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First published online 29 March 2007
doi: 10.1242/dev.002014
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Meeting Review |
1 Gladstone Institute of Cardiovascular Disease and Department of Pediatrics and
University of California, San Francisco, CA 94158, USA.
2 Cardiovascular Research Institute and Department of Biochemistry, University
of California, San Francisco, CA 94158, USA.
* Author for correspondence (e-mail: bbruneau{at}gladstone.ucsf.edu)
SUMMARY
At a recent Keystone symposium on `Molecular Pathways in Cardiac Development and Disease' in Colorado, significant advances in the understanding of heart development were discussed. The identification and isolation of cardiovascular progenitors, their modulation by secreted factors, and some tantalizing insights into cardiac regeneration were some of the highlights of what was characterized by some as a renaissance in cardiovascular development.
Introduction
When cardiovascular biologists recently gathered in Breckenridge, Colorado for the Keystone Symposium on `Molecular Pathways in Cardiac Development and Disease' (organized by Kenneth Chien, Eric Olson and Ketty Schwartz), it was clear that significant change had taken place in the field. Kenneth Chien (Harvard Medical School, Boston, MA, USA) put it best when he declared in his opening comments that we were witnessing a renaissance in cardiovascular biology. Indeed, if the era of descriptive anatomy was our medieval times, then, with the findings discussed at this conference, we are certainly entering the age of enlightenment. What profound discoveries are ushering in this new age? Largely, it is the much-anticipated and late-coming identification of cardiovascular precursors, and the understanding of their differentiation and allocation to different segments of the forming cardiovascular system. Greatly related to this is the understanding of how these precursors can be identified and expanded ex vivo, which has finally brought some hope towards the long-sought goals of cardiac regeneration. From a developmental biologist's perspective, this also has provided vindication for those who professed that, to regenerate a tissue, one must understand how it develops in the first place!
Progenitors netted
The identification and harnessing of cardiovascular lineages was one of the
big topics of discussion. Until recently, it was assumed that the heart arises
from a crescent of differentiating mesoderm cells that somehow expand to form
the nascent heart (e.g. Srivastava and
Olson, 2000
). This dogma came under serious scrutiny in a series
of papers that showed that the origin of a large portion of the developing
heart lay outside of the cardiac crescent (reviewed by
Buckingham et al., 2005). This
idea seemed almost heretical at the time, but shortly thereafter it received
crucial support with the molecular identification of one of the controlling
forces behind this `second heart field' (SHF), a transcription factor named
Isl1 (Cai et al., 2003).
Indeed, Isl1 is expressed in the SHF, and lineage-tracing experiments
showed that the descendants of Isl1-expressing cells populate most of
the heart, except for the free wall of the left ventricle. Furthermore, mice
lacking Isl1 fail to develop a right ventricle or outflow tract,
which are largely derived from the SHF. More recently, it has been shown that
Isl1-expressing precursor cells persist in late fetal and perinatal
life, suggesting that they might function as myoblast equivalents, poised to
regenerate a diseased or damaged heart
(Laugwitz et al., 2005).
At the Breckenridge conference, Chien started things off by taking these
findings a significant step forward. His team identified, based on the
expression of Isl1, an early multipotential embryonic cardiovascular
precursor cell that could differentiate into cardiac myocytes, smooth muscle
cells and endothelial cells (Moretti et
al., 2006) (Fig.
1). These progenitors could be isolated from mouse embryos, as
well as from embryonic stem (ES) cells differentiated into embryoid bodies.
Importantly, clonal analysis showed that a single Isl1-positive cell could
give rise to all three cardiovascular lineages. They then further defined the
cardiac lineage as being positive for Isl1, for the transcription factor
Nkx2-5 and for the receptor Flk1 (also known as Kdr - Mouse Genome
Informatics). This echoed another paper that was published last year, which
also identified Flk1+ multipotent cardiovascular progenitors
(Kattman et al., 2006),
although contrasted with a third paper that identified early bipotential
precursors as an Nkx2-5+/Sca-1+ population
(Wu et al., 2006). Thus, the
concepts and tools of hematopoietic developmental biology, namely multipotent
lineage progenitors and cell-sorting technologies, appear to have been
successfully adopted by cardiovascular biologists, and we now finally have a
handle on the developmental origin and the differentiation sequence of
multiple cardiovascular lineages. Brian Black (UCSF, San Francisco, CA, USA)
presented results of experiments aimed at understanding the molecular control
of the lineage transition from SHF progenitors to cardiac myocytes, in the
context of the developing organism. It was known that an enhancer from the
Mef2c gene controls its own expression specifically in the anterior
portion of the SHF (known as the anterior heart field, or AHF), and that this
transcriptional regulation is dependent on Isl1-binding sites
(Dodou et al., 2004). A
puzzling aspect of these findings is the apparent lack of overlap between
Mef2c expression (which includes the AHF and also its derivatives)
and Isl1 expression (which includes the SHF but not its derivatives).
