DEVELOPMENTAL DYNAMICS 231:655– 659, 2004
SHOP TALK
Een Geslaagd Jubileum! The 10th Weinstein Developmental Cardiovascular Conference in Leiden Corey H. Mjaatvedt,1* Scott Klewer,2 Andy Wessels,1 and Robert G. Gourdie1 Received 25 June 2004; Accepted 25 June 2004
The 10th annual Weinstein Conference was held May 13–16, 2004, at Leeuwenhorst Conference Center in the Netherlands outside of Leiden. The Weinstein meeting, founded by Dr. Constance Weinstein and subsequently named in her honor by the attendees, is one of the leading annual gatherings of researchers studying heart development. Basic research scientists and research clinicians were brought together from around the world to share their latest data and concepts of heart development. This year the meeting was sponsored by the Leiden University Medical Center in collaboration with the Medical University of South Carolina in Charleston and was attended by approximately 270 participants. The meeting began Friday evening with a welcoming reception followed by the keynote lecture by Margaret E. Buckingham of the Pasteur Institute. Dr. Buckingham’s lecture entitled, “Programming Cells to Form Muscle: The Cellular Contribution to Cardiogenesis,” reviewed her recently published work on lineage analysis of the anterior heart field in the mouse by a retrospective clonal analysis ap-
proach that takes advantage of an infrequent and spontaneous mitotic recombination of an inactivated nlaacZ reporter into an active nlacZ reporter in mouse (Meilhac et al., 2004). The analysis demonstrated that two lineages arise in the heart fields from a common progenitor. These two lineages then contribute unequally to specific regions of the developing heart. The left ventricle and the outflow tract seem to derive from a single lineage, whereas other regions derive from both. During questions and discussion, Roger Markwald proposed that the two lineages are derived at earlier stages from the primitive lateral heart fields and then are sorted into the separate lineages. The observation that some chambers derive from more than one lineage is consistent with earlier concepts that suggested the primitive heart forms initially in a segmental manner, but the mature chambers of the heart result from cellular interactions and remodeling of these primitive segments (Mjaatvedt et al., 1999). The cellular and molecular interactions leading to this specification of precardiac lineages will no doubt be a hot topic of future research in this area.
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SESSIONS The platform sessions began the next morning and were divided into six categories: Cardiogenesis and Chamber Formation, the Conduction System, Stem Cells and Regenerative Medicine, the Outflow Tract, Epithelial Mesenchymal Transformation, and Function and Imaging. Below, we have summarized some of the trends and novel findings reported during these sessions.
Cardiogenesis and Chamber Formation An appropriate, almost traditional, format of the Weinstein meeting is to start at the beginning of heart formation with a session on Cardiogenesis and Chamber Formation. The role of transcription factors SRF and NFATc1 and the Notch and Wnt signaling pathways in regulating gene expression and cardiac cell fates was explored in this session. Robert Schwartz described his work on the regulation of cardiovascular lineages by the differential phosphorylation of SRF MADS Box. Bin Zhou showed that NFATc1 activates genes such as DSCR1, Egr, and PCNA in an endothelial cell line (REN) and, surprisingly, to suppress
Department of Cell Biology and Anatomy, Medical University of South Carolina, Charleston, South Carolina Departments of Pediatrics and Cell Biology/Anatomy, Steele Memorial Children’s Research Center, University of Arizona, Tucson, Arizona *Correspondence to: Corey H. Mjaatvedt, Department of Cell Biology and Anatomy, Medical University of South Carolina, 173 Ashley Avenue, P.O. Box 250508, Charleston, SC 29425. E-mail:
[email protected] 2
DOI 10.1002/dvdy.20169 Published online 16 September 2004 in Wiley InterScience (www.interscience.wiley.com).
© 2004 Wiley-Liss, Inc.
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some genes known to function in epithelial/mesenchymal transformations such as Egfr and MMP3, suggesting that the semilunar valves may form by unique molecular mechanisms. Yusuke Watanabe showed that an expression of a constitutively active form of Notch-1 in the cardiac lineage of transgenic mice results in the specific up-regulation of Notch target genes and defects in trabeculation. Robert J. Garriock reported that Wnt11-related gene (Wnt11R) is the closest ortholog to chicken and mammalian Wnt11. Wnt11R activates JNK kinase and is sufficient but not necessary for induction of cardiac markers. Wnt11R seems to modulate myocardial tissue organization rather than specification of lineage. Several zebrafish models of defective cardiac contractility were described by Hope Coleman who evaluated the effects of aberrant contractility on heart morphogenesis. In these mutants, ventricular contractility is more important for atrioventricular (AV) valve formation than for atrial function. Atrial function in turn leads to irregular ventricular morphogenesis. An interesting highlight of this session was the somewhat controversial topic of atrial chamber formation. Antoon Moorman showed a series of three-dimensional (3D) reconstructions of the developing mouse heart based on gene expression profiles that strongly suggested that the pulmonary veins develop within an expression domain demarcating atrial myocardium, supporting earlier studies (Webb et al., 1998; Wessels et al., 2000). Marco De Ruiter, however, presented data that were pointing to an origin of the pulmonary veins within the tissues of the sinus venosus.
