Pharyngeal Mesoderm Research Articles

To Activate or Not to Activate

In a recent study, Watanabe et al identify a novel enhancer in the fgf10 locus that is necessary and sufficient for expression in anterior second heart field. Their results suggest a competitive mechanism in which Isl1 and Nkx2-5 bind the enhancer to activate or repress this enhancer, respectively. In the last decade, new insights into heart development have demonstrated the existence of a second heart field, which gives rise to the second heart lineage.1,2 The first heart lineage is defined as the first cells to differentiate within the early cardiac crescent and forming heart tube, gives rise to a majority of cells within the left ventricle, and contributes to both atria. The second heart field progenitors actively proliferate and are progressively added to the heart to give rise to the right ventricle, outflow tracts, and to a majority of cells within the atria. As second heart field progenitors enter the heart, proliferation is downregulated concomitant with differentiation. Gene networks which regulate the transition from second heart field progenitor to differentiated myocyte have been the subject of intense scrutiny, particularly for anterior second heart field progenitors, which will contribute to the anterior pole of the heart.3–5 Pioneering work with an nLacZ transgene fortuitously integrated into the Fgf10 locus revealed the mammalian anterior second heart field.6 Expression of endogenous Fgf10 occurs first in the second heart field medial to the differentiating cells of the first lineage in the cardiac crescent. Its expression was later observed in the anterior second heart field comprising pharyngeal mesoderm dorsal and anterior to the heart, including core mesoderm of the first 2 pharyngeal arches.7,8 Fgf10 cooperates with Fgf8 to drive proliferation of the second heart field.3 To shed new light on gene networks regulating the transition …

Fate mapping identifies the origin of SHF/AHF progenitors in the chick primitive streak.

Heart development depends on the spatio-temporally regulated contribution of progenitor cells from the primary, secondary and anterior heart fields. Primary heart field (PHF) cells are first recruited to form a linear heart tube; later, they contribute to the inflow myocardium of the four-chambered heart. Subsequently cells from the secondary (SHF) and anterior heart fields (AHF) are added to the heart tube and contribute to both the inflow and outflow myocardium. In amniotes, progenitors of the linear heart tube have been mapped to the anterior-middle region of the early primitive streak. After ingression, these cells are located within bilateral heart fields in the lateral plate mesoderm. On the other hand SHF/AHF field progenitors are situated anterior to the linear heart tube, however, the origin and location of these progenitors prior to the development of the heart tube remains elusive. Thus, an unresolved question in the process of cardiac development is where SHF/AHF progenitors originate from during gastrulation and whether they come from a region in the primitive streak distinct from that which generates the PHF. To determine the origin and location of SHF/AHF progenitors we used vital dye injection and tissue grafting experiments to map the location and ingression site of outflow myocardium progenitors in early primitive streak stage chicken embryos. Cells giving rise to the AHF ingressed from a rostral region of the primitive streak, termed region ‘A’. During development these cells were located in the cranial paraxial mesoderm and in the pharyngeal mesoderm. Furthermore we identified region ‘B’, located posterior to ‘A’, which gave rise to progenitors that contributed to the primary heart tube and the outflow tract. Our studies identify two regions in the early primitive streak, one which generates cells of the AHF and a second from which cardiac progenitors of the PHF and SHF emerge.

