Nkx2-5 defines distinct scaffold and recruitment phases during formation of the murine cardiac Purkinje fiber network.

The ventricular conduction system coordinates heartbeats by rapid propagation of electrical activity through the Purkinje fiber (PF) network. PFs share common progenitors with contractile cardiomyocytes, yet the mechanisms of segregation and network morphogenesis are poorly understood. Here, we apply genetic fate mapping and temporal clonal analysis to identify murine cardiomyocytes committed to the PF lineage as early as E7.5. We find that a polyclonal PF network emerges by progressive recruitment of conductive precursors to this scaffold from a pool of bipotent progenitors. At late fetal stages, the segregation of conductive cells increases during a phase of rapid recruitment to build the definitive PF network through a non-cell autonomous mechanism. We also show that PF differentiation is impaired in Nkx2-5 haploinsufficient embryos leading to failure to extend the scaffold. In particular, late fetal recruitment fails, resulting in PF hypoplasia and persistence of bipotent progenitors. Our results identify how transcription factor dosage regulates cell fate divergence during distinct phases of PF network morphogenesis.

Specialized impulse conduction pathway in the alligator heart.

Mammals and birds have a specialized cardiac atrioventricular conduction system enabling rapid activation of both ventricles. This system may have evolved together with high heart rates to support their endothermic state (warm-bloodedness) and is seemingly lacking in ectothermic vertebrates from which first mammals then birds independently evolved. Here, we studied the conduction system in crocodiles (Alligator mississippiensis), the only ectothermic vertebrates with a full ventricular septum. We identified homologues of mammalian conduction system markers (Tbx3-Tbx5, Scn5a, Gja5, Nppa-Nppb) and show the presence of a functional atrioventricular bundle. The ventricular Purkinje network, however, was absent and slow ventricular conduction relied on trabecular myocardium, as it does in other ectothermic vertebrates. We propose the evolution of the atrioventricular bundle followed full ventricular septum formation prior to the development of high heart rates and endothermy. In contrast, the evolution of the ventricular Purkinje network is strongly associated with high heart rates and endothermy.

Cellular Physiology and Clinical Manifestations of Fascicular Arrhythmias in Normal Hearts.

Fascicular ventricular arrhythmias represent a spectrum of ventricular tachycardias dependent on the specialized conduction system. Although they are more common in structurally abnormal hearts, there is an increasing body of literature describing their role in normal hearts. In this review, the authors present data from both basic and clinical research that explore the current understanding of idiopathic fascicular ventricular arrhythmias. Evaluation of the cellular electrophysiology of the Purkinje cells shows clear evidence of enhanced automaticity and triggered activity as potential mechanisms of arrhythmias. Perhaps more importantly, heterogeneity in conduction system velocity and refractoriness of the left ventricular conduction system in animal models are in line with clinical descriptions of re-entrant fascicular arrhythmias in humans. Further advances in our understanding of the conduction system will help bridge the current gap between basic science and clinical fascicular arrhythmias.

Specification of the mouse cardiac conduction system in the absence of Endothelin signaling.

Coordinated contraction of the heart is essential for survival and is regulated by the cardiac conduction system. Contraction of ventricular myocytes is controlled by the terminal part of the conduction system known as the Purkinje fiber network. Lineage analyses in chickens and mice have established that the Purkinje fibers of the peripheral ventricular conduction system arise from working myocytes during cardiac development. It has been proposed, based primarily on gain-of-function studies, that Endothelin signaling is responsible for myocyte-to-Purkinje fiber transdifferentiation during avian heart development. However, the role of Endothelin signaling in mammalian conduction system development is less clear, and the development of the cardiac conduction system in mice lacking Endothelin signaling has not been previously addressed. Here, we assessed the specification of the cardiac conduction system in mouse embryos lacking all Endothelin signaling. We found that mouse embryos that were homozygous null for both ednra and ednrb, the genes encoding the two Endothelin receptors in mice, were born at predicted Mendelian frequency and had normal specification of the cardiac conduction system and apparently normal electrocardiograms with normal QRS intervals. In addition, we found that ednra expression within the heart was restricted to the myocardium while ednrb expression in the heart was restricted to the endocardium and coronary endothelium. By establishing that ednra and ednrb are expressed in distinct compartments within the developing mammalian heart and that Endothelin signaling is dispensable for specification and function of the cardiac conduction system, this work has important implications for our understanding of mammalian cardiac development.

