Janynke F. Brons

Transcription Factor Tbx3 Is Required for the Specification of the Atrioventricular Conduction System

The cardiac conduction system consists of distinctive heart muscle cells that initiate and propagate the electric impulse required for coordinated contraction. The conduction system expresses the transcriptional repressor Tbx3, which is required for vertebrate development and controls the formation of the sinus node. In humans, mutations in Tbx3 cause ulnar–mammary syndrome. Here, we investigated the role of Tbx3 in the molecular specification of the atrioventricular conduction system. Expression analysis revealed early delineation of the atrioventricular bundle and proximal bundle branches by Tbx3 expression in human, mouse, and chicken. Tbx3-deficient mice, which die between embryonic day 12.5 and 15.5, ectopically expressed genes for connexin (Cx)43, atrial natriuretic factor (Nppa), Tbx18, and Tbx20 in the atrioventricular bundle and proximal bundle branches. Cx40 was precociously upregulated in the atrioventricular bundle of Tbx3 mutants. Moreover, the atrioventricular bundle and branches failed to exit the cell cycle in Tbx3 mutant embryos. Finally, Tbx3-deficient embryos developed outflow tract malformations and ventricular septal defects. These data reveal that Tbx3 is required for the molecular specification of the atrioventricular bundle and bundle branches and for the development of the ventricular septum and outflow tract. Our data suggest a mechanism in which Tbx3 represses differentiation into ventricular working myocardium, thereby imposing the conduction system phenotype on cells within its expression domain.

The Tbx2+ Primary Myocardium of the Atrioventricular Canal Forms the Atrioventricular Node and the Base of the Left Ventricle

The primary myocardium of the embryonic heart, including the atrioventricular canal and outflow tract, is essential for septation and valve formation. In the chamber-forming heart, the expression of the T-box transcription factor Tbx2 is restricted to the primary myocardium. To gain insight into the cellular contributions of the Tbx2+ primary myocardium to the components of the definitive heart, genetic lineage tracing was performed using a novel Tbx2Cre allele. These analyses revealed that progeny of Tbx2+ cells provide an unexpectedly large contribution to the Tbx2-negative ventricles. Contrary to common assumption, we found that the embryonic left ventricle only forms the left part of the definitive ventricular septum and the apex. The atrioventricular node, but not the atrioventricular bundle, was found to derive from Tbx2+ cells. The Tbx2+ outflow tract formed the right ventricle and right part of the ventricular septum. In Tbx2-deficient embryos, the left-sided atrioventricular canal was found to prematurely differentiate to chamber myocardium and to proliferate at increased rates similar to those of chamber myocardium. As a result, the atrioventricular junction and base of the left ventricle were malformed. Together, these observations indicate that Tbx2 temporally suppresses differentiation and proliferation of primary myocardial cells. A subset of these Tbx2Cre-marked cells switch off expression of Tbx2, which allows them to differentiate into chamber myocardium, to initiate proliferation, and to provide a large contribution to the ventricles. These findings imply that errors in the development of the early atrioventricular canal may affect a much larger region than previously anticipated, including the ventricular base.

Gene Expression Profiling of the Forming Atrioventricular Node Using a Novel Tbx3-Based Node-Specific Transgenic Reporter

The atrioventricular (AV) node is a recurrent source of potentially life-threatening arrhythmias. Nevertheless, limited data are available on its developmental control or molecular phenotype. We used a novel AV nodal myocardium–specific reporter mouse to gain insight into the gene programs determining the formation and phenotype of the developing AV node. In this reporter, green fluorescent protein (GFP) expression was driven by a 160-kbp bacterial artificial chromosome with Tbx3 and flanking sequences. GFP was selectively active in the AV canal of embryos and AV node of adults, whereas the Tbx3-positive AV bundle and sinus node were devoid of GFP, demonstrating that distinct regulatory sequences and pathways control expression in the components of the conduction system. Fluorescent AV nodal and complementary Nppa-positive chamber myocardial cell populations of embryonic day 10.5 embryos and of embryonic day 17.5 fetuses were purified using fluorescence-activated cell sorting, and their expression profiles were assessed by genome-wide microarray analysis, providing valuable information concerning their molecular identities. We constructed a comprehensive list of sodium, calcium, and potassium channel genes specific for developing nodal or chamber myocardium. Furthermore, the data revealed that the AV node and the chamber (working) myocardium phenotypes diverge during development but that the functional gene classes characterizing both subtypes are maintained. One of the repertoires identified in the AV node–specific gene profiles consists of multiple neurotrophic factors and semaphorins, not yet appreciated to play a role in nodal development, revealing shared characteristics between nodal and nervous system development.

Expression and requirement of T-box transcription factors Tbx2 and Tbx3 during secondary palate development in the mouse

Formation of the mammalian secondary palate is a highly regulated and complex process. Impairment of the underlying cellular and molecular programs often results in cleft palate, a common birth defect in mammals. Here we report that Tbx2 and Tbx3, two closely related genes encoding T-box transcription factors, are expressed in the mesenchyme of the mouse palatal structures during development. Mice homozygous mutant for Tbx2 and mice double heterozygous for Tbx2 and Tbx3 exhibit a cleft palate phenotype arguing for an important contribution of Tbx2 and Tbx3 to palatogenesis. In Tbx2-deficient embryos, the bilateral primordial palatal shelves form but are smaller and retarded in the outgrowth process. They do not make contact but retain the potential to fuse. Development of other craniofacial structures appears normal, suggesting that impaired palate formation in Tbx2-mutant mice is caused by a primary defect in the palatal shelf mesenchyme. This is further supported by increased cell proliferation and apoptosis accompanied by increased expression of Bmp4 and CyclinD1 in Tbx2-deficient palatal shelves. Hence, Tbx2 and Tbx3 function overlappingly to control growth of the palatal shelf mesenchyme.

Dissection of the promoter of the HAP4 gene in S. cerevisiae unveils a complex regulatory framework of transcriptional regulation.

In S. cerevisiae, the heteromeric Hap2/3/4/5 complex is necessary for induced transcription of a large number of genes involved in oxidative metabolism on non‐fermentable carbon sources. The Hap4p subunit is the activator subunit and at the same time also the regulatory part of the complex, since it is the only one whose level is regulated by carbon source itself. HAP4 promoter analysis shows a 265 bp activating region at position −1006/−741 bp upstream of the ATG start codon. Specific and differential protein‐binding to a 30 nt CSRE‐like sequence within this region was observed with extracts from repressing and inducing carbon sources. Carbon source‐dependent activation mediated by the 265 bp fragment, as well as protein binding to the 30 nt CSRE‐like region, is dependent on the presence of CAT8 function, unveiling a complex framework by which the expression of the HAP4 gene is coordinated. Copyright