Multiple essential roles for primary cilia in heart development

BackgroundThe primary cilium is a microtubule-based, plasma membrane-ensheathed protrusion projecting from the basal bodies of almost all cell types in the mammalian body. In the past several years a plethora of papers has indicated a crucial role for primary cilia in the development of a wide variety of organs. We have investigated heart development in cobblestone, a hypomorphic allele of the gene encoding the intraflagellar transport protein Ift88, and uncovered a number of the most common congenital heart defects seen in newborn humans.MethodsWe generated serial sections of mutant cobblestone and wild type embryos in the region encompassing the heart and the cardiac outflow tract. The sections were further processed to generate three-dimensional reconstructions of these structures, and immunofluorescence confocal microscopy, transmission electron microscopy, and in situ hybridization were used to examine signal transduction pathways in the relevant areas. Whole mount in situ hybridization was also employed for certain developmental markers.ResultsIn addition to an enlarged pericardium and failure of both ventricular and atrial septum formation, the cobblestone mutants displayed manifold defects in outflow tract formation, including persistent truncus arteriosus, an overriding aorta, and abnormal transformation of the aortic arches. To discern the basis of these anomalies we examined both the maintenance of primary cilia as well as endogenous and migratory embryonic cell populations that contribute to the outflow tract and atrioventricular septa. The colonization of the embryonic heart by cardiac neural crest occurred normally in the cobblestone mutant, as did the expression of Sonic hedgehog. However, with the loss of primary cilia in the mutant hearts, there was a loss of both downstream Sonic hedgehog signaling and of Islet 1 expression in the second heart field, a derivative of the pharyngeal mesoderm. In addition, defects were recorded in development of atrial laterality and ventricular myocardiogenesis. Finally, we observed a reduction in expression of Bmp4 in the outflow tract, and complete loss of expression of both Bmp2 and Bmp4 in the atrioventricular endocardial cushions. Loss of BMP2/4 signaling may result in the observed proliferative defect in the endocardial cushions, which give rise to both the atrioventricular septa as well as to the septation of the outflow tract.ConclusionsTaken together, our results potentially identify a novel link between Sonic hedgehog signaling at the primary cilium and BMP-dependent effects upon cardiogenesis. Our data further point to a potential linkage of atrioventricular septal defects, the most common congenital heart defects, to genes of the transport machinery or basal body of the cilia.

Pharyngeal mesoderm regulatory network controls cardiac and head muscle morphogenesis

The search for developmental mechanisms driving vertebrate organogenesis has paved the way toward a deeper understanding of birth defects. During embryogenesis, parts of the heart and craniofacial muscles arise from pharyngeal mesoderm (PM) progenitors. Here, we reveal a hierarchical regulatory network of a set of transcription factors expressed in the PM that initiates heart and craniofacial organogenesis. Genetic perturbation of this network in mice resulted in heart and craniofacial muscle defects, revealing robust cross-regulation between its members. We identified Lhx2 as a previously undescribed player during cardiac and pharyngeal muscle development. Lhx2 and Tcf21 genetically interact with Tbx1, the major determinant in the etiology of DiGeorge/velo-cardio-facial/22q11.2 deletion syndrome. Furthermore, knockout of these genes in the mouse recapitulates specific cardiac features of this syndrome. We suggest that PM-derived cardiogenesis and myogenesis are network properties rather than properties specific to individual PM members. These findings shed new light on the developmental underpinnings of congenital defects.

Organogenesis of the vertebrate heart

Organogenesis of the vertebrate heart involves a complex sequence of events initiating with specification and differentiation of myocardial and endocardial cells in anterior lateral mesoderm shortly after gastrulation, followed by formation and rightward looping of the early heart tube. During looping, the heart tube elongates by addition of second heart field progenitor cells from adjacent pharyngeal mesoderm at the arterial and venous poles. Progressive differentiation is controlled by intercellular signaling events between pharyngeal mesoderm, foregut endoderm, and neural crest-derived mesenchyme. Regulated patterns of myocardial gene expression and proliferation within the embryonic heart drive morphogenesis of atrial and ventricular chambers, while cardiac cushions, precursors of the definitive valves, form in the atrioventricular and outflow regions. In amniotes, separate systemic and pulmonary circulatory systems arise by septation and remodeling events that divide the atria and ventricles into left and right chambers. Cardiac neural crest cells play a key role in dividing the arterial pole of the heart into the ascending aorta and pulmonary trunk. During the remodeling phase the definitive cardiac conduction system, that coordinates the heartbeat, is established. In addition, the epicardium, critical for regulated ventricular growth and development of the coronary vasculature, spreads over the surface of the heart as an epithelium from which cells invade the myocardium to give rise to diverse cell types including fibroblasts and smooth muscle cells. Cardiogenesis thus involves highly coordinated development of multiple cell types and insight into the different lineage contributions and molecular regulation of each of these steps is expanding rapidly. WIREs Dev Biol 2013, 2:17-29. doi: 10.1002/wdev.68 For further resources related to this article, please visit the WIREs website.

Tetralogy of Fallot Associated With Invasive Adrenocortical Tumor in an Adult Woman

Migration of cardiac neural crest cells into the pharyngeal arches and the pharyngeal and splanchnic mesoderm contributes to the development of the cardiac outflow tract. The adrenal cortex is derived from the splanchnic mesoderm. Neuroblastoma is more prevalent in patients with congenital heart disease than in the general population, because both originate from embryonal neural crest-derived cells. Similarly, and in light of recent embryological findings, abnormal development or migration of splanchnic mesoderm, possibly due to an underlying genetic defect, could contribute to the association of adrenocortical carcinoma and tetralogy of Fallot. We present the case of a cardiologically asymptomatic 49-year-old woman with total correction of tetralogy of Fallot in the first year of life.

