial proportion are
reminiscent of those observed in embryonic OFT
(Fijnvandraat et al., 2003a;
Fijnvandraat et al., 2003b).
ES cells generated during the derivation of the responder mouse strain
containing the Tre2-Hand1 transgene responder construct were stably
transfected with a tTA driver under the control of a TK promoter to generate a
line that precociously overexpressed Hand1 (see Fig. S2A in the
supplementary material). Hand1 expression is first detected in
wild-type EBs at day 4 of differentiation
(Smart et al., 2002). The
Hand1-overexpressing line expressed the Hand1 responder
transgene precociously, prior to endogenous Hand1 expression, between
day 0 and 4 of differentiation (see Fig. S2B, inset i, in the supplementary
material). Overexpression was maintained throughout differentiation up to day
14 (see Fig. S2B in the supplementary material), at which stage Hand1
was expressed five- to sixfold higher in the Hand1-overexpressing
cell line than in the control line (see Fig. S2B, inset ii, in the
supplementary material).
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) in terms
of their inherent ability to form cardiomyocyte contractile foci and real-time
expression of cardiac markers. Hand1-overexpressing EBs revealed a
significant delay and reduction in the formation of foci compared with wild
type, whereas Hand1-null EBs formed a much higher proportion of
contractile foci, up to fivefold higher at day 10 of differentiation
(Fig. 10A). To demonstrate
that the beating foci observed were cardiomyocytes, and to confirm that the
Hand1-overexpressing line was deficient in forming cardiomyocytes,
cells were dispersed and isolated from EBs at day 14 and immunostained for
sarcomeric myosin using the MF20 monoclonal antibody. Whereas cardiomyocytes
associated with beating foci were readily identified in Hand1-null,
wild-type and control cell lines (see Fig. S2C in the supplementary material),
no cardiomyocytes were identified in the Hand1-overexpressing line,
confirming the deficiency in cardiomyocyte differentiation in the
gain-of-function line.
The levels of contractile foci were supported by the gene-expression
profiles for each cell line (Fig.
10B). Commitment to a cardiac fate was evident in
Hand1-overexpressing EBs from the corresponding expression of
Mef2c, Cx40 and Irx4, and Mlc2v as markers of
chamber myocardium. Moreover, expression of Irx4 and Mlc2v
at levels comparable to control were indicative of progression towards
ventricular-like differentiation (Fig.
10B). However, the significant reduction in the expression of
Nkx2.5, Gata4 and the pan-cardiac marker 
-cardiac
actin (Fig. 10B) suggests an
overall reduction in cardiomyocyte differentiation. As the
ventricular-specific markers (Irx4 and Mlc2v) appeared
normal, this suggests that there are no significant changes in EB-derived
cardiomyocytes that resemble early chamber myocardium and that the reduced
differentiation is possibly attributable to the OFT component of the
EB-derived cardiomyocytes (Fijnvandraat et
al., 2003a). This is consistent with the identification of
independent populations of EB-derived cardiomyocytes, the gene expression
profiles and electrophysiological activity of which are characteristic of
either embryonic OFT or early chamber myocardium
(Fijnvandraat et al., 2003a;
Fijnvandraat et al.,
2003b).
The observation of Nkx2.5 inhibition following Hand1 gain of
function suggests that, although Nkx2.5 precedes Hand1
expression in the developing heart in vivo and is thought to be higher up the
transcriptional hierarchy (Biben and
Harvey, 1997), Hand1 is able to feedback onto Nkx2.5 and
modulate not only its expression levels but subsequent induced myocardial
differentiation. Hand1-null EBs, by contrast, revealed significantly
elevated Nkx2.5 levels; in fact, all of the cardiac genes examined
(with the exception of Cx40) were highly upregulated in response to
Hand1 loss of function (Fig.
10B). This is consistent with the observation of significantly
increased contractile cardiomyocyte foci in Hand1-null EBs
(Fig. 10A). An overall
observation that Hand1 overexpression affected a restricted cohort of
the cardiac markers examined compared to loss of Hand1 is almost
certainly explained by the fact that the two parent ES-cell lines differed
considerably in their profiles for the markers in question (data not shown).
The differences in cardiomyocyte differentiation between the
Hand1-null and Hand1-overexpressing cell lines indicate a
non-redundant role for Hand1 in maintaining equilibrium between cardiomyocyte
proliferation versus differentiation.
Cyclin D2 and its associated kinase, Cdk4, are downstream of Hand1
The altered levels and rates of differentiation in the Hand1-null
and Hand1-overexpressing cell lines suggested that these lines may
have associated alterations in cell-proliferation levels. Furthermore, as
there is elevated proliferation in the OFT in Hand1-overexpressing
embryos, accompanied by a decrease in differentiation, it follows that, in the
event of Hand1 overexpression, there may be a reduction in exit from
the cell cycle, resulting in continued proliferation at the expense of
differentiation.