How could the Isl1-binding sites regulate the expression of Mef2c
beyond the domain of Isl1 expression? It turns out that Isl1 binding may
effect epigenetic changes at the chromatin level, presumably allowing the
persistent binding of subsequent transcriptional regulators, such as Gata4
and, probably, others. The theme of chromatin remodeling was further explored
by Benoit Bruneau (Gladstone Institute of Cardiovascular Disease, San
Francisco, CA, USA.), who showed that the de novo activation of cardiac genes
by cardiac DNA-binding transcription factors, such as Tbx5 and Nkx2-5, largely
depends on the recruitment of chromatin-remodeling complexes.
|
). These findings were extended to show that this mechanism is
likely to be relevant to human congenital heart defects, which can be caused
by NKX2-5 mutations. A Wnt turnaround: promoting cardiac differentiation
Another major focus of the meeting was on the signals that are important
for the differentiation of cardiovascular precursors into their final state.
Particular attention was paid to the differentiation of precursors into
cardiac myocytes. A surprising consensus to emerge at the meeting was that the
secreted Wnt family of signaling molecules are crucial positive modulators of
cardiac differentiation. This was rather surprising because, once again, it
was going against `firmly established' dogma; evidence from frog- and
chicken-embryo manipulation, and from the culture of P19 embryonal carcinoma
cells, have all converged towards one conclusion: that Wnts signaling via the
canonical ß-catenin-dependent pathway are negative regulators of cardiac
differentiation (Marvin et al.,
2001; Pandur et al.,
2002; Schneider and Mercola,
2001; Tzahor and Lassar,
2001). However convincing, these findings are at odds with the
positive role of Wnt signaling in Drosophila cardiogenesis
(Park et al., 1996;
Wu et al., 1995).
At the Keystone meeting in Breckenridge, several investigators presented
evidence that Wnts, in fact, can act as positive regulators of cardiogenesis
via the canonical pathway. Deepak Srivastava (Gladstone Institute of
Cardiovascular Disease, San Francisco, CA, USA) and Michael Schneider (Baylor
College of Medicine, Houston, TX, USA) showed that Wnts, or the activation of
the Wnt signaling pathway, could promote cardiogenesis in cultured ES cells
that are allowed to differentiate into embryoid bodies. These findings have
gained support from a recent publication that describes a positive and
negative timing-dependent role for Wnts in cardiogenesis from ES cells
(Naito et al., 2006). Thus,
Wnts may play a dual role in cardiac development by acting as positive
regulators at first and then switching to a negative-regulatory role
subsequently. Interestingly, Schneider presented evidence that seemed to
demonstrate that the pro-cardiogenic action of Wnts in embryoid bodies that
have been derived from ES cells is, in fact, partly due to cell non-autonomous
effects of Wnts, perhaps via the induction of Sox17 and Sox17-dependent genes
in adjacent endodermal cells (Liu et al.,
2007). However, other studies performed in vivo have indicated
that Wnts might be acting in this context in a cell-autonomous manner. As
discussed by Srivastava and Edward Morrisey (University of Pennsylvania,
Philadelphia, PA, USA), the deletion of ß-catenin in cardiac precursors
results in the loss of cardiac structures, whereas the activation of
ß-catenin in the same cells leads to the formation of ectopic cardiac
tissue. In vivo, Srivastava reported, ß-catenin appears to function not
in cell specification or migration, but rather in the expansion of the SHF
progenitors, consistent with findings in ES cells.
Regenerating the heart?
The discussions about progenitors and their regulation by extrinsic factors
prompted thoughts of cardiac regeneration. However, very little evidence shows
that this might be possible. One exciting recent development has been the
identification of the mechanisms by which the zebrafish heart can regenerate.
