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Genetic Networks in Cardiac Development and Stem Cell Biology
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A major focus of GICD’s efforts is on determining the molecular and cellular cues that instruct a stem or progenitor cell to adopt the cardiac cell fate and then differentiate into cardiac myocytes. GICD investigators use mouse and human embryonic stem cell lines in an attempt to harness the potential of stem cell biology for cardiovascular therapeutics. Scientists in this group are also studying the three-dimensional morphogenesis of the heart to determine the underlying mechanisms of cardiac malformations that affect newborns and often have consequences later in adulthood. This information will be vital to future attempts to fashion embryonic stem cells into functioning organs. Numerous model organisms are utilized, including mouse, chick, zebrafish, and fruit flies, along with tools of molecular biology and biochemistry. The investigators in this area are Benoit G. Bruneau, PhD, Bruce R. Conklin, MD, Deepak Srivastava, MD, and Shinya Yamanaka, MD, PhD.
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| Laboratory of Benoit Bruneau |
Heart development begins with the differentiation of committed precursors and continues through early postnatal life with gene programs relevant to adult heart disease. The Bruneau lab focuses on the transcriptional regulation of these processes and on the role of chromatin remodeling factors in cardiogenesis.
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Mouse models of congenital heart disease. A normal heart is on the left; a mutant heart is on the right. The mutant heart has persistent truncus arteriosus (PTA), a failure of outflow tract septation into the aorta and pulmonary artery, and a small right ventricle (rv), both considered severe congenital heart defects.
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Chromatin remodeling in heart development. Left: the process by which Baf60c, a chromatin remodeling factor, interacts with human disease-causing DNA-binding factors (e.g., Nkx2-5 and Tbx5) to bring the chromatin remodeling enzyme Brg1 to precardiac genes; subsequent chromatin remodeling activates genes in the heart. Right: reducing the function of Baf60c in the mouse results in severe defects in heart formation, and a partial reduction causes congenital heart defects.
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| Laboratory of Bruce R. Conklin |
The Conklin laboratory studies how hormone receptors coordinate the development and function of complex tissues such as the cardiovascular system. Their research is focused on the largest known family of receptors for hormones and drugs which are the G protein–coupled receptors (GPCRs), which are encoded by over 700 human genes. The laboratory combines genetic knockouts, designer GPCRs and bioinformatics approaches to gain a basic understanding of hormone signaling in mice and pluripotent embryonic stem (ES) cells.
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New receptors to rewire signaling pathways. The Conklin laboratory has engineered GPCRs called RASSLs (receptors activated by small synthetic ligands) that are unresponsive to endogenous natural hormones, but can still be activated by synthetic small-molecule drugs. We have successfully expressed RASSLs in a wide variety of tissues, controlling responses such as heart rate. These first RASSLs have already been used as tools to examine GPCR signaling in complex systems, including bone development taste, and olfaction. In recent years RASSLs have been developed to activate all the major GPCR pathways. RASSLs are now used in many laboratories worldwide to answer basic questions in neurobiology, endocrinology and cardiovascular studies. |
Bioinformatics, GenMAPP. Our pathway-oriented bioinformatics effort has produced a free, publicly distributed software package, GenMAPP (Gene Map Annotator and Pathway Profiler). GenMAPP is now used by researchers world-wide (>16,000 unique registrations, in 40 countries, >300 publications citing the program). We are expanding this open source program to allow genome-wide, pathway-oriented analysis for twenty species, for all types of functional genomic data, such as genetic variation, disease association studies, and analysis of functional genomic experiments. The software and more information are available at www.GenMAPP.org. |
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Stem cells as a model system. Our functional genomic experiments focus on GPCR signaling pathways in pluripotent mouse and human ES cell-derived cardiac myocytes (right). The Conklin laboratory is also studying ES cell differentiation at the level of gene transcription and alternative splicing that guide cell transition from ES cells to myocytes.
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| Laboratory of Deepak Srivastava |
The Srivastava laboratory focuses on transcriptional and translational regulation of key pathways in cardiac progenitor cell differentiation and cardiac morphogenesis. The scientists have discovered the genetic causes of several forms of familial congenital heart disease and elucidated the disease mechanisms. Recently, the Srivastava laboratory showed that several of the regulatory factors known as microRNAs (miRNAs) have critical roles in cardiac development and postnatal heart function. They use these and other pathways regulating early cardiac commitment to manipulate decisions of mouse and ES cells to differentiate or proliferate, a key step toward therapeutic application of ES cells for treatment of heart disease. |
Genetic knockout of the muscle-specific miRNA, miR-1-2, revealed roles for miR-1 in heart morphogenesis, cardiac cell cycle, and adult heart rhythm. Left: Sections through wild-type (WT) or miR-1-2–/– mouse hearts at embryonic day 15.5 (E15.5) show a ventricular septal defect (arrowhead) in a miR-1-2–/– embryo, a common human cardiac birth defect and a cause of death in some miR-1-2–/– embryos. Middle: miR-1-2–/– hearts continued to initiate nuclear division after birth, when cardiac cell proliferation has ceased in WT hearts (green-labeled cells, arrows). Right: ECGs of adult WT or miR-1-2–/– mice revealed heart rhythm defects, consistent with the sudden death observed in these animals.
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| Roles of miR-1, miR-133, and Wnt in cardiac cell specification and differentiation. Wnt activates beta-catenin to regulate gene expression and many cellular processes. Left: Loss of beta-catenin in progenitors of the right ventricle (rv) and outflow tract (ot) in mice reduced the size of these structures; while an excess of beta-catenin increased ot and rv size without affecting the left ventricle (lv). The ot and left ventricle are marked in blue. As shown in the model (right), our studies using mouse embryos, mouse ES cells, or human ES cells have revealed both inductive and suppressive roles for the muscle-specific microRNAs, miR-1, and miR-133, and the signaling molecule Wnt in various cardiac cell lineage decisions. |
Genetic mapping to identify human disease genes. In this example, the Srivastava laboratory identified a five-generation family with aortic valve disease. The disease ranges from grossly malformed leaflets identified at birth to an age-dependent degenerative disease of the valve involving severe and premature calcification of the aortic valve. This is the third most common form of heart disease and resembles the calcification observed in atherosclerosis. Genomewide scanning revealed a severe point mutation in the NOTCH1 gene, which causes both the malformation and a failure to repress an osteogenic (bone) gene program in the fibrous valve tissue over a lifetime. The disease mechanism is being studied in mice and will also be studied in patient-derived human embryonic stem cells as these become available (see work of Dr. Yamanaka).
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