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First published online 17 December 2008
doi: 10.1242/dev.030007
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1 Molecular Medicine Unit, UCL Institute of Child Health, London WC1N 1EH,
UK.
2 The Randall Centre of Cell and Molecular Biophysics and The Cardiovascular
Division, King's College, London SE1 1UL, UK.
3 Molecular Haematology and Cancer Biology Unit, UCL Institute of Child Health,
London WC1N 1EH, UK.
4 Bloomsbury Centre for Bioinformatics, Department of Computer Science,
University College London, Gower Street, London WC1E 6BT, UK.
5 Department of Genetics and Tumor Cell Biology, St Jude Children's Research
Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.
6 Division of Haematology, The Hanson Institute, Adelaide, South Australia 5000,
Australia.
7 Institute of Biosciences and Technology, Texas A&M University Health
Science Center, Houston, TX 77030, USA.
8 Massachusetts General Hospital Cardiovascular Research Center, Boston, MA
02114, USA.
9 Department of Cell Biology, Harvard Medical School, and the Harvard Stem Cell
Institute, Cambridge, MA 02138, USA.
* Author for correspondence (e-mail: p.riley{at}ich.ucl.ac.uk)
Accepted 21 November 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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-actinin, N-RAP and zyxin, which
collectively function to maintain an actin--actinin interaction as the
fundamental association of the sarcomere. Aspects of abnormal heart
development and the manifestation of a subset of muscular-based disease have
previously been attributed to mutations in key structural proteins. Our study
reveals an essential requirement for direct transcriptional regulation of
sarcomere integrity, in the context of enabling foetal cardiomyocyte
hypertrophy, maintenance of contractile function and progression towards
inherited or acquired myopathic disease.
Key words: Prox1, Mouse, Heart development, Myocardium, Sarcomere, Hypertrophy, Myopathy, N-RAP (Nrap), Zyxin
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Pyle and Solaro, 2004).
There is an absolute requirement for cardiac function during embryogenesis
in mammals and as such the sarcomeric components are expressed very early in
development and are correctly localised in the myofibrils by the time the
linear heart tube begins to contract
(Ehler et al., 1999). As
development progresses, the heart increases in mass not only by cardiomyocyte
hyperplasia, but also through a recently recognised foetal phase of
physiological hypertrophy (Hirschy et al.,
2006), a process that is dependent upon myofibril disassembly,
reassembly (Ahuja et al., 2004)
and elongation (Hirschy et al.,
2006).
Despite insight into the morphogenetic events that accompany
myofibrillogenesis and hypertrophic growth, very little is known about the
precise regulation of these processes during development. Furthermore, the
importance of appropriate assembly and maintenance of the myofibrillar
apparatus is underscored by the fact that defects in the terminal
differentiation and arrangement of contractile protein filaments are
associated with a number of cardiac myopathies
(Engel, 1999;
Gregorio and Antin, 2000;
Seidman and Seidman,
2001).
The homeobox transcription factor Prox1 is essential for murine lymphatic,
hepatocyte, retinal and pancreatic development
(Dyer et al., 2003;
Harvey et al., 2005;
Sosa-Pineda et al., 2000;
Wigle and Oliver, 1999).
Multiple lines of evidence suggest a role for Prox1 during cardiac
morphogenesis. Prox1 is expressed in the developing heart
(Oliver et al., 1993;
Rodriguez-Niedenfuhr et al.,
2001; Tomarev et al.,
1996; Wigle and Oliver,
1999) and embryos deficient in Prox1 die at 
E14.5, a critical
time point when lethality often results from grossly reduced cardiac
performance (Dyson et al.,
1995). Moreover, in a specific genetic background, a proportion of
Prox1-heterozygote mice fail to survive and become cyanotic soon
after birth (Harvey et al.,
2005), a phenotype that is consistent with impaired blood
circulation and heart defects.
Here we reveal how Prox1 functions transcriptionally upstream of sarcomere
assembly, myofibril organisation and foetal cardiomyocyte growth. We provide
evidence that Prox1 activity is required for the normal expression and
localisation of multiple sarcomeric components and that it directly regulates
the genes encoding -actinin, N-RAP (nebulin-related anchoring protein,
Nrap) and zyxin, which are essential structural proteins required for
stabilising actin within the thin filaments and ultimately for establishing
cardiomyocyte elongation and coordinated muscle contraction. These results,
therefore, provide important insight into the molecular mechanisms that govern
the ultrastructure and growth of cardiac muscle during development and
highlight how transcriptional misregulation of myofibril assembly may underlie
cardiac hypotrophy and myopathic disease.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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),
Prox1loxP (Harvey et
al., 2005), Nkx2.5CreKI (Moses
et al., 2001) and MLC2vCreKI
(Chen et al., 1998).
