Sylvia M. Evans

Isl1 identifies a cardiac progenitor population that proliferates prior to differentiation and contributes a majority of cells to the heart.

Hearts of mice lacking Isl1, a LIM homeodomain transcription factor, are completely missing the outflow tract, right ventricle, and much of the atria. isl1 expression and lineage tracing of isl1-expressing progenitors demonstrate that Isl1 is a marker for a distinct population of undifferentiated cardiac progenitors that give rise to the cardiac segments missing in isl1 mutants. Isl1 function is required for these progenitors to contribute to the heart. In isl1 mutants, isl1-expressing progenitors are progressively reduced in number, and FGF and BMP growth factors are downregulated. Our studies define two sets of cardiogenic precursors, one of which expresses and requires Isl1 and the other of which does not. Our results have implications for the development of specific cardiac lineages, left-right asymmetry, cardiac evolution, and isolation of cardiac progenitor cells.

Postnatal isl1+ cardioblasts enter fully differentiated cardiomyocyte lineages

The purification, renewal and differentiation of native cardiac progenitors would form a mechanistic underpinning for unravelling steps for cardiac cell lineage formation, and their links to forms of congenital and adult cardiac diseases. Until now there has been little evidence for native cardiac precursor cells in the postnatal heart. Herein, we report the identification of isl1+ cardiac progenitors in postnatal rat, mouse and human myocardium. A cardiac mesenchymal feeder layer allows renewal of the isolated progenitor cells with maintenance of their capability to adopt a fully differentiated cardiomyocyte phenotype. Tamoxifen-inducible Cre/lox technology enables selective marking of this progenitor cell population including its progeny, at a defined time, and purification to relative homogeneity. Co-culture studies with neonatal myocytes indicate that isl1+ cells represent authentic, endogenous cardiac progenitors (cardioblasts) that display highly efficient conversion to a mature cardiac phenotype with stable expression of myocytic markers (25%) in the absence of cell fusion, intact Ca2+-cycling, and the generation of action potentials. The discovery of native cardioblasts represents a genetically based system to identify steps in cardiac cell lineage formation and maturation in development and disease.

Multipotent Embryonic Isl1+ Progenitor Cells Lead to Cardiac, Smooth Muscle, and Endothelial Cell Diversification

Cardiogenesis requires the generation of endothelial, cardiac, and smooth muscle cells, thought to arise from distinct embryonic precursors. We use genetic fate-mapping studies to document that isl1(+) precursors from the second heart field can generate each of these diverse cardiovascular cell types in vivo. Utilizing embryonic stem (ES) cells, we clonally amplified a cellular hierarchy of isl1(+) cardiovascular progenitors, which resemble the developmental precursors in the embryonic heart. The transcriptional signature of isl1(+)/Nkx2.5(+)/flk1(+) defines a multipotent cardiovascular progenitor, which can give rise to cells of all three lineages. These studies document a developmental paradigm for cardiogenesis, where muscle and endothelial lineage diversification arises from a single cell-level decision of a multipotent isl1(+) cardiovascular progenitor cell (MICP). The discovery of ES cell-derived MICPs suggests a strategy for cardiovascular tissue regeneration via their isolation, renewal, and directed differentiation into specific mature cardiac, pacemaker, smooth muscle, and endothelial cell types.

A myocardial lineage derives from Tbx18 epicardial cells

Understanding the origins and roles of cardiac progenitor cells is important for elucidating the pathogenesis of congenital and acquired heart diseases. Moreover, manipulation of cardiac myocyte progenitors has potential for cell-based repair strategies for various myocardial disorders. Here we report the identification in mouse of a previously unknown cardiac myocyte lineage that derives from the proepicardial organ. These progenitor cells, which express the T-box transcription factor Tbx18, migrate onto the outer cardiac surface to form the epicardium, and then make a substantial contribution to myocytes in the ventricular septum and the atrial and ventricular walls. Tbx18-expressing cardiac progenitors also give rise to cardiac fibroblasts and coronary smooth muscle cells. The pluripotency of Tbx18 proepicardial cells provides a theoretical framework for applying these progenitors to effect cardiac repair and regeneration.

Myofibroblasts revert to an inactive phenotype during regression of liver fibrosis

Myofibroblasts produce the fibrous scar in hepatic fibrosis. In the carbon tetrachloride (CCl4) model of liver fibrosis, quiescent hepatic stellate cells (HSC) are activated to become myofibroblasts. When the underlying etiological agent is removed, clinical and experimental fibrosis undergoes a remarkable regression with complete disappearance of these myofibroblasts. Although some myofibroblasts apoptose, it is unknown whether other myofibroblasts may revert to an inactive phenotype during regression of fibrosis. We elucidated the fate of HSCs/myofibroblasts during recovery from CCl4- and alcohol-induced liver fibrosis using Cre-LoxP–based genetic labeling of myofibroblasts. Here we demonstrate that half of the myofibroblasts escape apoptosis during regression of liver fibrosis, down-regulate fibrogenic genes, and acquire a phenotype similar to, but distinct from, quiescent HSCs in their ability to more rapidly reactivate into myofibroblasts in response to fibrogenic stimuli and strongly contribute to liver fibrosis. Inactivation of HSCs was associated with up-regulation of the anti-apoptotic genes Hspa1a/b, which participate in the survival of HSCs in culture and in vivo.

