Marina Campione

T-Box Transcription Factor Tbx2 Represses Differentiation and Formation of the Cardiac Chambers

Specific regions of the embryonic heart tube differentiate into atrial and ventricular chamber myocardium, whereas the inflow tract, atrioventricular canal, inner curvatures, and outflow tract do not. These regions express Tbx2, a transcriptional repressor. Here, we tested its role in chamber formation. The temporal and spatial pattern of Tbx2 mRNA and protein expression in mouse hearts was found to be complementary to that of chamber myocardium‐specific genes Nppa, Cx40, Cx43, and Chisel, and was conserved in human. In vitro, Tbx2 repressed the activity of regulatory fragments of Cx40, Cx43, and Nppa. Hearts of transgenic embryos that expressed Tbx2 in the prechamber myocardium completely failed to form chambers and to express the chamber myocardium‐specific genes Nppa, Cx40, and Chisel, whereas other cardiac genes were normally expressed. These findings provide the first evidence that Tbx2 is a determinant in the local repression of chamber‐specific gene expression and chamber differentiation. Developmental Dynamics 229:763–770, 2004.

Pitx2 isoforms: involvement of Pitx2c but not Pitx2a or Pitx2b in vertebrate left–right asymmetry

During vertebrate left-right development the homeobox gene Pitx2 serves as a mediator between transient nodal signaling in the left lateral plate mesoderm (l-LPM) and asymmetric organ morphogenesis. Misexpression of Pitx2 in chick and frog led to alteration of organ situs. Here we report the presence of different Pitx2 isoforms in mouse and frog. Pitx2c but not Pitx2a or Pitx2b was asymmetrically expressed in the l-LPM, heart and gut, and was specifically induced by nodal in Xenopus animal cap explant cultures and whole embryos. Pitx2c induced its own transcription, suggesting a maintenance mechanism following the down-regulation of nodal in the l-LPM. Pitx2c thus represents the left-specific isoform involved in vertebrate left-right asymmetry.

Multiple Transcriptional Domains, With Distinct Left and Right Components, in the Atrial Chambers of the Developing Heart

During heart development, 2 fast-conducting regions of working myocardium balloon out from the slow-conducting primary myocardium of the tubular heart. Three regions of primary myocardium persist: the outflow tract, atrioventricular canal, and inflow tract, which are contiguous throughout the inner curvature of the heart. The contribution of the inflow tract to the definitive atrial chambers has remained enigmatic largely because of the lack of molecular markers that permit unambiguous identification of this myocardial domain. We now report that the genes encoding atrial natriuretic factor, myosin light chain (MLC) 3F, MLC2V, and Pitx-2, and transgenic mouse lines expressing nlacZ under the control of regulatory sequences of the mouse MLC1F/3F gene, display regionalized patterns of expression in the atrial component of the developing mouse heart. These data distinguish 4 broad transcriptional domains in the atrial myocardium: (1) the atrioventricular canal that will form the smooth-walled lower atrial rim proximal to the ventricles; (2) the atrial appendages; (3) the caval vein myocardium (systemic inlet); and (4) the mediastinal myocardium (pulmonary inlet), including the atrial septa. The pattern of expression of Pitx-2 reveals that each of these transcriptional domains has a distinct left and right component. This study reveals for the first time differential gene expression in the systemic and pulmonary inlets, which is not shared by the contiguous atrial appendages and provides evidence for multiple molecular compartments within the atrial chambers. Furthermore, this work will allow the contribution of each of these myocardial components to be studied in congenitally malformed hearts, such as those with abnormal venous return.