Role of the His-Purkinje system in the genesis of cardiac arrhythmia.

Although a plethora of earlier studies focused on the histology and action potential characteristics of Purkinje fibers, only recently has the His-Purkinje system been found to play a major role in the genesis of cardiac arrhythmias. The anatomic complexity of the left ventricular conduction system appears to favor reentrant arrhythmias in both diseased and healthy hearts. Macroreentrant circuits between the right and left bundles as well as between the left ventricular fascicles are amenable to cure by ablative techniques. Similarly, fascicular tachycardias occurring in individuals without structural cardiac disease appear to involve macroreentrant circuits between fascicles and associated strands (false tendons?). Exciting newer discoveries strongly implicate the Purkinje system as the cause of ventricular arrhythmias in patients with short-coupled premature ventricular complexes and in those with catecholaminergic polymorphous ventricular tachycardia. The role of the His-Purkinje system in the genesis and maintenance of ventricular fibrillation is yet another frontier for fertile investigation. A rich variety of cardiac arrhythmias appears to involve the ventricular specialized conduction system and may be amenable to ablative therapy.

Epicardium-derived cells are important for correct development of the Purkinje fibers in the avian heart.

During embryonic development, the proepicardial organ (PEO) grows out over the heart surface to form the epicardium. Following epithelial-mesenchymal transformation, epicardium-derived cells (EPDCs) migrate into the heart and contribute to the developing coronary arteries, to the valves, and to the myocardium. The peripheral Purkinje fiber network develops from differentiating cardiomyocytes in the ventricular myocardium. Intrigued by the close spatial relationship between the final destinations of migrating EPDCs and Purkinje fiber differentiation in the avian heart, that is, surrounding the coronary arteries and at subendocardial sites, we investigated whether inhibition of epicardial outgrowth would disturb cardiomyocyte differentiation into Purkinje fibers. To this end, epicardial development was inhibited mechanically with a membrane, or genetically, by suppressing epicardial epithelial-to-mesenchymal transformation with antisense retroviral vectors affecting Ets transcription factor levels (n=4, HH39-41). In both epicardial inhibition models, we evaluated Purkinje fiber development by EAP-300 immunohistochemistry and found that restraints on EPDC development resulted in morphologically aberrant differentiation of Purkinje fibers. Purkinje fiber hypoplasia was observed both periarterially and at subendocardial positions. Furthermore, the cells were morphologically abnormal and not aligned in orderly Purkinje fibers. We conclude that EPDCs are instrumental in Purkinje fiber differentiation, and we hypothesize that they cooperate directly with endothelial and endocardial cells in the development of the peripheral conduction system.

Induction and patterning of the Purkinje fibre network.

Impulse-conducting Purkinje cells differentiate from myocytes during embryogenesis. In the embryonic chicken heart, this conversion of contractile myocytes into conduction cells occurs subendocardially and periarterially. The unique sites of Purkinje fibre differentiation suggest that a shear stress-induced paracrine signal from the endocardium and arterial beds may induce adjacent myocytes to differentiate into conduction cells. Consistent with this model, Purkinje fibre marker genes can be induced in cultured embryonic myocytes by endothelin (ET), an endothelial cell-derived signalling peptide. This inductive response is, however, gradually lost from myocytes as embryos develop, and mature myocytes express only genes characteristic of hypertrophy in response to ET. In vivo, active ET is produced, through proteolytic processing, from its precursor by ET-converting enzyme 1 (ECE1) and triggers signalling by binding to its receptors, ETA and ETB. In the embryonic heart, the expression of these ET signalling components changes dynamically, defining the site and timing of Purkinje fibre differentiation within the ventricular myocardium during chick embryogenesis.

Hemodynamics is a key epigenetic factor in development of the cardiac conduction system.