To provide further evidence for elevated levels of cell proliferation in a
Hand1 gain-of-function background and mechanistic insight into how Hand1 might
promote increased cell division, we examined levels of cyclin D2 and the
associated cyclin-dependent kinase, Cdk4, in Hand1-overexpressing
EB-derived cardiomyocytes. As cardiomyocytes develop, they lose the ability to
proliferate and there appears to be a block in the gap phases of the cell
cycle, most notably at G1, consistent with the terminal differentiation of
these cells (Brooks et al.,
1998). Progression through G1 is dependent upon the D-type cyclins
and, although many different stimuli induce cell proliferation through
different signalling pathways, activation of one or more of the D-type cyclins
is necessary for most, if not all, of these pathways
(Busk et al., 2005). In
Hand1-overexpressing EBs, we observed significantly elevated protein
levels of both cyclin D2 and Cdk4 compared with wild type; this was
reciprocated by reduced levels for both proteins in EBs that were either
heterozygous or homozygous mutant for Hand1
(Fig. 10C,D). The differences
in cyclin D2 and Cdk4 levels observed between Hand1 gain- and loss-of-function
EBs appears to reflect the significant differences in numbers of
differentiated cardiomyocytes/beating foci observed between the two
populations (5% in the Hand1-overexpressing EBs versus 90% in the
Hand1-null EBs) following 14 days of differentiation
(Fig. 10A). Therefore, Hand1
activity influences cyclin D expression and associated CDK activity, both of
which are hallmarks of cell-cycle entry and pivotal for continued cell
proliferation.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Hemberger et al., 2004),
whereas, in the heart, overexpression results in expansion of the
proliferative pool of undifferentiated cardiomyocyte precursors.
An important potential caveat to the findings of this study resides in the
interpretation of the result of overexpression of Hand1 as being
either a gain-of-function or a dominant-negative effect, taking into account
the reported role of Hand1 as part of heterodimeric complex
(Firulli et al., 2000;
Scott et al., 2000). Moreover,
an additional consideration is whether, at high concentrations, specificity of
Hand1 downstream effects may be lost resulting in an impact on non-endogenous
Hand1 target genes. Although Hand1 functions as a heterodimer with E-factors
in vitro, according to the classic bHLH paradigm
(Firulli et al., 2000;
Scott et al., 2000), it can
also function as a homodimer (Hill and
Riley, 2004; Scott et al.,
2000) and, indeed, a tethered Hand1 homodimer allele, generated by
homologous recombination in ES cells, can rescue the Hand1-null phenotype
(Hu et al., 2006). Therefore,
a dominant-negative effect on heterodimer formation can be ruled out as almost
certainly lacking functional significance in vivo. The specificity issue is
one that can be levied at any loss- or gain-of-function model, as the effects
can be wide ranging and are often indirect. In this case, the Hand1
expression is actually only twofold above controls
(Fig. 1). Therefore, we are
working throughout with concentrations of Hand1 where significant non-specific
effects are unlikely; this is borne out by restriction of the phenotype
exclusively to areas of Hand1 expression.
|
). It is, therefore, plausible that Hand1 crucially
controls this differentiation process in the OFT myocardium, balancing levels
of cell proliferation and differentiation to maintain appropriate OFT
remodelling. In the event of Hand1 overexpression in the OFT, the
balance shifts towards continued proliferation, resulting in increased cell
number, abnormal OFT development and subsequent aberrant morphogenesis of the
heart. Interestingly, a shortening of the OFT was noted in the
cardiac-specific knockout of Hand1
(McFadden et al., 2005) and,
although this was not investigated further, it has been suggested that the OFT
defect may be secondary to an observed LV hypoplasia
(Buckingham et al., 2005;
McFadden et al., 2005). Our
results indicate that Hand1 directly affects the development of the OFT and
suggest that shortening of the OFT in cardiac-specific Hand1-null
embryos may well be a primary defect of reduced cardiomyocyte
proliferation.
Hand1 gain of function acts to promote continued proliferation of
existing precursors in the distal OFT, enabling them to maintain an early
phenotype whereby cardiomyocytes proliferate even as they begin to
differentiate, and subsequently prevents further differentiation associated
with cell-cycle exit. Conversely, in a Hand1-null background the
heart tube fails to loop, the OFT is hypoplastic and the presumptive ventricle
is thin walled (Riley et al.,
1998). Moreover, Hand1-null ES cells differentiated in
vitro give rise to a significantly higher proportion of beating foci compared
with wild type and this is associated with a significant upregulation in
markers of cardiomyocyte differentiation. Therefore, upstream regulation of
Hand1 levels in the expanding heart tube represents a switch between
cardiomyocyte proliferation and differentiation during organogenesis, and
Hand1 itself can regulate the progenitor pool in the developing OFT in a
manner entirely distinct from signals intrinsic to and emanating from the
AHF.
Overexpression of Hand1 in the LV appears to promote further
proliferation, thus preventing the outer curvature from expanding and
ballooning ventrally as secondary myocardium to form the ventricular chamber.
