Rationale: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. Objective: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. Methods and Results: A retrospective clonal analysis using the ␣-cardiac actin nlaacZ/؉ mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using Key Words: conduction system Ⅲ cell lineage Ⅲ clonal analysis Ⅲ nlaacZ Ⅲ Purkinje fibers T he cardiac conduction system controls the generation and propagation of electric activity through the heart to coordinate cardiac contraction. Atrial contraction is initiated by the sinoatrial node or pacemaker. After a delay mediated by the atrioventricular node (AVN), the ventricular conduction system (VCS) ensures rapid propagation of electric activity to the ventricular apex. 1 The VCS is comprised of a central component, the atrioventricular (AV or His) bundle and right and left bundle branches, and a peripheral component composed of a dense ellipsoid network of Purkinje fibers. These structures have been well characterized in adult mouse and human hearts by their specific histological and electrophysiological properties. 2,3 However, despite the clinical importance of the VCS in regulating cardiac rhythm, important questions remain as to the origin and the mode of development of the mammalian VCS. 4 Existing views of VCS development are largely based on data obtained in avian embryos, despite major anatomic differences within the VCS between birds and mammals. In the avian system, lineage analysis using replication defective retroviral labeling has demonstrated that conductive cells share common progenitors with working cardiomyocytes and that the VCS develops by a process of induction and recruitment of myocytes through endothelial derived signals. 5,6 According to this model, conductive myocytes are nonproliferative and subsequent growth of the conduction system occurs by accretional recruitment of new myocytes (or ingrowth). However, in the chick, a perivascular network of Purkinje fibers is localized deep in the myocardium in proximity to coronary vessels, whereas in the mouse, as in humans, Purkinje fibers are present only at the subendocardial ventricular surface 1,7,8 and it is unclear whether the chick model is applicable to the mammalian VCS. In mammals an outgrowth model has been proposed for development of the central VCS, by which conductive cells develop independently of the working myocardium from a pool of conductive Original

SummaryThe homeobox transcription factors NKX2-5 and MEIS1 are essential for vertebrate heart development and normal physiology of the adult heart. We show that, during cardiac differentiation, the two transcription factors have partially overlapping expression patterns, with the result that as cardiac progenitors from the anterior heart field differentiate and migrate into the cardiac outflow tract, they sequentially experience high levels of MEIS1 and then increasing levels of NKX2-5. Using the Popdc2 gene as an example, we also show that a significant proportion of target genes for NKX2-5 contain a binding motif recognized by NKX2-5, which overlaps with a binding site for MEIS1. Binding of the two factors to such overlapping sites is mutually exclusive, and this provides a simple regulatory mechanism for spatial and temporal synchronization of a common pool of targets between NKX2-5 and MEIS1.

The precise origins of myocardial progenitors and their subsequent contribution to the developing heart has been an area of considerable activity within the field of cardiovascular biology. How these progenitors are regulated and what signals are responsible for their development are, however,much less well understood. Clearly, not only is there a need to identify factors that regulate the transition from proliferation of cardioblasts to differentiation of cardiac muscle, but it is also necessary to identify factors that maintain an adequate pool of undifferentiated myocyte precursors as a prerequisite to preventing organ hypoplasia and congenital heart disease. Here, we report how upregulation of the basic helix-loop-helix (bHLH)transcription factor Hand1, restricted exclusively to Hand1-expressing cells, brings about a significant extension of the heart tube and extraneous looping caused by the elevated proliferation of cardioblasts in the distal outflow tract. This activity is independent of the further recruitment of extracardiac cells from the secondary heart field and permissive for the continued differentiation of adjacent myocardium. Culture studies using embryonic stem (ES) cell-derived cardiomyocytes revealed that,in a Hand1-null background, there is significantly elevated cardiomyocyte differentiation, with an apparent default mesoderm pathway to a cardiomyocyte fate. However, Hand1 gain of function maintains proliferating precursors resulting in delayed and significantly reduced cardiomyocyte differentiation that is mediated by the prevention of cell-cycle exit, by G1 progression and by increased cell division. Thus, this work identifies Hand1 as a crucial cardiac regulatory protein that controls the balance between proliferation and differentiation in the developing heart, and fills a significant gap in our understanding of how the myocardium of the embryonic heart is established.