Embryonic and foetal Islet-1 positive cells in human hearts are also positive to c-Kit

During embryogenesis, the mammalian heart develops from a primitive heart tube originating from two bilateral primary heart fields located in the lateral plate mesoderm. Cells belongings to the pre-cardiac mesoderm will differentiate into early cardiac progenitors, which express early transcription factors which are also common to the Isl-1 positive cardiac progenitor cells isolated from the developing pharyngeal mesoderm and the foetal and post-natal mice hearts. A second population of cardiac progenitor cells positive to c-Kit has been abundantly isolated from adult hearts. Until now, these two populations have been considered two different sets of progenitor cells present in the heart in different stages of an individual life. In the present study we collected embryonic, foetal and infant hearts, and we tested the hypotheses that c-Kit positive cells, usually isolated from the adult heart, are also present in the intra-uterine life and persist in the adult heart after birth, and that foetal Isl-1 positive cells are also positive to c-Kit. Using immunohistochemistry we studied the temporal distribution of Isl-1 positive and c-Kit/CD105 double positive cells, and by immunofluorescence and confocal analysis we studied the co-localization of c-Kit and Isl-1 positive cells. The results indicated that cardiomyocytes and interstitial cells were positive for c-Kit from the 9th to the 19h gestational week, that cells positive for both c-Kit and CD105 appeared in the interstitium at the 17h gestational week and persisted in the postnatal age, and that the Isl-1 positive cells were a subset of the c-Kit positive population.

Identification of a Tbx1/Tbx2/Tbx3 genetic pathway governing pharyngeal and arterial pole morphogenesis

The 22q11.2 deletion syndrome (22q11.2DS) is the most common microdeletion disorder and is characterized by abnormal development of the pharyngeal apparatus and heart. Cardiovascular malformations (CVMs) affecting the outflow tract (OFT) are frequently observed in 22q11.2DS and are among the most commonly occurring heart defects. The gene encoding T-box transcription factor 1 (Tbx1) has been identified as a major candidate for 22q11.2DS. However, CVMs are generally considered to have a multigenic basis and single-gene mutations underlying these malformations are rare. The T-box family members Tbx2 and Tbx3 are individually required in regulating aspects of OFT and pharyngeal development. Here, using expression and three-dimensional reconstruction analysis, we show that Tbx1 and Tbx2/Tbx3 are largely uniquely expressed but overlap in the caudal pharyngeal mesoderm during OFT development, suggesting potential combinatorial requirements. Cross-regulation between Tbx1 and Tbx2/Tbx3 was analyzed using mouse genetics and revealed that Tbx1 deficiency affects Tbx2 and Tbx3 expression in neural crest-derived cells and pharyngeal mesoderm, whereas Tbx2 and Tbx3 function redundantly upstream of Tbx1 and Hh ligand expression in pharyngeal endoderm and bone morphogenetic protein- and fibroblast growth factor-signaling in cardiac progenitors. Moreover, in vivo, we show that loss of two of the three genes results in severe pharyngeal hypoplasia and heart tube extension defects. These findings reveal an indispensable T-box gene network governing pharyngeal and OFT development and identify TBX2 and TBX3 as potential modifier genes of the cardiopharyngeal phenotypes found in TBX1-haploinsufficient 22q11.2DS patients.

Mesodermal retinoic acid signaling regulates endothelial cell coalescence in caudal pharyngeal arch artery vasculogenesis

Disruption of retinoic acid signaling causes a variety of pharyngeal arch artery and great vessel defects, as well as malformations in many other tissues, including those derived from the pharyngeal endoderm. Previous studies implied that arch artery defects in the context of defective RA signaling occur secondary to pharyngeal pouch segmentation defects, although this model has never been experimentally verified. In this study, we examined arch artery morphogenesis during mouse development, and the role of RA in this process. We show in normal embryos that the arch arteries form by vasculogenic differentiation of pharyngeal mesoderm. Using various genetic backgrounds and tissue-specific mutation approaches, we segregate pharyngeal arch artery and pharyngeal pouch defects in RA receptor mutants, and show that RA signal transduction only in pharyngeal mesoderm is required for arch artery formation. RA does not control pharyngeal mesodermal differentiation to endothelium, but instead promotes the aggregation of endothelial cells into nascent vessels. Expression of VE-cadherin was substantially reduced in RAR mutants, and this deficiency may underlie the arch artery defects. The consequences of disrupted mesodermal and endodermal RA signaling were restricted to the 4th and 6th arch arteries and to the 4th pharyngeal pouch, respectively, suggesting that different regulatory mechanisms control the formation of the more anterior arch arteries and pouches.