The His-Purkinje system (HPS) is a network of conduction cells responsible for coordinating the contraction of the ventricles. Earlier studies using bipolar electrodes indicated that the functional maturation of the HPS in the chick embryo is marked by a topological shift in the sequence of activation of the ventricle. Namely, at around the completion of septation, an immature base-to-apex sequence of ventricular activation was reported to convert to the apex-to-base pattern characteristic of the mature heart. Previously, we have proposed that hemodynamics and/or mechanical conditioning may be key epigenetic factors in development of the HPS. We thus hypothesized that the timing of the topological shift marking maturation of the conduction system is sensitive to variation in hemodynamic load. Spatiotemporal patterns of ventricular activation (as revealed by high-speed imaging of fluorescent voltage-sensitive dye) were mapped in chick hearts over normal development, and following procedures previously characterized as causing increased (conotruncal banding, CTB) or reduced (left atrial ligation, LAL) hemodynamic loading of the embryonic heart. The results revealed that the timing of the shift to mature activation displays striking plasticity. CTB led to precocious emergence of mature HPS function relative to controls whereas LAL was associated with delayed conversion to apical initiation. The results from our study indicate a critical role for biophysical factors in differentiation of specialized cardiac tissues and provide the basis of a new model for studies of the molecular mechanisms involved in induction and patterning of the HPS in vivo.

The role of the epicardium and neural crest as extracardiac contributors to coronary vascular development.

At species-specific times in embryonic development, the pro-epicardial organ appears as an outcropping of the mesothelial body wall, near the sinus venosus-liver region. The pro-epicardial vesicles attach to the myocardium, flatten, and join to form the epicardium. The epicardium shows epithelial-mesenchymal transformation: cells detach from the epithelium, fill the subepicardial space, and invade the heart tube. Epicardium-derived cells migrate as far as the core of the endocardial cushions, which differentiate into the atrioventricular valve leaflets. In the cardiac wall, other epicardium-derived cells differentiate into interstitial fibroblasts and adventitial and smooth muscle cells of the coronary arteries. Using neural crest tracings in mouse embryos (Wnt1-Cre-lacZ), we studied the patterning of cardiac neural crest cells during development. Participation of neural crest cells in the formation of the vascular media could not be excluded, although epicardium-derived cells have hitherto been considered responsible for formation of the coronary arterial smooth muscle cells. The endothelial cells of the coronary network derive mostly from the endothelium of the sinus venosus-liver region by vasculogenesis and angiogenesis. However, an epicardium-derived cell origin of some endothelial cells cannot be ruled out. The coronary vasculature is closely related to the differentiating Purkinje network, but isolated epicardium-derived cells are also associated with Purkinje cells. After ablating the pro-epicardial organ in quail embryos, we found severe malformations in the myocardial architecture, leading to the hypothesis that epicardium-derived cells give instructive signals to the myocardium for proper differentiation of the compact and the trabeculated compartments.

Coronary arteriogenesis and differentiation of periarterial Purkinje fibers in the chick heart: is there a link?

In the following review, we outline the cellular ontogeny and time course of coronary artery development within the vertebrate heart. Our eventual focus will be the potential role of arteriogenesis in the differentiation of a subset of specialized conduction cells in the chick heart. We begin by briefly outlining early heart formation, showing how the outermost layer of the looped, tube heart--the epicardium--is of extracardiac origin and provides the progenitor cells to the entire vascular bed. Subsequently, we summarize the events of coronary arterial development that follow epicardialization. Finally, we discuss work in the chick that indicates how arteries form pioneering, directional conduits through ventricular tissue, adjacent to which myocardial cells differentiate to form the most peripheral component of the avian conduction system--a network of periarterial Purkinje fibers.

Competency of embryonic cardiomyocytes to undergo Purkinje fiber differentiation is regulated by endothelin receptor expression.

Purkinje fibers of the cardiac conduction system differentiate from heart muscle cells during embryogenesis. In the avian heart, Purkinje fiber differentiation takes place along the endocardium and coronary arteries. To date, only the vascular cytokine endothelin (ET) has been demonstrated to induce embryonic cardiomyocytes to differentiate into Purkinje fibers. This ET-induced Purkinje fiber differentiation is mediated by binding of ET to its transmembrane receptors that are expressed by myocytes. Expression of ET converting enzyme 1, which produces a biologically active ET ligand, begins in cardiac endothelia, both arterial and endocardial, at initiation of conduction cell differentiation and continues throughout heart development. Yet, the ability of cardiomyocytes to convert their phenotype in response to ET declines as embryos mature. Therefore, the loss of responsiveness to the inductive signal appears not to be associated with the level of ET ligand in the heart. This study examines the role of ET receptors in this age-dependent loss of inductive responsiveness and the expression profiles of three different types of ET receptors, ET(A), ET(B) and ET(B2), in the embryonic chick heart. Whole-mount in situ hybridization analyses revealed that ET(A) was ubiquitously expressed in both ventricular and atrial myocardium during heart development, while ET(B) was predominantly expressed in the atrium and the left ventricle. ET(B2) expression was detected in valve leaflets but not in the myocardium. RNase protection assays showed that ventricular expression of ET(A) and ET(B) increased until Purkinje fiber differentiation began. Importantly, the levels of both receptor isotypes decreased after this time. Retrovirus-mediated overexpression of ET(A) in ventricular myocytes in which endogenous ET receptors had been downregulated, enhanced their responsiveness to ET, allowing them to differentiate into conduction cells. These results suggest that the developmentally regulated expression of ET receptors plays a crucial role in determining the competency of ventricular myocytes to respond to inductive ET signaling in the chick embryo.