Although previous work had shown that, unlike in mammalian hearts, this
regeneration was possible (Poss et al.,
2002), the mechanisms underlying this phenomenon remained rather
mysterious. Kenneth Poss (Duke University, Durham, NC, USA) presented recent
evidence that resident cardiovascular precursors can be recruited to the site
of injury to repopulate the missing tissue, and that a recapitulation of the
developmental stages of cardiogenesis takes place during this process
(Lepilina et al., 2006).
Furthermore, he demonstrated that the initial step in the response to injury
is a reprogramming of the entire epicardium, followed by
epithelial-mesenchymal transformation of these cells and subsequent
neovascularization (Lepilina et al.,
2006). The location and identity of dormant regenerative
precursors remains mysterious, although Poss presented new data that suggests
that cellular and molecular mechanisms important for cardiac regeneration also
help to mediate the growth and homeostatic maintenance of the adult heart.
Whether similar events can be forced upon mammalian myocardium remains to be
seen. However, promising results were presented by Paul Riley (UCL Institute
of Child Health, London, UK), who showed that Thymosin ß4, an
actin-binding protein, is essential for the formation of the coronary
vasculature from the epicardium, and that exogenous Thymosin ß4 could
induce the recruitment of adult epicardial cells to stimulate
neovascularization (Smart et al.,
2007). These findings provide an exciting potential means by which
to help an injured or ischemic myocardium to repair, as had been suggested
from previous findings that have shown that Thymosin ß4 has a beneficial
effect on infarcted hearts in mice
(Bock-Marquette et al., 2004).
These results also indicate a mechanism by which injured myocardium could be
revascularized, which is perhaps an important step in facilitating
regeneration.
Transcriptional and post-transcriptional regulation
Significant advances in our understanding of the transcriptional and
post-transcriptional regulation of heart development and maturation were also
presented at this meeting. Important new insights into the transcriptional
control of the development of the cardiac conduction system - the specialized
cells that propagate cardiac impulses - were presented by Ivan Moskowitz
(University of Chicago, Chicago, IL, USA), who described the identification of
the helix-loop-helix factor Id2 as an essential regulator of the patterning
and function of the cardiac conduction system. Id2 was identified as lying
downstream of Tbx5 and Nkx2-5, a pair of transcription factors that have
previously been shown to influence arrhythmias in humans and mice when
haploinsufficient (Jay et al.,
2004; Mori and Bruneau,
2004; Moskowitz et al.,
2004; Schott et al.,
1998). Indeed, Moskowitz showed that compound haploinsufficiency
of Tbx5 and Nkx2-5 appears to completely abrogate
ventricular conduction-system specification.
Post-transcriptional regulation has finally entered its age of
enlightenment with regard to heart development, where microRNAs and their
regulatory mechanisms have been painted onto the cardiac canvas. Srivastava
presented work on the loss-of-function of a muscle-restricted microRNA,
miR-1-2 (also known as Mirn1-2 - Mouse Genome Informatics),
which has previously been implicated in heart development
(Zhao et al., 2005). Mice
lacking miR-1-2 have a range of phenotypes, including: cell cycle
dysregulation, which results in the hyper-proliferation of cardiac myocytes;
structural defects that include ventricular septal defects; and postnatal
electrophysiological defects. This range of defects reflects the probable role
of microRNAs in modulating multiple targets. The essential role of microRNAs
in cardiac development was also strongly reinforced by Eric Olson (UT
Southwestern, Dallas, TX, USA), who also presented exciting new data
concerning microRNA-regulated events in the post-natal maintenance of the
heart, including the altered expression of several microRNA genes in stressed
hearts and their role in the pathological response of the heart to hypertrophy
(van Rooij et al., 2006).
Conclusion
The molecular picture of the heart, and how this organ develops and functions, is far from being fully painted; but, if the initial brush strokes from our current renaissance in cardiac biology are anything to go by, then our interpretation of this vital organ, once sketched so elegantly by da Vinci himself in 1510, will truly be worth comparing to the works of the great Leonardo himself.
ACKNOWLEDGMENTS
We thank D. Srivastava for critical reading of the manuscript, and all of the presenters cited for comments and clarifications.
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