Revitalizing Our View of Heart Development

paper by Kelly et al., from Margaret Buckingham's group at the Pasteur Institute in Paris, changed the way we think about the developing mammalian heart, as well as the origins of congenital heart disease. Prior to this work, classical studies showing that the heart tube grows in part by the transformation of a noncardiac epithelium into cardiomyocytes had lapsed into obscurity. Kelly et al. and other papers have reinstated the notion that heart growth and morphogenesis are highly dynamic and driven by the recruitment of multipotent heart progenitor cells to a primitive scaffold. It was as if a light went on in the heart development field. This and subsequent work, much of it from Buckingham and colleagues, allows us to understand the cellular and molecular nature of heart growth and to see the origins of congenital heart disease in the aberrant behavior of progenitor cell populations. This PaperPick refers to The Arterial Pole of the Mouse Heart Forms from Fgf10-Expressing Cells in Pharyngeal Mesoderm, by R.G. Kelly, N.A. Brown, and M.E. Buckingham, published in September 2001. Video Abstract Robert Kelly discusses the historical context for the work, outlines how research into the second heart field has progressed in the interim, and highlights the evolutionary and biomedical implications of the resulting changes in our understanding of cardiac development.

Redundant and dosage sensitive requirements for Fgf3 and Fgf10 in cardiovascular development

Heart development requires contributions from, and coordinated signaling interactions between, several cell populations, including splanchnic and pharyngeal mesoderm, postotic neural crest and the proepicardium. Here we report that Fgf3 and Fgf10, which are expressed dynamically in and near these cardiovascular progenitors, have redundant and dosage sensitive requirements in multiple aspects of early murine cardiovascular development. Embryos with Fgf3 −/+ ;Fgf10 −/− , Fgf3 −/− ;Fgf10 −/+ and Fgf3 −/− ;Fgf10 −/− genotypes formed an allelic series of increasing severity with respect to embryonic survival, with double mutants dead by E11.5. Morphologic analysis of embryos with three mutant alleles at E11.5–E13.5 and double mutants at E9.5–E11.0 revealed multiple cardiovascular defects affecting the outflow tract, ventricular septum, atrioventricular cushions, ventricular myocardium, dorsal mesenchymal protrusion, pulmonary arteries, epicardium and fourth pharyngeal arch artery. Assessment of molecular markers in E8.0–E10.5 double mutants revealed abnormalities in each progenitor population, and suggests that Fgf3 and Fgf10 are not required for specification of cardiovascular progenitors, but rather for their normal developmental coordination. These results imply that coding or regulatory mutations in FGF3 or FGF10 could contribute to human congenital heart defects.

Latent TGF-β binding protein 3 identifies a second heart field in zebrafish

The four-chambered mammalian heart develops from two fields of cardiac progenitor cells (CPCs) distinguished by their spatiotemporal patterns of differentiation and contributions to the definitive heart [1–3]. The first heart field differentiates earlier in lateral plate mesoderm, generates the linear heart tube and ultimately gives rise to the left ventricle. The second heart field (SHF) differentiates later in pharyngeal mesoderm, elongates the heart tube, and gives rise to the outflow tract (OFT) and much of the right ventricle. Because hearts in lower vertebrates contain a rudimentary OFT but not a right ventricle [4], the existence and function of SHF-like cells in these species has remained a topic of speculation [4–10]. Here we provide direct evidence from Cre/Lox-mediated lineage tracing and loss of function studies in zebrafish, a lower vertebrate with a single ventricle, that latent-TGFβ binding protein 3 (ltbp3) transcripts mark a field of CPCs with defining characteristics of the anterior SHF in mammals. Specifically, ltbp3+ cells differentiate in pharyngeal mesoderm after formation of the heart tube, elongate the heart tube at the outflow pole, and give rise to three cardiovascular lineages in the OFT and myocardium in the distal ventricle. In addition to expressing Ltbp3, a protein that regulates the bioavailability of TGFβ ligands [11], zebrafish SHF cells co-express nkx2.5, an evolutionarily conserved marker of CPCs in both fields [4]. Embryos devoid of ltbp3 lack the same cardiac structures derived from ltbp3+ cells due to compromised progenitor proliferation. Additionally, small-molecule inhibition of TGFβ signaling phenocopies the ltbp3-morphant phenotype whereas expression of a constitutively active TGFβ type I receptor rescues it. Taken together, our findings uncover a requirement for ltbp3-TGFβ signaling during zebrafish SHF development, a process that serves to enlarge the single ventricular chamber in this species.