Negative Autoregulation of the Organizer-specific Homeobox Gene goosecoid

The homeobox gene goosecoid has been implicated to play a central role in the Spemann organizer tissue of the vertebrate embryo. Misexpression of goosecoid on the ventral side of a Xenopus laevis gastrula embryo was shown to result in a partial duplication of the primary body axis, reminiscent of the Spemann organizer graft. Normal embryonic development thus requires tight temporal and spatial control of genes instrumental for organizer function. In the present study we investigated the transcriptional control of goosecoid gene expression. Sequence analysis of the mouse and human promoter region revealed the presence of two palindromic binding elements for homeobox genes of the prd type to which goosecoidbelongs. We show that Goosecoid protein can bind to these sitesin vitro. By using reporter gene constructs of the human and mouse promoter, we demonstrate that Goosecoid can act as a repressor of its own promoter activity in transient co-transfection experiments in mouse P19 cells and in Xenopus embryos. Autorepression depends on the presence of the homeodomain and is mediated through the prd element more proximal to the transcriptional start site. Our results suggest a role forgoosecoid in restricting organizer activity in the vertebrate gastrula embryo.

Current Perspectives in Cardiac Laterality

The heart is the first organ to break symmetry in the developing embryo and onset of dextral looping is the first indication of this event. Looping is a complex process that progresses concomitantly to cardiac chamber differentiation and ultimately leads to the alignment of the cardiac regions in their final topology. Generation of cardiac asymmetry is crucial to ensuring proper form and consequent functionality of the heart, and therefore it is a highly regulated process. It has long been known that molecular left/right signals originate far before morphological asymmetry and therefore can direct it. The use of several animal models has led to the characterization of a complex regulatory network, which invariably converges on the Tgf-β signaling molecule Nodal and its downstream target, the homeobox transcription factor Pitx2. Here, we review current data on the cellular and molecular bases of cardiac looping and laterality, and discuss the contribution of Nodal and Pitx2 to these processes. A special emphasis will be given to the morphogenetic role of Pitx2 and to its modulation of transcriptional and functional properties, which have also linked laterality to atrial fibrillation.

A novel role of the organizer gene Goosecoid as an inhibitor of Wnt/PCP-mediated convergent extension in Xenopus and mouse.

Goosecoid (Gsc) expression marks the primary embryonic organizer in vertebrates and beyond. While functions have been assigned during later embryogenesis, the role of Gsc in the organizer has remained enigmatic. Using conditional gain-of-function approaches in Xenopus and mouse to maintain Gsc expression in the organizer and along the axial midline, neural tube closure defects (NTDs) arose and dorsal extension was compromised. Both phenotypes represent convergent extension (CE) defects, arising from impaired Wnt/planar cell polarity (PCP) signaling. Dvl2 recruitment to the cell membrane was inhibited by Gsc in Xenopus animal cap assays and key Wnt/PCP factors (RhoA, Vangl2, Prickle, Wnt11) rescued Gsc-mediated NTDs. Re-evaluation of endogenous Gsc functions in MO-mediated gene knockdown frog and knockout mouse embryos unearthed PCP/CE-related phenotypes as well, including cartilage defects in Xenopus and misalignment of inner ear hair cells in mouse. Our results assign a novel function to Gsc as an inhibitor of Wnt/PCP-mediated CE. We propose that in the organizer Gsc represses CE as well: Gsc-expressing prechordal cells, which leave the organizer first, migrate and do not undergo CE like the Gsc-negative notochordal cells, which subsequently emerge from the organizer. In this model, Gsc provides a switch between cell migration and CE, i.e. cell intercalation.

Cardiovascular development: Toward biomedical applicability†

The adult heart displays a low proliferation capacity, compromising its function if exposed to distinct biological insults. Interestingly, the observation that an increasing number of cell types display an unpredicted cellular plasticity has opened new therapeutical avenues. In this review we will summarize the current knowledge of non-resident stem cells that can be putatively used for cardiac regeneration. At present, bone marrow stem cells have been extensively studied as a cellular source to heal the heart; however, their myocardial contribution is highly limited. Experimental studies have demonstrated that skeletal myoblasts can engraft into the heart, although, unfortunately, they lead to myocardial uncoupling. Embryonic stem cells can spontaneously generate cardiomyocytes that exhibit a variety of electrophysiological phenotypes. Several constrains should nonetheless be overcome before entering the clinical arena, such as the ability to direct and control the generation of cardiomyocytes into a single myocardial lineage.