Secondary myocardium has a distinct molecular profile to that of the primary
myocardium of the linear heart tube. Once this program is initiated, secondary
myocardium is thought to balloon out from the outer curvature of the primary
heart tube to form the four chambers of the heart
(Christoffels et al., 2000).
The downregulation of markers of secondary myocardium, such as Anf
and chisel (Christoffels et al.,
2000), in Hand1-overexpressing embryos may, therefore,
explain the failure of the ventricle to balloon out, as differentiation into
secondary myocardium is simply not initiated. In this case, Hand1 may be
controlling the balance between differentiation and proliferation in the
ventricular chamber such that, in the event of Hand1 overexpression
in the presumptive LV, as in the OFT, the balance shifts towards
proliferation, resulting in an increased number of cells and reduced
cardiomyocyte differentiation with the associated down-regulation of
differentiation markers. Conversely, in tetraploid-rescued Hand1-null
embryos, the thin myocardial wall of the LV in the absence of Hand1 can now be
explained by a shift in balance towards decreased proliferation, rather than
by secondary effects, as previously thought
(Riley et al., 1998;
Riley et al., 2000). The fact
that the LV is a derivative of the primary heart field suggests that Hand1
uniquely impacts on derivatives of both the primary (LV) and secondary (OFT)
lineages in a cell-autonomous manner. This is further supported by the
extension of the heart tube observed in Hand1-overexpression embryos
as early as E8.0, which suggests that elevated Hand1 levels exert a
proliferative effect on the cells arising from the primary heart field. It
remains to be seen, however, whether the failure in the ballooning and lack of
ventricular lumen is primarily linked to an over-proliferation of ventricular
myocardium or is secondary to the elevated haemodynamic load and increased
fluid pressure arising from the extended heart tube.
|
The observation that Hand1 lies upstream of cyclin D2 is important
in light of recent studies implicating both genes in adult-onset
cardiovascular disease. Adult transgenic mice expressing cyclin D2 in the
myocardium show increased levels of basal cardiomyocyte cell-cycle activity
(Pasumarthi and Field, 2002)
and this increase in activity appears to result in a progressive reduction of
infarct size and improved cardiac function
(Rubart and Field, 2006).
Additionally, it has been demonstrated that cyclin D2 represses hypertrophy by
forcing cardiomyocytes through the cell cycle, resulting in cell
proliferation. This suggests that cyclin D2 levels directly determine whether
cells grow by hypertrophy or proliferation
(Busk et al., 2005).
Hand1 has been shown to be downregulated in human ischemic and
dilated cardiomyopathies (Natarajan et
al., 2001), and also following the induction of cardiac
hypertrophy in adult rodent hearts
(Thattaliyath et al., 2002).
Furthermore, a study on cardiac lineage protein 1 (Clp1;
Hexim1 - Mouse Genome Informatics)-null mice suggested that an
observed downregulation of Hand1 in the hearts of Clp1
embryos directly promoted a foetal form of cardiac hypertrophy
(Huang et al., 2004). The
downregulation of Hand1 during cardiac hypertrophy may, therefore,
result in the downregulation of cyclin D2, which in turn is a likely
contributing factor towards hypertrophic growth. This suggests that Hand1 and
cyclin D2 are capable of modifying cardiac responses in the myocardium to
external stimuli and that, collectively, they may play an important role
during hypertrophic signalling.
Disruption in the balance of cell number during heart development, via
over-proliferation and associated failure in differentiation, clearly leads to
an inability to maintain appropriate morphogenesis. Conversely, failure to
maintain an adequate pool of undifferentiated myocyte precursors results in
organ hypoplasia, as is observed in mice lacking Hand1, and almost
certainly contributes to the aetiology of congenital heart disease (CHD). OFT
anomalies account for 
30% of all cases of CHD
(Kelly and Buckingham, 2002).
Reduced OFT length is associated with defective alignment with the
atrioventricular junction and abnormal rotation of OFT myocardium, which in
turn leads to abnormal ventricular septation and OFT anomalies such as double
outlet right ventricle and persistent truncus arteriosus. Autonomous,
Hand1-induced proliferation of cardiomyocyte precursors during heart-tube
elongation provides insight into directing progenitor cells along a path
towards integrated myocardium, which is of direct relevance to cell-based
therapies for cardiovascular disease and cell-autonomous strategies for
cardiac regenerative medicine.
Two potential approaches for deriving cell-based therapies are the transplantation of stem cells from an external source and the activation of resident stem cells within the heart. Clearly, for these to be viable options there is a need for further understanding of the precise molecular pathways involved in directing cardiomyocyte proliferation and differentiation, and how to generate greater numbers of functional cardiomyocytes. These studies collectively reveal that Hand1 is involved in maintaining the correct balance between proliferation and differentiation in the developing heart, suggesting that manipulation of Hand1 levels could potentially affect both of these avenues of therapeutic research.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/133/22/????/DC1
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ACKNOWLEDGMENTS |
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