Abstract-The sinus node (or sinoatrial node [SAN]), the pacemaker of the heart, is a functionally and structurally heterogeneous tissue, which consists of a large "head" within the right caval vein myocardium and a "tail" along the terminal crest. Here, we investigated its cellular origin and mechanism of formation. Using genetic lineage analysis and explant assays, we identified T-box transcription factor Tbx18-expressing mesenchymal progenitors in the inflow tract region that differentiate into pacemaker myocardium to form the SAN. We found that the head and tail represent separate regulatory domains expressing distinctive gene programs. Tbx18 is required to establish the large head structure, as seen by the existence of a very small but still functional tail piece in Tbx18-deficient fetuses. In contrast, Tbx3-deficient embryos formed a morphologically normal SAN, which, however, aberrantly expressed Cx40 and other atrial genes, demonstrating that Tbx3 controls differentiation of SAN head and tail cardiomyocytes but also demonstrating that Tbx3 is not required for the formation of the SAN structure. Our data establish a functional order for Tbx18 and Tbx3 in SAN formation, in which Tbx18 controls the formation of the SAN head from mesenchymal precursors, on which Tbx3 subsequently imposes the pacemaker gene program. Key Words: heart development Ⅲ progenitors Ⅲ Hcn4 Ⅲ Cx43 Ⅲ transgenic mice T he sinoatrial node (SAN) or sinus node is the most upstream component of the cardiac conduction system. As the primary pacemaker, it serves to initiate and control the rate of electric impulses for the ordered stimulation and contraction of the cardiac chambers. The critical importance of the SAN is reflected in dysfunctions that arise on aging and disease, including sick sinus syndrome, leading to implantation in about one-half of all pacemaker recipients in the United States. 1 The SAN consists of a small group of primitive variably sized myocytes with little contractile filaments and intermingled fibroblasts at the junction of the right venous entrance and the atrium. 2 They form an elongated "comma-shaped" structure that is subdivided into a large "head" in the right superior caval vein bordering the atrium, and a "tail" along the terminal crest. 3,4 The leading pacemaker usually originates from a small number of cells within the SAN, but its location shifts under altered physiological conditions, a phenomenon that likely contributes to both controllability and stability of pacemaker activity. 2,5,6 For example, ␤-adrenergic stimulation in rat causes dominant pacemaker activity to shift cranially into the SAN head region, 7 supporting the notion that functional specialization correlates with structural regionalization in the SAN.The mechanisms underlying the functional regionalization, as well as the pathological mechanisms underlying SAN dysfunction, are only insufficiently understood. Histological studies initially indicated that the mouse SAN forms at approximately embryonic day (E)10 to E11 from myocardium of t...

The thymus is continuously seeded by progenitors derived from hematopoietic stem cells, which reside in the BM. These progenitors migrate via the blood stream into the thymus, where they adopt a T cell fate, proliferate, and diff erentiate into mature functional T cells. This differentiation process is characterized by multiple developmental stages. The earliest thymic progenitors lack surface expression of CD4 and CD8 and are therefore referred to as doublenegative (DN) thymocytes. They subsequently up-regulate both CD4 and CD8 coreceptors (double positive [DP]) before undergoing positive and negative selection, and maturing to CD4 and CD8 single-positive (SP) thymocytes that emigrate to the periphery. Immature DN thymocytes can be subdivided into four subpopulations according to the surface expression of CD117, CD44, and CD25. The most immature thymocyte progenitors (DN1) express CD117 and CD44 and are negative for CD25, followed by the DN2 population, which upregulates CD25, and the DN3 cells, which downregulate CD117 and CD44 before generating DN4 thymocytes lacking expression of all three markers ( 1, 2 ).Over the last decade, many reports highlighted the importance of the evolutionarily conserved Notch cascade for the lymphoid system ( 3 ). Mammals possess 4 Notch receptors (N1 -4), which are activated by two classes of Thymic T cell lineage commitment is dependent on Notch1 (N1) receptor -mediated signaling. Although the physiological ligands that interact with N1 expressed on thymic precursors are currently unknown, in vitro culture systems point to Delta-like 1 (DL1) and DL4 as prime candidates. Using DL1 -and DL4-lacZ reporter knock-in mice and novel monoclonal antibodies to DL1 and DL4, we show that DL4 is expressed on thymic epithelial cells (TECs), whereas DL1 is not detected. The function of DL4 was further explored in vivo by generating mice in which DL4 could be specifi cally inactivated in TECs or in hematopoietic progenitors. Although loss of DL4 in hematopoietic progenitors did not perturb thymus development, inactivation of DL4 in TECs led to a complete block in T cell development coupled with the ectopic appearance of immature B cells in the thymus. These immature B cells were phenotypically indistinguishable from those developing in the thymus of conditional N1 mutant mice. Collectively, our results demonstrate that DL4 is the essential and nonredundant N1 ligand responsible for T cell lineage commitment. Moreover, they strongly suggest that N1-expressing thymic progenitors interact with DL4-expressing TECs to suppress B lineage potential and to induce the fi rst steps of intrathymic T cell development.

Abstract-The venous pole of the mammalian heart is a structurally and electrically complex region, yet the lineage and molecular mechanisms underlying its formation have remained largely unexplored. In contrast to classical studies that attribute the origin of the myocardial sinus horns to the embryonic venous pole, we find that the sinus horns form only after heart looping by differentiation of mesenchymal cells of the septum transversum region into myocardium. The myocardial sinus horns and their mesenchymal precursor cells never express Nkx2-5, a transcription factor critical for heart development. In addition, lineage studies show that the sinus horns do not derive from cells previously positive for Nkx2-5. In contrast, the sinus horns express the T-box transcription factor gene Tbx18. Mice deficient for Tbx18 fail to form sinus horns from the pericardial mesenchyme and have defective caval veins, whereas the pulmonary vein and atrial structures are unaffected. Our studies define a novel heart precursor population that contributes exclusively to the myocardium surrounding the sinus horns or systemic venous tributaries of the developing heart, which are a source of congenital malformation and cardiac arrhythmias. Key Words: sinus horns Ⅲ congenital heart defect Ⅲ Nkx2-5 Ⅲ Tbx18 Ⅲ morphogenesis Ⅲ recruitment T he systemic venous return of the heart consists of multiple anatomical components including the proximal myocardial part of the right superior and inferior caval veins, the coronary sinus (the persisting left caval vein in the mouse), and the sinus venarum. These structures are thought to be the mature counterparts of the right and left sinus horns in the embryo, which are the myocardial parts of the common cardinal veins upstream of the venous valves that bulge into the pericardial cavity. These, in turn, are presumed to derive from the embryonic venous pole or inflow tract of the forming heart and the common cardinal veins. 1,2 Developmental disorders of the heart, which include malformations of the pulmonary and systemic venous returns, 3,4 represent the most common human birth defects. 5,6 In addition, several specific components of the venous returns are found to be the origin of arrhythmias. 7-9 Recent 3D reconstruction and genetic analyses have greatly improved our insight into the morphogenesis of the systemic and pulmonary venous returns. 2,3,10 Nevertheless, the cellular origin of the components of the systemic and pulmonary venous return and the genetic mechanisms underlying their formation are not known.The embryonic heart of amniotes initially represents a tube consisting of precursor cells for most of the left ventricle and a small portion of the atria. Outflow tract, right ventricle, and large portions of the atria are only subsequently recruited from a second lineage of mesenchymal cells. 11-13 Nkx2-5, which encodes a homeobox transcription factor, is expressed in both the first and second lineages of the heart and plays pivotal roles in early and late steps of cardiogenesis. 13,14 Using ...