Prox1loxP/loxP mice were crossed with
Nkx2.5CreKI;Prox1loxP or
MLC2vCreKI;Prox1loxP to generate
Nkx2.5CreKI;Prox1loxP/loxP (Prox1Nkx)
or MLC2vCreKI;Prox1loxP/loxP
(Prox1MLC) embryos, respectively. Embryos and hearts were
dissected in PBS, fixed in 4% paraformaldehyde (PFA), dehydrated, embedded in
paraffin and sectioned at 15 µm. Sections were stained with Haematoxylin
and Eosin.
Immunohistochemistry and TUNEL assays
Embryos were treated as above and sectioned at 10 µm.
Immunohistochemistry was performed using anti-Prox1 (Reliatech) and
anti-phosphohistone H3 (Upstate) antibodies, developed using a standard
streptavidin-HRP method and counterstained with Haematoxylin. Terminal
deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) assays
were performed according to the manufacturer's protocols (Promega).
Immunofluorescence and confocal microscopy
E10.5-14.5 whole-mount hearts were dissected in PBS and fixed overnight in
4% PFA. E18.5 hearts were dissected, embedded in paraffin and sectioned as
above. Immunofluorescence and confocal microscopy were performed as described
previously (Ehler et al.,
1999). Samples were analysed on a Zeiss LSM 510 confocal
microscope equipped with argon and helium neon lasers using a 63x/1.4
lens. Image processing was performed using Zeiss software and Photoshop
(Adobe). Cell outlines were traced on sections (blinded to genotype), using
ImageJ
(http://rsb.info.nih.gov/ij)
to assist in assessing cell shape and calculating cell area as indicators of
foetal hypertrophy.
Antibodies
The following antibodies were used: sarcomeric -actinin (clone
EA-53; Sigma), β-catenin, vinculin and smooth muscle -actin (clone
1A4; Sigma), cardiac -actin (Progen), titin (T12; gift of Prof. D.
Fürst, Institut für Zellbiologie, Universität Bonn, Germany),
desmin (clone D33; Dako), MHC (A4.1025; DSHB), cardiac MyBP-C (gift of Prof.
T. Obinata, Department of Biology, Chiba University, Japan), CD31 (Pecam1; BD
Pharmingen), Prox1 (Reliatech) and Gapdh (Chemicon). For western blots, rabbit
anti-sarcomeric -actinin antibody was used (gift of Prof. D.
Fürst).
Transmission electron microscopy
E13.5/E18.5 hearts were dissected and fixed in 3% glutaraldehyde (EM grade,
Agar Scientific), 0.1 M sodium cacodylate, 5 mM CaCl2 (pH 7.4).
Hearts were processed in a Lynx automated tissue processor (Australian
Biomedical) and embedded in resin. All sectioning was performed on a Reichert
Ultracut S ultramicrotome. Sections were imaged using a Philips CM 10
transmission electron microscope and images collected using Kodak Megaview II
and SIS Keenview digital imaging systems and SIS software. Four
Prox1Nkx and four control hearts were examined for each
stage.
Western blotting
E13.5 heart lysates were prepared in RIPA buffer. Western blotting was
performed using standard methods. Scanning densitometry was performed and
signal quantified using Scion Image (Scion Corporation) and ImageJ.
Quantitative real-time (qRT) PCR analysis
mRNA was isolated from E12.5 hearts using the Micro FastTrack 2.0 Kit
(Invitrogen) according to the manufacturer's instructions. Reverse
transcription was performed using Superscript III reverse transcriptase
(Invitrogen) according to the manufacturer's instructions. qRT-PCR analysis
was performed on an ABI 7000 Sequence Detector (Applied Biosystems) using SYBR
Green (Quantitect SYBR Green PCR Kit, Qiagen). Data were normalised to
Hprt1 expression and analysed using DART-PCR
(Peirson et al., 2003).
P-values were obtained using Student's t-test
(n=9). Primers for qRT-PCR (Table
1) were obtained from Primer Bank
(http://pga.mgh.harvard.edu/primerbank)
or designed using Primer Express (version 2.0, Applied Biosystems).
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), using a digoxigenin-labelled antisense riboprobe specific
for Nppa (Kuo et al.,
1997).
