Laurent Dupays

Hand1 regulates cardiomyocyte proliferation versus differentiation in the developing heart.

The precise origins of myocardial progenitors and their subsequent contribution to the developing heart has been an area of considerable activity within the field of cardiovascular biology. How these progenitors are regulated and what signals are responsible for their development are, however, much less well understood. Clearly, not only is there a need to identify factors that regulate the transition from proliferation of cardioblasts to differentiation of cardiac muscle, but it is also necessary to identify factors that maintain an adequate pool of undifferentiated myocyte precursors as a prerequisite to preventing organ hypoplasia and congenital heart disease. Here, we report how upregulation of the basic helix-loop-helix (bHLH) transcription factor Hand1, restricted exclusively to Hand1-expressing cells, brings about a significant extension of the heart tube and extraneous looping caused by the elevated proliferation of cardioblasts in the distal outflow tract. This activity is independent of the further recruitment of extracardiac cells from the secondary heart field and permissive for the continued differentiation of adjacent myocardium. Culture studies using embryonic stem (ES) cell-derived cardiomyocytes revealed that, in a Hand1-null background, there is significantly elevated cardiomyocyte differentiation, with an apparent default mesoderm pathway to a cardiomyocyte fate. However, Hand1 gain of function maintains proliferating precursors resulting in delayed and significantly reduced cardiomyocyte differentiation that is mediated by the prevention of cell-cycle exit, by G1 progression and by increased cell division. Thus, this work identifies Hand1 as a crucial cardiac regulatory protein that controls the balance between proliferation and differentiation in the developing heart, and fills a significant gap in our understanding of how the myocardium of the embryonic heart is established.

Biphasic Development of the Mammalian Ventricular Conduction System

Rationale: The ventricular conduction system controls the propagation of electric activity through the heart to coordinate cardiac contraction. This system is composed of specialized cardiomyocytes organized in defined structures including central components and a peripheral Purkinje fiber network. How the mammalian ventricular conduction system is established during development remains controversial. Objective: To define the lineage relationship between cells of the murine ventricular conduction system and surrounding working myocytes. Methods and Results: A retrospective clonal analysis using the &agr;-cardiac actinnlaacZ/+ mouse line was carried out in three week old hearts. Clusters of clonally related myocytes were screened for conductive cells using connexin40-driven enhanced green fluorescent protein expression. Two classes of clusters containing conductive cells were obtained. Mixed clusters, composed of conductive and working myocytes, reveal that both cell types develop from common progenitor cells, whereas smaller unmixed clusters, composed exclusively of conductive cells, show that proliferation continues after lineage restriction to the conduction system lineage. Differences in the working component of mixed clusters between the right and left ventricles reveal distinct progenitor cell histories in these cardiac compartments. These results are supported by genetic fate mapping using Cre recombinase revealing progressive restriction of connexin40-positive myocytes to a conductive fate. Conclusions: A biphasic mode of development, lineage restriction followed by limited outgrowth, underlies establishment of the mammalian ventricular conduction system.

The effect of connexin40 deficiency on ventricular conduction system function during development

AIMS The aim of this study was to characterize ventricular activation patterns in normal and connexin40-deficient mice in order to dissect the role of connexin40 in developing the conduction system. METHODS AND RESULTS We performed optical mapping of epicardial activation between ED9.5-18.5 and analysed ventricular activation patterns and times of left ventricular activation. Mouse embryos deficient for connexin40 were compared with normal and heterozygous littermates. Morphology of the primary interventricular ring (PIR) was delineated with the help of T3-LacZ transgene. Four major types of ventricular activation patterns characterized by primary breakthrough in different parts of the heart were detected during development: PIR, left ventricular apex, right ventricular apex, and dual right and left ventricular apices. Activation through PIR was frequently present at the early stages until ED12.5. From ED14.5, the majority of hearts showed dual left and right apical breakthrough, suggesting functionality of both bundle branches. Connexin40-deficient embryos showed initially a delay in left bundle branch function, but the right bundle branch block, previously described in the adults, was not detected in ED14.5 embryos and appeared only gradually with 80% penetrance at ED18.5. CONCLUSION The switch of function from the early PIR conduction pathway to the mature apex to base activation is dependent upon upregulation of connexin40 expression in the ventricular trabeculae. The early function of right bundle branch does not depend on connexin40. Quantitative analysis of normal mouse embryonic ventricular conduction patterns will be useful for interpretation of effects of mutations affecting the function of the cardiac conduction system.

Tbx2 misexpression impairs deployment of second heart field derived progenitor cells to the arterial pole of the embryonic heart

Tbx2 is a member of the T-box family of transcription factors that play important roles during heart development. In the embryonic heart tube, Tbx2 is expressed in non-chamber myocardium (outflow tract and interventricular canal) and has been shown to block chamber formation. We have developed a genetic system to conditionally misexpress Tbx2 in the embryonic mouse heart at early stages of development. We show that Tbx2 expression throughout the myocardium of the heart tube both represses proliferation and impairs secondary heart field (SHF) progenitor cell deployment into the outflow tract (OFT). Repression of proliferation is accompanied by the upregulation of Ndrg2 and downregulation of Ndrg4 expression, both genes believed to be involved in cell growth and proliferation. Impaired deployment of SHF cells from the pharyngeal mesoderm is accompanied by downregulation of the cell adhesion molecules Alcam and N-cadherin in the anterior part of the embryonic heart. Tbx2 misexpression also results in downregulation of Tbx20 within the OFT, indicating complex and region-specific transcriptional cross-regulation between the two T-box genes.