Pharyngeal mesoderm development during embryogenesis: implications for both heart and head myogenesis

The pharyngeal mesoderm (PM), located in the head region of the developing embryo, recently triggered renewed interest as the major source of cells contributing to broad regions of the heart as well as to the head musculature. What exactly is PM? In this review, we describe the anatomical and molecular characteristics of this mesodermal population and its relationship to the first and second heart fields in chick and mouse embryos. The regulatory network of transcription factors and signalling molecules that regulate PM development is also discussed. In addition, we summarize recent studies into the evolutionary origins of this tissue and its multipotential contributions to both cardiac and pharyngeal muscle progenitors.

Mechanisms of T-box gene function in the developing heart

The multi-chambered mammalian heart arises from a simple tube by polar elongation, myocardial differentiation and morphogenesis. Members of the large family of T-box (Tbx) transcription factors have been identified as crucial players that act in distinct subprogrammes during cardiac regionalization. Tbx1 and Tbx18 ensure elongation of the cardiac tube at the anterior and posterior pole, respectively. Tbx1 acts in the pharyngeal mesoderm to maintain proliferation of mesenchymal precursor cells for formation of a myocardialized and septated outflow tract. Tbx18 is expressed in the sinus venosus region and is required for myocardialization of the caval veins and the sinoatrial node. Tbx5 and Tbx20 function in the early heart tube and independently activate the chamber myocardial gene programme, whereas Tbx2 and Tbx3 locally repress this programme to favour valvuloseptal and conduction system development. Here, we summarize that these T-box factors act in different molecular circuits and control target gene expression using diverse molecular strategies including binding to distinct protein interaction partners.

Clonal analysis reveals common lineage relationships between head muscles and second heart field derivatives in the mouse embryo

Head muscle progenitors in pharyngeal mesoderm are present in close proximity to cells of the second heart field and show overlapping patterns of gene expression. However, it is not clear whether a single progenitor cell gives rise to both heart and head muscles. We now show that this is the case, using a retrospective clonal analysis in which an nlaacZ sequence, converted to functional nlacZ after a rare intragenic recombination event, is targeted to the alpha(c)-actin gene, expressed in all developing skeletal and cardiac muscle. We distinguish two branchiomeric head muscle lineages, which segregate early, both of which also contribute to myocardium. The first gives rise to the temporalis and masseter muscles, which derive from the first branchial arch, and also to the extraocular muscles, thus demonstrating a contribution from paraxial as well as prechordal mesoderm to this anterior muscle group. Unexpectedly, this first lineage also contributes to myocardium of the right ventricle. The second lineage gives rise to muscles of facial expression, which derive from mesoderm of the second branchial arch. It also contributes to outflow tract myocardium at the base of the arteries. Further sublineages distinguish myocardium at the base of the aorta or pulmonary trunk, with a clonal relationship to right or left head muscles, respectively. We thus establish a lineage tree, which we correlate with genetic regulation, and demonstrate a clonal relationship linking groups of head muscles to different parts of the heart, reflecting the posterior movement of the arterial pole during pharyngeal morphogenesis.

Analysis of a Hand1 hypomorphic allele reveals a critical threshold for embryonic viability

Loss-of-function analysis of the basic helix-loop-helix (bHLH) transcription factor Hand1 indicates critical roles in development. In an effort to generate a Hand1 cDNA knock-in reporter mouse, we generated two hypomorphic alleles, which extend embryonic survival to between embryonic day (E) 10.5 and E12.5. Heart morphogenesis appears largely normal; however, hypomorphic mice display thin left ventricular myocardium and reduction in pharyngeal mesoderm. Caudal defects, large allantois, and thickened yolk sac are observed and consistent with systemic Hand1 gene deletion. Hand1 mRNA is expressed at 30% of wild-type littermates and known Hand1-dependent genes show intermediate expression compared with wild-type and Hand1 null mice. Interestingly, putative bHLH partners, Hand2 and Twist1, show altered expression in both Hand1 null and hypomorphic backgrounds and intercrossing the Hand1 hypomorphic mice onto the Hand2 systemic null background exacerbates the cardiac and lateral mesoderm phenotypes. Together, these data define a critical threshold of Hand1 expression that is necessary for embryonic survival.