ChIP-on-chip
E12.5 hearts were dissected in PBS containing 0.3% Triton X-100 and
cross-linked for 3 hours at room temperature in 1.8% formaldehyde, homogenised
in lysis buffer and sonicated. Sixty micrograms of chromatin lysate was used
per immunoprecipitation with 10 µg anti-Prox1 antibody (Reliatech) in ChIP
dilution buffer at 4°C overnight. A no-antibody `immunoprecipitation' was
performed as a control. Immune complexes were pulled down with Protein A/G
beads, washed, resuspended in TE (10 mM Tris, 5 mM EDTA, pH 8.0), the
cross-links reversed overnight at 65°C and the DNA purified. DNA (10 ng)
was blunt-ended and unidirectional adapters were ligated overnight at
16°C. Adapter-ligated DNA was amplified by PCR. Experimental conditions,
buffer composition, adapter sequences and PCR conditions are available on
request. ChIP and no-antibody samples were checked by qRT-PCR for enrichment
of a positive control, Fgfr3 (a previously identified in vitro target
of Prox1) (Shin et al., 2006),
and against a negative control, Cyp7a1
(Qin et al., 2004). Amplified
DNA (7.5 µg) was fragmented and end labelled using the GeneChip WT
Double-Stranded DNA Terminal Labelling Kit, hybridised to GeneChip Mouse
Promoter 1.0R Arrays, and then stained using the GeneChip Hybridisation, Wash
and Stain Kit (all Affymetrix). ChIP data were analysed using CisGenome
(v1.0_beta;
http://www.biostat.jhsph.edu/~hji/cisgenome/index.htm).
Three independent ChIP and no-antibody control reactions were performed.
Standard PCR to confirm ChIP of the enhancer elements was carried out using
the primers listed in Table
1.
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). Briefly, in vitro translated Prox1, or nuclear extracts
from mouse P19Cl6 cells (Habara-Ohkubo,
1996) transfected with Flag-Prox1, were incubated with
32P-labelled oligonucleotide in binding buffer at room temperature
for 30 minutes. Overlapping 60 bp oligonucleotides, which spanned the entire
Prox1-binding elements of Actn2, Nrap and Zyx as identified
by ChIP-on-chip, were used in preliminary binding reactions to narrow down
each element to within a single 60 bp oligonucleotide (sequences highlighted
in red in Fig. S9 in the supplementary material). Unlabelled oligonucleotide
(10-fold excess) was used in the competition binding assays. Anti-Flag (M2;
Sigma; 4.6 µg) was used to supershift bound Prox1 in the transfected P19Cl6
cell extracts. Each binding reaction was run on an 8% polyacrylamide gel,
which was dried and subjected to autoradiography.
Reporter transactivation assays
P19CL6 cells were maintained in standard P19 culture conditions
(McBurney et al., 1982).
Transfections were carried out using Effectene reagent as described previously
(Hill and Riley, 2004).
Briefly, duplicate wells of P19CL6 cells were transfected with a reporter in
which luciferase was located downstream of putative Prox1-binding elements
from Actn2, Nrap or Zyx, and a chick -cardiac actin
minimal promoter (Hill and Riley,
2004), either with or without pcDNA3-Prox1 (250 ng) and a
β-actin-β-galactosidase (β-gal) expressing plasmid to normalise
luciferase activity for transfection efficiency. Luciferase and β-gal
activity were assayed 48 hours post-transfection as described
(Hill and Riley, 2004).
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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E10.5 (Fig. 1). At
E12.5 and E14.5, Prox1 expression was notable in the interventricular septum
and myocardium (Fig. 1B-E), but
was excluded from the smooth muscle of the outflow tract (not shown).
Furthermore, Prox1 was expressed in the outer endocardial layer of the
atrioventricular endocardial cushion leaflets
(Fig. 1E,F) and the mitral
valve leaflets (Fig. 1F,H), but
was absent from cushion mesenchyme (Fig.
1F,G). Confocal analysis confirmed the predicted nuclear
localisation of Prox1 in atrial and ventricular myocytes (see Fig. S1A-D in
the supplementary material).
Heart development is abnormal in Prox1-null mice
Mice that are homozygous null for Prox1 die between E14.5 and E15
(Wigle and Oliver, 1999). The
cause of lethality in Prox1-null embryos has not been determined. To
address whether loss of cardiac expression of Prox1 is a potential
contributing factor, we initially examined surviving post-natal day 5 (P5)
Prox1-heterozygous mice and observed, at a gross anatomical level,
that the hearts were reduced in size (by an average of 30%) as compared with
those of wild-type littermate controls (see Fig. S2A in the supplementary
material). Histological analysis revealed a range of cardiac anomalies
including hypoplastic ventricular walls, loss of muscle striation, a
disorganised interventricular septum and abnormally persistent muscle
surrounding the aorta (see Fig. S2B,D in the supplementary material). Analysis
of Prox1-null embryos at E13.5 and E14.5 revealed that mutant hearts
were significantly smaller (up to 50%) than those of wild-type littermates
(see Fig. S2E,F in the supplementary material) and, consistent with the
phenotype in the P5 heterozygotes, we observed myocardial disarray, including
a disrupted interventricular septum (see Fig. S2G,H in the supplementary
material).