Spatiotemporal and tissue specific distribution of apoptosis in the developing chick heart.

To investigate spatial and temporal distributions of apoptosis in the embryonic chick heart and its relation to different tissue types, we examined apoptosis in the embryonic chick heart from Hamburger and Hamilton stage 17 through 3 days after hatching. MF20 antibody, alpha-smooth muscle actin (SMA) antibody and EAP-300 antibody were applied to delineate specific cell types. During early development of the embryonic chick heart, very few apoptotic cells were detected. The first distinctive zone of apoptosis was observed in the outflow tract at stage 25. This focus was most prominent during septation of the pulmonary artery from the aorta (i.e., between stages 28 and 29), and diminished to virtually background level by stage 32, except in the subconal regions. Subsequently, remarkable apoptosis appeared in the atrioventricular cushions by stage 26, peaked at stages 29-31, and dropped significantly thereafter. Characteristic distribution patterns of apoptotic cells were also detected in the cardiac conduction tissues, including the His bundle, the bundle branches, and the ventricular trabeculae. After stage 36, cell death dropped to background level, except in developing coronary vessels. MF20 and TUNEL double staining revealed that apoptosis in cardiomyocytes was limited to a few specific regions, much less than in cushion tissues. SMA and TUNEL double staining demonstrated that vascular structures were the major foci of apoptosis from stage 40 to 44, whereas adjacent perivascular Purkinje cells displayed significantly less cell death at these stages. The characteristic spatiotemporal locations of apoptosis parallel the morphologic changes and tissue differentiation during heart development, suggesting that apoptosis is crucial to the transformation of the heart from a simple tube to a complex multichambered pump.

Elevated expression of Nkx-2.5 in developing myocardial conduction cells.

A number of different phenotypes emerge from the mesoderm-derived cardiomyogenic cells of the embryonic tubular heart, including those comprising the cardiac conduction system. The transcriptional regulation of this phenotypic divergence within the cardiomyogenic lineage remains poorly characterized. A relationship between expression of the transcription factor Nkx-2.5 and patterning to form cardiogenic mesoderm subsequent to gastrulation is well established. Nkx-2.5 mRNA continues to be expressed in myocardium beyond the looped, tubular heart stage. To investigate the role of Nkx-2.5 in later development, we have determined the expression pattern of Nkx-2.5 mRNA by in situ hybridization in embryonic chick, fetal mouse, and human hearts, and of Nkx-2.5 protein by immunolocalization in the embryonic chick heart. As development progresses, significant nonuniformities emerge in Nkx-2.5 expression levels. Relative to surrounding force-generating ("working") myocardium, elevated Nkx-2.5 mRNA signal becomes apparent in the specialized cells of the conduction system. Similar differences are found in developing chick, human, and mouse fetal hearts, and nuclear-localized Nkx-2.5 protein is prominently expressed in differentiating chick conduction cells relative to adjacent working myocytes. This tissue-restricted expression of Nkx-2.5 is transient and correlates with the timing of spatio-temporal recruitment of cells to the central and the peripheral conduction system. Our data represent the first report of a transcription factor showing a stage-dependent restriction to different parts of the developing conduction system, and suggest some commonality in this development between birds and mammals. This dynamic pattern of expression is consistent with the hypothesis that Nkx-2.5, and its level of expression, have a role in regulation and/or maintenance of specialized fate selection by embryonic myocardial cells.

Growth arrest specific gene 1 is a positive growth regulator for the cerebellum.