Conduction System One of the first genes identified in humans to cause inherited cardiovascular defects was Nkx2-5 (Schott et al., 1998; Benson et al., 1999). Subsequent work in humans and other species demonstrated that Nkx-2.5 is specifically up-regulated in conduction tissues (Thomas et al., 2001). In the Conduction System session, Magali Theveniau-Ruissy presented her work on targeted reporter genes
used to probe conduction system development in the Nkx2-5 ⫹/⫺ mouse. By crossing the Nkx2-5⫹/⫺ mice with Cx40eGFP/⫹ mice, they found that EGFP strands were reduced in the His–Purkinje system of the ventricles, particularly in the apex, confirming the results of another recent study (Jay et al., 2004). However, the study went further to show that the defective phenotype arises during the first four neonatal days (P0 –P4), suggesting that Nkx2-5 functions during a late maturation phase of the cardiac conduction system. Continuing this theme of linkage between development and disease, Vinciane Gaussin presented data that mice with a targeted deletion of the bone morphogenetic protein receptor ALK3 had structural and functional changes in the AV myocardium compatible with Wolf– Parkinson–White syndrome in humans. The anatomical substrate for conduction tracts that bypass the AV node, as in WPW, were also a focus of the presentation by Monique Jongbloed, who showed 3D reconstructions of the distribution of LacZ-positive cells in embryos from the CCS-LacZ mouse. Benoit Bruneau described his data from an Irx5 knockout mouse that indicated Irx5 may be a transcriptional regulator of transmural distributions of ion channels responsible for repolarization. Transcription factors were also addressed by Willem Hoogaars from the Amsterdam group, who described work consistent with a model in which continuous repression of chamber phenotype by Tbx3 occurs in regions of the heart in which central conduction system components form. In other work of relevance to the conduction system, Diego Franco reported the use of the nLaacZ mouse to show that conduction cells and working myocytes in mouse share a common clonal origin, confirming earlier retroviral clonal analyses undertaken in chick (Cheng et al., 1999).
Stem Cells and Regenerative Medicine Stem cells and regenerative therapies offer unprecedented opportunities for treating many diseases, in-
cluding cardiovascular diseases that have their origins in the embryo. The third platform session, i.e., Stem cells and Regenerative Medicine, explored this topic from both a basic science and clinical perspective. Drs. Douwe Atsma and Kai Wollert both provided fascinating looks at the current status and implication of stem cell technology for cardiovascular medicine. Their clinical safety and feasibility studies involving small groups of European patients with end-stage heart failure demonstrate improved cardiac function and no toxicity after intracoronary delivery of autologous bone marrow stem cells. Consistent with mouse studies by Loren Field showing that stem cells do not readily acquire a cardiac phenotype, these noncontrolled human studies demonstrate only a small percentage of injected cells actually enter the myocardium. The authors speculate that observed benefits from stem cell therapy may result from exogenous “software rather than hardware.” In other words, stem cell delivery of essential soluble factors, rather than new myocardial cells per se, may lead to improved cardiac function in heart failure patients. In this regard, Nadia Rosenthal presented work showing that a locally expressed isoform of IGF-1 could recruit or enhance the ability of myogenic progenitors to regenerate muscle in transgenic mice. A highlight of this session was the Einthoven Foundation lecture and presentation of the Einthoven medal awarded to Prof. Peter Carmeliet. Dr. Carmeliet presented his work on the role vascular endothelial growth factor (VEGF) and family members PIGF and VEGF-B play in normal and abnormal development of the vascular system (Carmeliet et al., 1999; Luttun et al., 2002, 2004; Autiero et al., 2003; Stalmans et al., 2003). In addition, Dr. Carmeliet described his genetic research that linked low levels VEGF to an increased risk of amyotrophic lateral sclerosis (ALS) in both mouse and humans (Oosthuyse et al., 2001; Lambrechts et al., 2003). Dr. Carmeliet hopes to translate this basic research knowledge of the VEGF gene into novel clinical applications that could have beneficial effects for patients with ALS.