Prox1 is an essential regulator of heart development
Conventional Prox1-null embryos have a lymphatic phenotype arising
from defects in endothelial cell budding as early as E11.5
(Wigle and Oliver, 1999),
which may adversely affect cardiovascular development. To confirm a primary
role for Prox1 in the developing heart, we generated a cardiac-specific
knockout of Prox1 by crossing a conditional homozygous floxed
Prox1 strain (Prox1loxP/loxP)
(Harvey et al., 2005) with two
cardiac-expressing Cre strains: Nkx2.5CreKI (designated
Prox1Nkx), which directs expression of Cre recombinase
throughout the majority of cardiomyocytes from E7.5
(Moses et al., 2001); and
MLC2vCreKI (designated Prox1MLC), in which Cre
is expressed specifically in ventricular cardiomyocytes from E8.75
(Chen et al., 1998). The two
Cre strains were employed, therefore, to target Prox1 globally
throughout the entire myocardium and in a subpopulation of ventricular
myocardium, thus acting as respective internal controls for a
cardiomyocyte-specific loss of Prox1 function.
Prox1 protein levels in Prox1Nkx and
Prox1MLC E13.5 hearts were analysed by western blot on
whole heart lysates, followed by densitometry. This confirmed a 3-fold
reduction in Prox1 protein levels in the cardiac-specific knockout hearts as
compared with littermate control hearts (Prox1loxP or Cre
allele only) (Fig. 2A).
Whole-mount and histological sections of Prox1Nkx and
Prox1MLC embryos were examined between E10.5 and E18.5. No
phenotype was evident between E10.5 and E12.5 (not shown), despite expression
of Prox1 in the heart at these stages. At E13.5 and E14.5, the
Prox1Nkx- and Prox1MLC-conditional
mutants recapitulated the cardiac phenotype of the conventional knockout
embryos described above. Prox1Nkx embryos were slightly
growth restricted (on average 5% smaller) compared with control littermates,
with cranial haemorrhaging. Both Prox1Nkx and
Prox1MLC embryos had oedema of varying severity
(Fig. 2B), indicative of
inadequate cardiac function. The hearts of both Prox1-conditional
mutants were up to 30% smaller than controls hearts
(Fig. 2C-E). Furthermore, in
Prox1Nkx hearts, the right atrium was reproducibly
expanded (up to 2-fold) and blood-filled, never appearing to empty
appropriately, irrespective of fixation stage during the cardiac cycle,
suggesting that there might be impaired blood flow through the heart
(Fig. 2D). Moreover, in the
Prox1MLC hearts, there was reduced expansion of the left
ventricle (25% reduction in chamber size) consistent with haemodynamic
obstruction (Fig. 2E).
Haematoxylin and Eosinstained frontal sections through E13.5 and E14.5 hearts
(Prox1Nkx, Fig.
2F-K; Prox1MLC, not shown) revealed small
thin-walled ventricles and disrupted myocardium with a highly disorganised
interventricular septum (Fig.
2H,I). Additionally, membranous ventricular septal defects (VSDs)
were observed in Prox1Nkx embryos
(Fig. 2J,K) that were unlikely
to be due to myocardial disruption, but possibly related to additional
endocardial cushion defects (not shown). By E18.5, the overall area of
ventricular myocardium was significantly smaller in
Prox1Nkx than control hearts, in particular that of the
right ventricle. Moreover, the myocardium was disorganised and less compacted
(reduced by up to 50%) and the surface of the ventricles was irregular,
although both compact and trabecular layers did appear to form
(Fig. 2L-N). Consistent with
the predominantly right-sided defects in Prox1Nkx embryos,
the pulmonary trunk was reduced in diameter, often being half the size of the
aorta, suggesting impaired outflow tract remodelling. At E18.5, the membranous
VSD was still evident in Prox1Nkx embryos, and we also
observed muscular VSDs (Fig.
2N) that were likely to result from the reduced level of
myocardial compaction in the ventricles.
|
). Analysis
of both Prox1Nkx and Prox1MLC mutant
hearts confirmed specificity of phenotype at the level of the myocardium, as
supported by immunostaining for markers of vascular endothelial and smooth
muscle cells (Pecam1 and smooth muscle -actin, respectively) that
revealed normal coronary vessel development (see Fig. S3A-D in the
supplementary material). Owing to the similarities in myocardial phenotype
observed in both models, the term Prox1-conditional will be used to
describe both the Prox1Nkx and
Prox1MLC hearts unless specifically stated otherwise.