Nkx2-5 Pathways and Congenital Heart Disease: Loss of Ventricular Myocyte Lineage Specification Leads to Progressive Cardiomyopathy and Complete Heart Block

Human mutations in Nkx2-5 lead to progressive cardiomyopathy and conduction defects via unknown mechanisms. To define these pathways, we generated mice with a ventricular-restricted knockout of Nkx2-5, which display no structural defects but have progressive complete heart block, and massive trabecular muscle overgrowth found in some patients with Nkx2-5 mutations. At birth, mutant mice display a hypoplastic atrioventricular (AV) node and then develop selective dropout of these conduction cells. Transcriptional profiling uncovered the aberrant expression of a unique panel of atrial and conduction system-restricted target genes, as well as the ectopic, high level BMP-10 expression in the adult ventricular myocardium. Further, BMP-10 is shown to be necessary and sufficient for a major component of the ventricular muscle defects. Accordingly, loss of ventricular muscle cell lineage specification into trabecular and conduction system myocytes is a new mechanistic pathway for progressive cardiomyopathy and conduction defects in congenital heart disease.

The Renewal and Differentiation of Isl1+ Cardiovascular Progenitors Are Controlled by a Wnt/β-Catenin Pathway

Isl1(+) cardiovascular progenitors and their downstream progeny play a pivotal role in cardiogenesis and lineage diversification of the heart. The mechanisms that control their renewal and differentiation are largely unknown. Herein, we show that the Wnt/beta-catenin pathway is a major component by which cardiac mesenchymal cells modulate the prespecification, renewal, and differentiation of isl1(+) cardiovascular progenitors. This microenvironment can be reconstituted by a Wnt3a-secreting feeder layer with ES cell-derived, embryonic, and postnatal isl1(+) cardiovascular progenitors. In vivo activation of beta-catenin signaling in isl1(+) progenitors of the secondary heart field leads to their massive accumulation, inhibition of differentiation, and outflow tract (OFT) morphogenic defects. In addition, the mitosis rate in OFT myocytes is significantly reduced following beta-catenin deletion in isl1(+) precursors. Agents that manipulate Wnt signals can markedly expand isl1(+) progenitors from human neonatal hearts, a key advance toward the cloning of human isl1(+) heart progenitors.

Ablation of Cypher, a PDZ-LIM domain Z-line protein, causes a severe form of congenital myopathy

Cypher is a member of a recently emerging family of proteins containing a PDZ domain at their NH2 terminus and one or three LIM domains at their COOH terminus. Cypher knockout mice display a severe form of congenital myopathy and die postnatally from functional failure in multiple striated muscles. Examination of striated muscle from the mutants revealed that Cypher is not required for sarcomerogenesis or Z-line assembly, but rather is required for maintenance of the Z-line during muscle function. In vitro studies demonstrated that individual domains within Cypher localize independently to the Z-line via interactions with α-actinin or other Z-line components. These results suggest that Cypher functions as a linker-strut to maintain cytoskeletal structure during contraction.

β-Catenin directly regulates Islet1 expression in cardiovascular progenitors and is required for multiple aspects of cardiogenesis

Recent studies have demonstrated that the LIM homeodomain transcription factor Islet1 (Isl1) marks pluripotent cardiovascular progenitor cells and is required for proliferation, survival, and migration of recently defined second heart field progenitors. Factors that are upstream of Isl1 in cardiovascular progenitors have not yet been defined. Here we demonstrate that β-catenin is required for Isl1 expression in cardiac progenitors, directly regulating the Isl1 promoter. Ablation of β-catenin in Isl1-expressing progenitors disrupts multiple aspects of cardiogenesis, resulting in embryonic lethality at E13. β-Catenin is also required upstream of a number of genes required for pharyngeal arch, outflow tract, and/or atrial septal morphogenesis, including Tbx2, Tbx3, Wnt11, Shh, and Pitx2. Our findings demonstrate that β-catenin signaling regulates proliferation and survival of cardiac progenitors.

Required, Tissue-Specific Roles for Fgf8 in Outflow Tract Formation and Remodeling

Fibroblast growth factor 8 (Fgf8) is a secreted signaling protein expressed in numerous temporospatial domains that are potentially relevant to cardiovascular development. However, the pathogenesis of complex cardiac and outflow tract defects observed in Fgf8-deficient mice, and the specific source(s) of Fgf8 required for outflow tract formation and subsequent remodeling are unknown. A detailed examination of the timing and location of Fgf8 production revealed previously unappreciated expression in a subset of primary heart field cells; Fgf8 is also expressed throughout the anterior heart field (AHF) mesoderm and in pharyngeal endoderm at the crescent and early somite stages. We used conditional mutagenesis to examine the requirements for Fgf8 function in these different expression domains during heart and outflow tract morphogenesis. Formation of the primary heart tube and the addition of right ventricular and outflow tract myocardium depend on autocrine Fgf8 signaling in cardiac crescent mesoderm. Loss of Fgf8 in this domain resulted in decreased expression of the Fgf8 target gene Erm, and aberrant production of Isl1 and its target Mef2c in the anterior heart field, thus linking Fgf8 signaling with transcription factor networks that regulate survival and proliferation of the anterior heart field. We further found that mesodermal- and endodermal-derived Fgf8 perform specific functions during outflow tract remodeling: mesodermal Fgf8 is required for correct alignment of the outflow tract and ventricles, whereas activity of Fgf8 emanating from pharyngeal endoderm regulates outflow tract septation. These findings provide a novel insight into how the formation and remodeling of primary and anterior heart field-derived structures rely on Fgf8 signals from discrete temporospatial domains.