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Outflow Tract The cardiac outflow tract (OFT) is involved in nearly one third of all live birth congenital heart defects. Since the first reports at the Weinstein meeting in Tucson (1999) that the outflow tract originates from an alternative anterior heart field, our understanding of the developmental origins, cellular contributions, and remodeling of the outflow tract of the heart has progressed significantly. The largest platform session in this year’s Weinstein Meeting, as well as the keynote lecture by Dr. Buckingham, reflected the growing interest in outflow tract development. Highlights of this session included the identification of new subpopulations of cell involved in avian OFT development, the roles of transforming growth factor beta (TGF) signaling, and genetic modifiers of del22q11 syndromes. Andy Wessels identified one subset of cells at the base of the semilunar valves using a new marker (alpha h1-calponin isoform) that specifically localizes to a population of cells undergoing a mesenchymal to myocardial transition, a process recently implicated in models of heart malformations. Simon Conway similarly reported a novel subset of “periostin expressing” cells at the base of the outflow tract that is reminiscent of neural crest (NCC). However, they do not correspond to neural crest lineage maps and may be a previously unrecognized population. Several investigators reported new findings concerning the role of growth factor signaling in the outflow tract. Michiko Watanabe reported that hypoxia-induced apoptosis of the proximal outflow tract triggered both hypoxia-induced factor 1 alpha (HIF-1alpha) and VEGF receptor 2. Of interest, HIF-1alpha was not restricted to apoptotic regions but was also found in the distal outflow where very little apoptosis is detected. The expression of VEGF receptor 2, however, was correlated with a lack of apoptosis in the distal myocardium. TGF- signaling through SMAD2 was reported to play an essential role in development of the aortic arch (Daniel Molin). Deleting of the type II Tgf receptor in neural crest lineage of
reporter (wnt1-Cre/TGFr2 floxed/ R26R) mouse embryos results in persistent truncus arteriosus, possibly due to the premature change from a mesenchymal to squamous cell morphology in neural crest– derived smooth muscle cells. TGF- signaling is also required to prevent interrupted aortic arch by preventing apoptosis in the fourth arch artery (Henry Sucov). The TGF2⫺/⫺ mouse shows higher than normal levels of NCC in the septum and semilunar valves along with a lack of myocardialization, suggesting that apoptosis, proliferation, and differentiation are affected by the loss of TGF2 ligand (Tom Doetschman). Cleary, TGF signals seem to be involved in multiple aspects of mesenchyme growth and remodeling in the outflow tract and this will continue to be an area of intense research. Other work reported on progress in understanding DiGeorge and other del22q11 syndromes. The T-box– containing gene Tbx1 has been identified as a candidate locus within the 22q11 deleted locus; however, other modifying genetic loci are likely to play a role. Anne Moon described her work on FGF8, showing evidence that FGF8 signaling is directly upstream of Crkl, a 22q11 gene product, suggesting that impaired FGF8 signaling may be of major importance in DiGeorge and other del22q11 syndromes. The role of FGF8 signaling was confirmed by a significant increase in the cardiovascular defects observed in the homeobox gene Gbx2⫺/⫺;Fgf8 ⫹/⫺ mutants reported by Noah Byrd and suggests that Gbx2 is a potential candidate 22qll modifying locus.
Epithelial/Mesenchymal Transformation Much of heart development can be thought of as a series of epithelial to mesenchymal transformations starting with gastrulation to form precardiac mesoderm, the transformation of cells to form the endocardial tube, endocardial transformation to cushion mesenchyme, epicardial to mesenchymal transformations to form coronary arteries, conduction system, and contribute to developing valves. As part of the Epithelial
Mesenchymal Transformation session, Brenda Rongish presented interesting work on the displacement of the extracellular matrix molecules, particularly fibrillin, defective in Marfan syndrome, during the cellular transformations associated with bilateral heart field fusion. Timelapse movies of fibrillin and fibronectin being swept into the fusing heart tubes made an interesting point that components of the extracellular matrix, like cells, have their own developmental fates that influence morphogenesis at relatively distant locations and times. Another presentation explored the downstream targets affected in cushion mesenchyme by inhibiting the gene Mox-1, required for cushion mesenchyme transformation and screening with cDNA chick heart arrays (Ray Runyan). Several genes were up-regulated, including HSP90, ANF, HAS2, and beta-1,4,-galactosyltransferase. Ankur Saxena presented data suggesting that regulation of migrating cells in heart cushions and the cerebellum may rely on the same signaling molecules, G-protein coupled receptor (CXCR4), stromal cell-derived factor 1 (SDF-1) and ephrin-B1, B2. The epicardial to mesenchymal transformation was explored by John Burch, who described the first identified epithelial to mesenchymal transformation response element that modulates a 10-fold increase in epicardial specific activity when rat epicardial cell are gown in an epithelial versus mesenchymal cells. Rob Dettman presented data, and an inventive slide movie, demonstrating that VCAM-1 binding to ␣4integrin inhibits mesenchyme formation at the epicardial surface possibly through changes in the actin cytoskeleton. Finally, evidence was presented (Ramon Munoz-Chapuli) supporting the hypothesis that WT-1 may control the balance between EPDC subpopulations effects on myocardium, vascular smooth muscle, or vascular endothelium.