Sarcomeric integrity is disrupted in Prox1-conditional hearts
At a gross level, as determined by low-resolution histological analyses,
loss of Prox1 function in the heart appeared to lead to myocardial disarray
that was manifested in disorganisation of the myofibrils. To investigate this
in more detail, the expression and localisation of sarcomeric markers was
analysed by high-resolution confocal microscopy following immunostaining of
whole-mount embryonic hearts. The relative distribution of the major
components of the sarcomere is illustrated in Fig. S4A (see Fig. S4A in the
supplementary material). In the first instance, colocalisation of Prox1 and
selected sarcomeric markers (actin and sarcomeric -actinin) was
confirmed in E13.5 control hearts (see Fig. S1A-D in the supplementary
material). Prox1-conditional E13.5 and E14.5 whole-mount hearts were
stained with phalloidin to visualise F-actin and the arrangement of the thin
filaments, and immunostained with antibodies to sarcomeric myosin heavy chain
(MHC) and cardiac myosin binding protein C (MyBP-C) to visualise the thick
filament architecture. Additionally, hearts were immunostained with antibodies
to sarcomeric -actinin, a crucial Z-disc protein that cross-links
sarcomeric actin and is involved in many signalling transduction pathways, to
myomesin, which localises to the M-band where it interacts with myosin and
titin providing elasticity and stability to the sarcomere, and to
β-catenin, which localises to the adherens junctions and demarcates
cell-cell contacts (Fig. 3; see
Fig. S4A and Fig. S5A-F in the supplementary material). In
Prox1-conditional hearts at E13.5, we consistently observed severe
global disruption of thin filament, thick filament, Z-disc and M-band
organisation in the ventricular myocardium as compared with littermate
controls (Fig. 3A-I,L).
Variation in the severity of disruption of actin filaments and -actinin
was evident (see Fig. S6 in the supplementary material), consistent with
mosaicism in the level of Prox1 knockdown
(Fig. 2A; a western analysis
for Prox1 expression with scanning densitometry normalised to Gapdh). We
classified overall phenotype severity based on the following three criteria as
revealed by the confocal ultrastructure analysis: (1) cardiomyocyte cell shape
(using ImageJ, see Materials and methods), (2) sarcomeric striation and (3)
myofibrillar organisation/cell alignment. Defects in one of these categories
were classed as mild, of which we recorded 14.3% of
Prox1Nkx (n=56) and 18.2% of
Prox1MLC (n=41) mutants; defects in all three
criteria were classed as severe, of which we recorded 85.7% of
Prox1Nkx (n=56) and 81.8% of
Prox1MLC (n=41) mutants at E13.5. Despite
variation in the severity of the phenotype, the nature of the specific defects
common to both the Prox1Nkx and
Prox1MLC mutants, presented as anomalies in muscle
ultrastructure, confirmed a cardiomyocyte-autonomous role for Prox1. The
severe phenotype was incompatible with embryonic survival, whereas incomplete
deletion of Prox1 resulting in the mild phenotype contributed to a
low incidence of survival of Prox1-conditional mutants to post-natal
stages (5/111 Prox1Nkx and 0/64
Prox1MLC). Additionally, we occasionally observed thin
filament and Z-disc disruption and loss of striation in
Prox1Nkx atrial myocardium (not shown), which is
consistent with the expression patterns of Prox1 and
Nkx2.5.
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-actinin. Desmin is an intermediate filament protein
that also interacts with sarcomeric -actinin at Z-discs, providing a
lateral connection between Z-discs of adjacent myofibrils (see Fig. S4A in the
supplementary material). Prox1-conditional E13.5 hearts were triple
stained for titin (Fig. 3J,M),
desmin (Fig. 3K,N) and
phalloidin (not shown), which demonstrated that loss of Prox1 results in a
severe disruption of Z-disc organisation. The full extent of sarcomeric
protein misregulation in Prox1-conditional hearts is summarised in
Fig. S4B (see Fig. S4B in the supplementary material).
Given that Prox1 is a transcription factor, we sought to determine whether
alterations in the expression levels of myofibril and Z-disc components might
underlie the ultrastructure defects. E12.5 or E13.5 Prox1-conditional
and control individual hearts were examined by quantitative real-time
(qRT)-PCR or western blotting, respectively. qRT-PCR was carried out for genes
encoding the sarcomere components that were shown to be mislocalised in
Prox1-conditional myocardium. -cardiac actin (Actc1),
sarcomeric -actinin (Actn2) and β-MHC (Myh7)
were significantly downregulated and MyBP-C (Mybpc3) was
significantly upregulated (Fig.