Postnatal cerebellum development involves the generation of granule cells and Bergmann glias (BGs). The granule cell precursors are located in the external germinal layer (EGL) and the BG precursors are located in the Purkinje layer (PL). BGs extend their glial fibers into the EGL and facilitate granule cells' inward migration to their final location. Growth arrest specific gene 1 (Gas1) has been implicated in inhibiting cell-cycle progression in cell culture studies (G. Del Sal et al., 1992, Cell 70, 595--607). However, its growth regulatory function in the CNS has not been described. To investigate its role in cerebellar growth, we analyzed the Gas1 mutant mice. At birth, wild-type and mutant mice have cerebella of similar size; however, mature mutant cerebella are less than half the size of wild-type cerebella. Molecular and cellular examinations indicate that Gas1 mutant cerebella have a reduced number of granule cells and BG fibers. We provide direct evidence that Gas1 is required for normal levels of proliferation in the EGL and the PL, but not for their differentiation. Furthermore, we show that Gas1 is specifically and coordinately expressed in both the EGL and the BGs postnatally. These results support Gas1 as a common genetic component in coordinating EGL cell and BG cell proliferation, a link which has not been previously appreciated.

Purkinje fibers of the avian heart express a myogenic transcription factor program distinct from cardiac and skeletal muscle.

A rhythmic heart beat is coordinated by conduction of pacemaking impulses through the cardiac conduction system. Cells of the conduction system, including Purkinje fibers, terminally differentiate from a subset of cardiac muscle cells that respond to signals from endocardial and coronary arterial cells. A vessel-associated paracrine factor, endothelin, can induce embryonic heart muscle cells to differentiate into Purkinje fibers both in vivo and in vitro. During this phenotypic conversion, the conduction cells down-regulate genes characteristic of cardiac muscle and up-regulate subsets of genes typical of both skeletal muscle and neuronal cells. In the present study, we examined the expression of myogenic transcription factors associated with the switch of the gene expression program during terminal differentiation of heart muscle cells into Purkinje fibers. In situ hybridization analyses and immunohistochemistry of embryonic and adult hearts revealed that Purkinje fibers up-regulate skeletal and atrial muscle myosin heavy chains, connexin-42, and neurofilament protein. Concurrently, a cardiac muscle-specific myofibrillar protein, myosin-binding protein-C (cMyBP-C), is down-regulated. During this change in transcription, however, Purkinje fibers continue to express cardiac muscle transcription factors, such as Nkx2.5, GATA4, and MEF2C. Importantly, significantly higher levels of Nkx2.5 and GATA4 mRNAs were detected in Purkinje fibers as compared to ordinary heart muscle cells. No detectable difference was observed in MEF2C expression. In culture, endothelin-induced Purkinje fibers from embryonic cardiac muscle cells dramatically down-regulated cMyBP-C transcription, whereas expression of Nkx2.5 and GATA4 persisted. In addition, myoD, a skeletal muscle transcription factor, was up-regulated in endothelin-induced Purkinje cells, while Myf5 and MRF4 transcripts were undetectable in these cells. These results show that during and after conversion from heart muscle cells, Purkinje fibers express a unique myogenic transcription factor program. The mechanism underlying down-regulation of cardiac muscle genes and up-regulation of skeletal muscle genes during conduction cell differentiation may be independent from the transcriptional control seen in ordinary cardiac and skeletal muscle cells.

In vivo induction of cardiac Purkinje fiber differentiation by coexpression of preproendothelin-1 and endothelin converting enzyme-1.

The rhythmic heart beat is coordinated by electrical impulses transmitted from Purkinje fibers of the cardiac conduction system. During embryogenesis, the impulse-conducting cells differentiate from cardiac myocytes in direct association with the developing endocardium and coronary arteries, but not with the venous system. This conversion of myocytes into Purkinje fibers requires a paracrine interaction with blood vessels in vivo, and can be induced in vitro by exposing embryonic myocytes to endothelin-1 (ET-1), an endothelial cell-associated paracrine factor. These results suggest that an endothelial cell-derived signal is capable of inducing juxtaposed myocytes to differentiate into Purkinje fibers. It remains unexplained how Purkinje fiber recruitment is restricted to subendocardial and periarterial sites but not those juxtaposed to veins. Here we show that while the ET-receptor is expressed throughout the embryonic myocardium, introduction of the ET-1 precursor (preproET-1) in the embryonic myocardium is not sufficient to induce myocytes to differentiate into conducting cells. ET converting enzyme-1 (ECE-1), however, is expressed preferentially in endothelial cells of the endocardium and coronary arteries where Purkinje fiber recruitment takes place. Retroviral-mediated coexpression of both preproET-1 and ECE-1 in the embryonic myocardium induces myocytes to express Purkinje fiber markers ectopically and precociously. These results suggest that expression of ECE-1 plays a key role in defining an active site of ET signaling in the heart, thereby determining the timing and location of Purkinje fiber differentiation within the embryonic myocardium.

