Generation of Mice With a Conditional Null Allele For Tbx2

The T-box transcription factor Tbx2 plays important roles in patterning and development, and has been implicated in cell-cycle regulation and cancer. Conventional disruption of Tbx2 results in abnormalities of the heart, limbs, eye and other structures, and early fetal lethality. To gain insight into the role of Tbx2 in different tissues and at different stages of development, we have generated a conditional null allele of Tbx2 by flanking Exon 2 with loxP sites (Tbx2(fl2)). Homozygous Tbx2(fl2) mice are viable and fertile, indicating that the Tbx2(fl2) allele is a fully functional Tbx2 allele. Cre-mediated recombination, using a ubiquitously active CMVCre line, results in deletion of Exon 2 and loss of protein expression. Embryos homozygous for the recombined allele (Tbx2(Delta 2)) show the same heart and limb defects as conventional Tbx2-deficient embryos. This Tbx2 conditional null allele will be a valuable tool to uncover tissue-specific roles of Tbx2 in development and disease. genesis 48:195-199, 2010. (C) 2010 Wiley-Liss, In

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 developmen

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 bas

Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria

The sinoatrial node initiates the heartbeat and controls the rate and rhythm of contraction, thus serving as the pacemaker of the heart. Despite the crucial role of the sinoatrial node in heart function, the mechanisms that underlie its specification and formation are not known. Tbx3, a transcriptional repressor required for development of vertebrates, is expressed in the developing conduction system. Here we show that Tbx3 expression delineates the sinoatrial node region, which runs a gene expression program that is distinct from that of the bordering atrial cells. We found lineage segregation of Tbx3-negative atrial and Tbx3-positive sinoatrial node precursor cells as soon as cardiac cells turn on the atrial gene expression program. Tbx3 deficiency resulted in expansion of expression of the atrial gene program into the sinoatrial node domain, and partial loss of sinoatrial node-specific gene expression. Ectopic expression of Tbx3 in mice revealed that Tbx3 represses the atrial phenotype and imposes the pacemaker phenotype on the atria. The mice displayed arrhythmias and developed functional ectopic pacemakers. These data identify a Tbx3-dependent pathway for the specification and formation of the sinoatrial node, and show that Tbx3 regulates the pacemaker gene expression program and phenotype