3O). Titin (Ttn) and desmin (Des) were
consistently downregulated, but to varying levels, which led to the changes
not being statistically significant (Fig.
3O). Nonetheless, these results confirm the extensive sarcomere
dysgenesis in the Prox1-conditional mutant hearts both in terms of
appropriate myofibril organisation and gene expression. Additionally, we
analysed sarcomere component protein levels in E13.5 individual hearts. In
accordance with the changes in gene expression, sarcomeric -actinin and
sarcomeric MHC were downregulated in Prox1-conditional hearts
(Fig. 3P). Levels of desmin,
β-catenin and vinculin were unchanged (not shown), which, alongside
confocal immunofluorescence for β-catenin (see Fig. S5A-F in the
supplementary material), confirmed that cell-cell contacts through
adherens-type junctions were unaltered between neighbouring
Prox1-conditional cardiomyocytes.
Subsequently, we examined the relationship between the level of Prox1
reduction and -actinin expression further. Individual heart samples
were analysed by both western blotting and immunostaining and we observed a
direct correlation between the degree of Prox1 knockdown and the reduction in
sarcomeric -actinin levels (Fig.
3O,P). This suggests that Prox1 activity might regulate the
expression of sarcomeric -actinin and, moreover, that a modest yet
significant reduction (0.7-fold) in -actinin is sufficient to
contribute to the myofibrillar disarray in the conditional mutant hearts
(Fig. 3I,L). Furthermore, this
analysis demonstrated a definitive association between the level of Prox1
expression and the severity of the myocardial phenotype
(Fig. 3O,P; see Fig. S6 in the
supplementary material).
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Prox1 is essential for physiological foetal hypertrophy
We next investigated whether Prox1 plays a role in either the initial
stages of myofibrillogenesis or in the maintenance of appropriate myofibril
structure throughout later stages of cardiac development. To this end, we
immunostained whole-mount Prox1-conditional hearts at E10.5, E11.5
and E12.5 for sarcomeric -actinin and phalloidin to pinpoint exactly
when the myocardial defects first arise. In the Prox1Nkx
mutants, Prox1 levels were reduced from early cardiac crescent stages
(E7.5) (Moses et al., 2001)
onwards, and yet at E10.5 and E11.5 there was no sarcomeric disruption and the
myofibrils appeared to have assembled correctly
(Fig. 5A,D), whereas by E12.5
the first signs of myofibril disorganisation were observed (see Fig. S7A-D in
the supplementary material). The fact that sarcomere defects do not manifest
until E13.5 and then persist throughout the rest of development suggests that
Prox1 is not required for the initiation of myofibrillogenesis but for the
subsequent maintenance of appropriate sarcomeric structure and stability. In
addition, we observed that in E13.5 Prox1-conditional hearts
cardiomyocyte morphology and distribution were noticeably altered: the
cardiomyocytes were more rounded in shape, disorganised and distributed in an
apparently random pattern as compared with the control hearts, in which the
cardiomyocytes had begun to elongate and align in parallel
(Fig. 3A-N). Consistent with
this, we observed a significant increase in -actinin expression in
wild-type hearts between E9.5 and E12.5, indicative of a requirement for
sarcomeric-dependent cell growth after the recruitment of cells and at the
onset of physiological hypertrophy (see Fig. S7E in the supplementary
material). When sections of E18.5 Prox1-conditional and control
hearts were examined by phalloidin staining and immunostaining for sarcomeric
-actinin we observed that a large proportion of cardiomyocytes were
still rounded at E18.5, having failed to undergo hypertrophic growth and
acquire the characteristic rod shape of mature cardiomyocytes
(Fig. 5C,F).
Hypertrophy in adult hearts is induced by a large variety of stimuli, but
the consistent end point is re-expression of a foetal gene programme,
including Nppa (Anf), Myh7 (β-MHC) and Nppb
(BNP) (Molkentin et al.,
1998). During development, Nppa, Myh7 and Nppb
are markers of myocardial differentiation and chamber expansion, but because
differentiation per se is accompanied by a concomitant increase in
cardiomyocyte cell growth, we examined the expression levels of Nppa
and Myh7 as surrogate markers of foetal hypertrophy. qRT-PCR on E12.5
hearts and in situ hybridisation on E13.5 embryo sections revealed that both
genes were significantly downregulated in Prox1Nkx hearts
(Fig. 5G-K), not only
indicating that was there markedly reduced foetal hypertrophic growth in the
absence of Prox1, but also suggesting that Prox1-conditional hearts
are not hypoplastic, but hypotrophic. Morphometric measurements of cell size
revealed that cardiomyocytes in Prox1Nkx hearts failed to
enlarge during development, confirming a defect in hypertrophic growth as
opposed to an alteration in cell shape
(Fig. 5L). Immunostaining for
phosphohistone H3, a marker of cells in mitosis, and TUNEL assays confirmed
that there was no decrease in proliferation or increase in apoptosis between
E10.5 and E13.5 (see Fig. S8 in the supplementary material). Therefore, in the
absence of Prox1, although laid down appropriately in early heart development,
the sarcomere structure is not maintained, resulting in myofibril disruption,
loss of striation and a failure of cardiomyocyte hypertrophic growth and
maturation (Fig. 5M).