Early Chordate Origins of the Vertebrate Second Heart Field

The vertebrate heart is formed from diverse embryonic territories, including the first and second heart fields. The second heart field (SHF) gives rise to the right ventricle and outflow tract, yet its evolutionary origins are unclear. We found that heart progenitor cells of the simple chordate Ciona intestinalis also generate precursors of the atrial siphon muscles (ASMs). These precursors express Islet and Tbx1/10, evocative of the splanchnic mesoderm that produces the lower jaw muscles and SHF of vertebrates. Evidence is presented that the transcription factor COE is a critical determinant of ASM fate. We propose that the last common ancestor of tunicates and vertebrates possessed multipotent cardiopharyngeal muscle precursors, and that their reallocation might have contributed to the emergence of the SHF.

The function of the Egr1 transcription factor in cartilage formation and adaptation to microgravity in zebrafish,Danio rerio

Summary Osteoporosis is one of the major concerns for an ageing human population and for passengers on long-term space flights. Teleosts represent a potentially interesting alternative for studying bone physiology. In zebrafish (Danio rerio), the cartilaginous elements that form the pharyngeal arches derive from cranial neural crest cells, whose proper patterning and morphogenesis require reciprocal interactions with other tissue types such as pharyngeal endoderm, ectoderm and mesoderm. We show how the zebrafish can be used to study the function of signal transduction pathways, such as the Fgf pathway, or that of particular genes, such as the zinc finger transcription factor Egr1, in pharyngeal skeleton formation and maintenance. We investigate the changes caused by microgravity and chemical treatments on zebrafish. We analyze early gene expression modification using whole genome microarray experiments and the long-term consequences by staining bone structures.

Second lineage of heart forming region provides new understanding of conotruncal heart defects

Abnormal heart development causes various congenital heart defects. Recent cardiovascular biology studies have elucidated the morphological mechanisms involved in normal and abnormal heart development. The primitive heart tube originates from the lateral-most part of the heart forming mesoderm and mainly gives rise to the left ventricle. Then, during the cardiac looping, the outflow tract is elongated by the addition of cardiogenic cells from the both pharyngeal and splanchnic mesoderm (corresponding to anterior and secondary heart field, respectively), which originate from the mediocaudal region of the heart forming mesoderm and are later located anteriorly (rostrally) to the dorsal region of the heart tube. Therefore, the heart progenitors that contribute to the outflow tract region are distinct from those that form the left ventricle. The knowledge that there are two different lineages of heart progenitors in the four-chambered heart provides new understanding of the morphological and molecular etiology of conotruncal heart defects.

Mechanisms of heart development in the Japanese lamprey, Lethenteron japonicum

Vertebrate hearts have evolved from undivided tubular hearts of chordate ancestors. One of the most intriguing issues in heart evolution is the abrupt appearance of multichambered hearts in the agnathan vertebrates. To explore the developmental mechanisms behind the drastic morphological changes that led to complex vertebrate hearts, we examined the developmental patterning of the agnathan lamprey Lethenteron japonicum. We isolated lamprey orthologs of genes thought to be essential for heart development in chicken and mouse embryos, including genes responsible for differentiation and proliferation of the myocardium (LjTbx20, LjTbx4/5, and LjIsl1/2A), establishment of left-right heart asymmetry (LjPitxA), and partitioning of the heart tube (LjTbx2/3A), and studied their expression patterns during lamprey cardiogenesis. We confirmed the presence of the cardiac progenitors expressing LjIsl1/2A in the pharyngeal and splanchnic mesoderm and the heart tube of the lamprey. The presence of LjIsl1/2A-positive cardiac progenitor cells in cardiogenesis may have permitted an increase of myocardial size in vertebrates. We also observed LjPitxA expression in the left side of lamprey cardiac mesoderm, suggesting that asymmetric expression of Pitx in the heart has been acquired in the vertebrate lineage. Additionally, we observed LjTbx2/3A expression in the nonchambered myocardium, supporting the view that acquisition of Tbx2/3 expression may have allowed primitive tubular hearts to partition, giving rise to multichambered hearts.