Expression of Slit and Robo Genes in the Developing Mouse Heart

Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest‐derived cells. Studies in Drosophila have shown that the Slit‐Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2‐5 is required for early ventral restriction of Slit3 and that the T‐box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit‐Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart. Developmental Dynamics 239:3303–3311, 2010.

Overexpression of the transcription factor Hand1 causes predisposition towards arrhythmia in mice

Elevated levels of the cardiac transcription factor Hand1 have been reported in several adult cardiac diseases but it is unclear whether this change is itself maladaptive with respect to heart function. To test this possibility, we have developed a novel, inducible transgenic system, and used it to overexpress Hand1 in adult mouse hearts. Overexpression of Hand1 in the adult mouse heart leads to mild cardiac hypertrophy and a reduction in life expectancy. Treated mice show no significant fibrosis, myocyte disarray or congestive heart failure, but have a greatly reduced threshold for induced ventricular tachycardia, indicating a predisposition to cardiac arrhythmia. Within 48 h, they show a significant loss of connexin43 protein from cardiac intercalated discs, with increased intercalated disc beta-catenin expression at protein and RNA levels. These changes are sustained during prolonged Hand1 overexpression. We propose that cardiac overexpression of Hand1 offers a useful mouse model of arrhythmogenesis and elevated HAND1 may provide one of the molecular links between the failing heart and arrhythmia.

iASPP, a previously unidentified regulator of desmosomes, prevents arrhythmogenic right ventricular cardiomyopathy (ARVC)-induced sudden death

Significance Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a disease that is selective to the right side of the heart and results in heart failure and sudden death. Genetic defects in desmosome components account for approximately 50% of human ARVC cases; in the other 50% of patients, however, the causes remain unknown. We show that inhibitor of apoptosis-stimulating protein of p53 (iASPP) is an important regulator of desmosomes. It interacts with desmoplakin and desmin in cardiomyocytes and regulates desmosome integrity and intermediate filaments. iASPP-deficient mice display pathological features of ARVC and die of sudden death. In human ARVC patients, cardiomyocytes exhibit reduced levels of iASPP at the cell junctions, suggesting that iASPP may be critical in ARVC pathogenesis. Desmosomes are anchoring junctions that exist in cells that endure physical stress such as cardiac myocytes. The importance of desmosomes in maintaining the homeostasis of the myocardium is underscored by frequent mutations of desmosome components found in human patients and animal models. Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a phenotype caused by mutations in desmosomal components in ∼50% of patients, however, the causes in the remaining 50% of patients still remain unknown. A deficiency of inhibitor of apoptosis-stimulating protein of p53 (iASPP), an evolutionarily conserved inhibitor of p53, caused by spontaneous mutation recently has been associated with a lethal autosomal recessive cardiomyopathy in Poll Hereford calves and Wa3 mice. However, the molecular mechanisms that mediate this putative function of iASPP are completely unknown. Here, we show that iASPP is expressed at intercalated discs in human and mouse postmitotic cardiomyocytes. iASPP interacts with desmoplakin and desmin in cardiomyocytes to maintain the integrity of desmosomes and intermediate filament networks in vitro and in vivo. iASPP deficiency specifically induces right ventricular dilatation in mouse embryos at embryonic day 16.5. iASPP-deficient mice with exon 8 deletion (Ppp1r13lΔ8/Δ8) die of sudden cardiac death, displaying features of ARVC. Intercalated discs in cardiomyocytes from four of six human ARVC cases show reduced or loss of iASPP. ARVC-derived desmoplakin mutants DSP-1-V30M and DSP-1-S299R exhibit weaker binding to iASPP. These data demonstrate that by interacting with desmoplakin and desmin, iASPP is an important regulator of desmosomal function both in vitro and in vivo. This newly identified property of iASPP may provide new molecular insight into the pathogenesis of ARVC.

Spatiotemporal regulation of enhancers during cardiogenesis

With the advance in chromatin immunoprecipitation followed by high-throughput sequencing, there has been a dramatic increase in our understanding of distal enhancer function. In the developing heart, the identification and characterisation of such enhancers have deepened our knowledge of the mechanisms of transcriptional regulation that drives cardiac differentiation. With next-generation sequencing techniques becoming widely accessible, the quantity of data describing the genome-wide distribution of cardiac-specific transcription factor and chromatin modifiers has rapidly increased and it is now becoming clear that the usage of enhancers is highly dynamic and complex, both during the development and in the adult. The identification of those enhancers has revealed new insights into the transcriptional mechanisms of how tissue-specific gene expression patterns are established, maintained, and change dynamically during development and upon physiological stress.

03-P093 Tbx2 modulates proliferation and elongation of the mouse embryonic heart tube

In vertebrates, retinal progenitor cells (RPCs) are specified at the end of the gastrulation within the anterior neural plate (ANP) as a single domain called the eye field. From that single domain, positioned medially within the ANP, the RPCs give rise to two optic vesicles, positioned more laterally. How this dramatic morphogenetic remodelling occurs is not well understood. We will show that soon after their specification, RPCs do not present a classical neuroepithelial appearance, with apical and basal sides; instead, RPCs seem to be randomly oriented and present a more mesenchymal morphology. Less than an hour later, the RPCs have organised into a neuroepithelium with all their apical sides oriented towards the lumen of the eye field. This reorganisation is followed by the splitting of the eye field and the evagination of the optic vesicles. We will discuss the potential role of the Wnt/PCP pathway and the transcription factor Rx3 in these cellular rearrangements as well as the role of the morphogenetic movements of surrounding tissues (prospective telencephalon and hypothalamus) in the splitting of the eye field.