Prox1 directly regulates fundamental components of the sarcomere
To gain specific insight into the molecular mechanism(s) by which Prox1
regulates myofibril organisation we sought to identify direct downstream
targets of Prox1 in the developing heart. Until now, no in vivo target of
Prox1 has been identified, nor has a definitive consensus Prox1 DNA-binding
site been determined. Therefore, we combined chromatin immunoprecipitation
(ChIP) against endogenous Prox1 with microarray analysis (ChIP-on-chip).
ChIP-on-chip revealed putative Prox1-bound enhancer regions of genes encoding
the Z-disc protein -actinin (Actn2) and the myofibrillar and
adherens junction proteins N-RAP (Nrap) and zyxin (Zyx),
both of which directly interact with -actinin in the Z-disc
(Fig. 6A). ChIP of the three
enhancer elements was confirmed by PCR (see Fig. S9A in the supplementary
material). We analysed the Actn2, Nrap and Zyx target
sequences for predicted transcription factor binding sites using MatInspector
Professional
(http://www.genomatix.de):
40-50 transcription factor binding sites were predicted per sequence but no
putative Prox1 or Prospero (the Drosophila homologue of Prox1)
binding sites were identified, although the Prox1 sites described to date are
highly degenerate and predicted based on a Prospero consensus
(Cook et al., 2003;
Lengler et al., 2005;
Shin et al., 2006). The lack
of a core consensus motif within the three Prox1-bound enhancer elements
identified for Actn2, Nrap and Zyx, despite the conservation
of each element across species, is consistent with results obtained from
unbiased screens of genome-wide conserved regulatory sequence variants
(Pennacchio et al., 2006).
The ChIP data were subsequently validated by EMSAs with overlapping probes from each of the three enhancer regions and in vitro translated Prox1 or lysates from mouse P19Cl6 cells overexpressing Prox1 (see Fig. S9B-D in the supplementary material). Competition gel shifts with unlabelled probe (Fig. 6B) and antibody supershifts (Fig. 6C) of the refined oligonucleotide sequences (60 bp) confirmed specific Prox1 binding to the enhancers within Actn2, Nrap and Zyx. Prox1-induced transcription via all three enhancer elements was demonstrated by cotransfection and reporter gene activation assays (Actn2, 15-fold activation; Nrap, 32-fold; Zyx, 9-fold) (Fig. 6D). qRT-PCR for Nrap and Zyx (Fig. 6E) confirmed reduced expression of these factors in a Prox1-deficient background, as was previously determined for Actn2 (Fig. 3O).
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Prox1 deficiency impacts directly on sarcomeric components that facilitate
Z-disc and thin filament interaction. Reduced expression of Nrap and
Zyx in Prox1-conditional hearts, two genes that are direct
transcriptional targets of Prox1, is highly significant in terms of
maintaining Z-disc stability. N-RAP has been proposed to act as a catalytic
scaffold for the association of thin filament actin and Z-disc -actinin
during myofibrillogenesis (Dhume et al.,
2006), and zyxin also interacts with -actinin to facilitate
actin assembly and organisation (Crawford
et al., 1992; Frank et al.,
2006). During myofibrillogenesis, N-RAP and zyxin are associated
with cell-cell contacts that make up the developing intercalated discs, which
ultimately mature during post-natal stages
(Perriard et al., 2003). Our
confocal and TEM observations that cell junctions (β-catenin-positive
adherens-type junctions and desmosomes) are appropriately established and
remain intact between neighbouring cardiomyocytes in
Prox1-conditional mutants, suggest that the role of Prox1 is
primarily to regulate Nrap and Zyx to facilitate
cross-linking between actin and -actinin in the Z-disc as one of the
fundamental associations of the sarcomere (see Fig. S4A in the supplementary
material). Actn2 is also implicated in this study as a direct target
of Prox1, and the modest, yet significant, reduction in the expression levels
of Actn2 in a Prox1 mutant background, correlating with the
Z-disc disruption, underlines the crucial role of -actinin in
maintaining sarcomere integrity. Moreover, as Zyx-null mice are
viable (Hoffman et al., 2003),
although their hearts have not been examined in detail for histological
defects, the phenotype we describe represents a cumulative effect on the
actin--actinin interaction: directly, via -actinin expression
and localisation, and through interactions with the co-factors N-RAP and
zyxin.