Induction of Purkinje fiber differentiation by coronary arterialization.

A synchronized heart beat is controlled by pacemaking impulses conducted through Purkinje fibers. In chicks, these impulse-conducting cells are recruited during embryogenesis from myocytes in direct association with developing coronary arteries. In culture, the vascular cytokine endothelin converts embryonic myocytes to Purkinje cells, implying that selection of conduction phenotype may be mediated by an instructive cue from arteries. To investigate this hypothesis, coronary arterial development in the chicken embryo was either inhibited by neural crest ablation or activated by ectopic expression of fibroblast growth factor (FGF). Ablation of cardiac neural crest resulted in approximately 70% reductions (P < 0.01) in the density of intramural coronary arteries and associated Purkinje fibers. Activation of coronary arterial branching was induced by retrovirus-mediated overexpression of FGF. At sites of FGF-induced hypervascularization, ectopic Purkinje fibers differentiated adjacent to newly induced coronary arteries. Our data indicate the necessity and sufficiency of developing arterial bed for converting a juxtaposed myocyte into a Purkinje fiber cell and provide evidence for an inductive function for arteriogenesis in heart development distinct from its role in establishing coronary blood circulation.

Development of the cardiac conduction system involves recruitment within a multipotent cardiomyogenic lineage.

The cardiac pacemaking and conduction system sets and maintains the rhythmic pumping action of the heart. Previously, we have shown that peripheral cells of the conduction network in chick (periarterial Purkinje fibers) are selected within a cardiomyogenic lineage and that this recruitment occurs as a result of paracrine cues from coronary arteries. At present, the cellular derivation of other elements of this specialized system (e.g. the nodes and bundles of the central conduction system) are controversial, with some proposing that the evidence supports a neurogenic and others a myogenic origin for these tissues. While such ontological questions remain, it is unlikely that progress can be made on the molecular mechanisms governing patterning and induction of the central conduction system. Here, we have undertaken lineage-tracing strategies based on the distinct properties of replication-incompetent adenoviral and retroviral lacZ-expressing constructs. Using these complementary approaches, it is shown that cells constituting both peripheral and central conduction tissues originate from cardiomyogenic progenitors present in the looped, tubular heart with no detectable contribution by migratory neuroectoderm-derived populations. Moreover, clonal analyses of retrovirally infected cells incorporated within any part of the conduction system suggest that such cells share closer lineage relationships with nearby contractive myocytes than with other, more distal elements of the conduction system. Differentiation birthdating by label dilution using [(3)H]thymidine also demonstrates the occurrence of ongoing myocyte conscription to conductive specialization and provides a time course for this active and localized selection process in different parts of the system. Together, these data suggest that the cardiac conduction system does not develop by outgrowth from a prespecified pool of 'primary' myogenic progenitors. Rather, its assembly and elaboration occur via processes that include progressive and localized recruitment of multipotent cardiomyogenic cells to the developing network of specialized cardiac tissues.

Changing activation sequence in the embryonic chick heart. Implications for the development of the His-Purkinje system.

In the mature heart, impulse propagation through the His-Purkinje system (HPS) is required for efficient ventricular contraction in an apex-to-base direction. However, the embryonic heart begins to contract as a myocardial tube without a specialized conduction system. To identify the developmental stage when the HPS begins to function, we mapped the ventricular depolarization sequence from microvolt-level electrograms recorded from embryonic myocardium using 50-micron extracellular electrodes, high-gain amplification, and signal-processing techniques. Analysis of left ventricular activation in 99 embryonic hearts revealed a transition in the activation sequence that was dependent on developmental stage. As the heart develops, a transition in the activation sequence occurred from the primitive base-to-apex pattern (in 20 of 33 hearts) at early stages (Hamburger-Hamilton stages 25 to 28) to the HPS-like apex-to-base pattern (12 of 17 hearts) late in development (stages 33 to 36). Immunohistological experiments (n = 10) also confirm that the expression pattern of two biochemical HPS markers changes in parallel with the change to the mature ventricular activation pattern. These data indicate that the ventricular activation sequence in the chick heart develops to a mature pattern at stages 29 to 31, suggesting that preferential conduction through the HPS begins shortly after ventricular septation is complete.