Function and Imaging The classic developmental concept that “form follows function” is especially true in heart development because cardiogenesis must proceed
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within the context of a working organ subject to changing blood flow, pressure, and shear stress. In the Function and Imaging session, Beerend Hierck presented a computation fluid dynamics model of the beating heart in an effort to identify within contraction phases “hotspots” of changing stress to be correlated with shifts in KLF-2, ET-1, and NOS-3 gene expression. Similarly, Ferdinand le Noble showed that hemodynamics also dynamically regulates arterial–venous patterning in the yolk sac by modulating the expression of genes such as ephrin-B2, neuropilin-1, and -2. Observations such as these point out the need for new techniques to model and test hypothetical links between gene expression and stress forces in vitro. In a remarkable example of cardiac biomimicry, Rich Goodwin described the engineering of small aligned collagen tubes that, when seeded with appropriate embryonic cell types, can be used to replicate heart morphogenesis, including the formation of leaflet-like structures from AV cushion tissue. Applying currently available medical imaging techniques, such as fiberoptic optical coherence tomography (Florence Rothenberg), magnetic resonance imaging of embryos (Shoumo Bhattacharya), and ultrasound/echocardiography showed great promise for rapid noninvasive screening of developmental cardiovascular defects in animal models. As an example, Cecilia W. Lo demonstrated the considerable progress made in combining ultrasound imaging with ENU mutagenesis in mice to rapidly screen for mutations causing congenital cardiovascular anomalies.
POSTER SESSIONS As with all previous Weinstein conferences, the poster sessions were at the true “heart” of the meeting. The poster session provides a particularly good forum for students and younger investigators to present their current research and receive feedback from future reviewers. Of the approximately 270 participants at the meeting, 94 were students. Each year several posters by younger investigators
are selected to receive special recognition at the awards dinner. The winning prize this year was awarded to William Pu for his work entitled “Regulation of Heart Development by GATA4: Distinct Roles of Endocardial and Myocardial Expression in Cardiac Morphogenesis.” Others who received special recognition were (in alphabetical order): Konstantinos Bilbilis, Ching-Pin Chang, Bianca C.W. Groenendijk, Katharine M. Hardy, and Cindy Martin. Some other highlights of the poster session were elegant lineage tracing experiments by de Lange and colleagues that clarified the origin and process of AV valve leaflet remodeling. Insights into regulatory domains responsible for AV canalspecific expression were identified through analysis of NFATC (Bin Zhou), TBX2 (Vincent Christoffels), and GATA (John Burch). Importantly, silencing domains may prove essential for regionally restricted cushion formation and epithelial to mesenchymal transformation. Several excellent posters focused on the interrelationship between cardiac structure and function during development. For example, Rob Poelman and colleagues examined molecular changes related to localized sheer stress and flows. John Foker demonstrated the importance of flow in ventricular chamber size in patients with unbalanced endocardial cushion defects. Technological advances in imaging were highlighted in several posters, including optical mapping of the conduction system (Dina Myers), rapid episonic fluorescence imaging capture (EFIC) for rapid 3D reconstruction of heart development (Vipul Mangal, Julie Rosenthal), Doppler velocimetry of vascular development (Sandra Stekelenburg-de Vos), and deep confocal imaging (David Sedmera). These exciting new imaging technologies will surely clarify future investigations of heart morphology.
CONCLUSION The meeting concluded with a dinner party and awards ceremony in Leiden at the impressive Taffeh Tem-
ple in the Museum of Antiquities. We applaud the organizers, Adriana C. Gittenberger-de Groot, Roger R. Markwald, Robert E. Poelmann, Marco C. DeRuiter, Andy Wessels, Tom Trusk, Marit Boot, Joke van Benten, Annalisa Angelini, Martin J. Schalij, and Debby Bridgers, and all the conference participants for making the 10th anniversary of the Weinstein Conference such a memorable event, and we look forward to next year’s meeting in Tucson, Arizona.
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