Perturbation of the actin--actinin association directly explains the
Z-disc anomalies in Prox1-null hearts. Although we cannot entirely
exclude additional effects of loss of Prox1 function, normal levels of
cardiomyocyte proliferation and apoptosis and the specificity of phenotype at
the level of sarcomeric maintenance suggest that the latter is the primary
defect in Prox1-conditional mutants. Moreover, thin filament-Z-disc
disruption will feedback directly onto the thin and thick filament arrangement
of the sarcomere, as observed at the level of TEM, resulting in a more global
disorganisation of myofibrils (see Fig. S4B in the supplementary material).
The latter might also explain the observed M-band defects, but equally these
might relate more directly to misregulation of Nrap in the
Prox1-mutant background, as N-RAP associates with the M-bands of
maturing myofibrils where it acts as a catalytic scaffold
(Lu et al., 2005).
Prox1 is not required for the initial stages of myofibrillogenesis because
the phenotypic defects do not begin to manifest until E12.5. Sarcomere
proteins are expressed immediately prior to the onset of beating and, once
integrated into mature myofibrils, they tend to have a relatively long
half-life that varies between 3 and 10 days
(Martin, 1981). Moreover,
between E8.25 and E10.5, the developing heart increases in mass primarily
through the addition of cells from the second cardiac lineage
(Zaffran et al., 2004).
Therefore, there may be little requirement for newly synthesised structural
proteins during these early stages of heart development. The initial
activation of the genes encoding sarcomeric proteins is clearly carried out by
alternate, as yet unidentified, transcriptional regulation pathways, with the
role of Prox1 confined to regulating sarcomere maintenance and stability from
E10.5 onwards, when all populations of cardiac cells have been acquired and
the developing heart continues to grow by a combination of both cardiomyocyte
hyperplasia and hypertrophy. The fact that we observed significantly impaired
hypertrophic growth following loss of Prox1 is secondary to the
primary defect of disrupted assembly of sarcomere proteins. Developing
cardiomyocytes elongate in a unidirectional manner by addition of sarcomeres
to the existing myofibrils, the timing of which corresponds precisely with the
onset of myocardial disruption and failure of the cells to elongate in
Prox1-conditional myocardium (see the model in
Fig. 5M).
|
-actinin,
N-RAP and zyxin, as direct targets of Prox1 suggests that misregulation of
essential sarcomere components and their interacting protein partners is the
primary cause of myofibril disruption in Prox1-conditional
myocardium. A number of other studies have described roles for transcription
factors in initiating or maintaining cardiac muscle ultrastructure during
development and disease, most notably serum response factor (Srf)
(Balza and Misra, 2006;
Nelson et al., 2005), Gata4,
Nkx2.5 and Mef2 (Akazawa and Komuro,
2003) and calcineurin (Ppp3ca)/Nfat
(Bourajjaj et al., 2008;
Heineke and Molkentin, 2006).
However, to the best of our knowledge, no study to date has demonstrated
direct transcriptional regulation of structural protein genes in vivo.
Aberrant terminal differentiation and improper assembly of contractile
protein filaments are associated with a number of cardiac myopathies
(Engel, 1999;
Gregorio and Antin, 2000;
Seidman and Seidman, 2001).
Many of these disorders are caused by mutations in components of the
myofibrillar apparatus itself, including β-MHC, troponins T and I, titin
and -tropomyosin (Alcalai et al.,
2008; Chang and Potter,
2005), or perturbations in the associated calcium-dependent
signalling pathways (Frey et al.,
2004; Molkentin et al.,
1998). However, a large proportion of cardiomyopathies remain
unexplained, with no mutations found in sarcomere or sarcomere-related
proteins. Our study not only provides novel insight into the transcriptional
regulation of cardiomyocyte ultrastructure and hypertrophy during development,
but also implicates Prox1 as a crucial regulatory factor that might underlie
the pathology of both inherited and acquired myopathic disease.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/3/495/DC1
|
Footnotes |
|---|
-actinin) and T. Obinata (MyBP-C) for antibodies. This work
was funded by the British Heart Foundation, the
Medical Research Council,
and by R01-HL073402 (G.O.) from the
National Institutes of
Health and by the American
Lebanese Syrian Associated Charities (ALSAC). Deposited in PMC
for release after 12 months. |
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