Lambertus J. Wisse

Double-Outlet Right Ventricle and Overriding Tricuspid Valve Reflect Disturbances of Looping, Myocardialization, Endocardial Cushion Differentiation, and Apoptosis in TGF-β2–Knockout Mice

Background—Transforming growth factor-β2 (TGF-β2) is a member of a family of growth factors with the potential to modify multiple processes. Mice deficient in the TGF-β2 gene die around birth and show a variety of defects of different organs, including the heart. Methods and Results—We studied the hearts of TGF-β2–null mouse embryos from 11.5 to 18.5 days of gestation to analyze the types of defects and determine which processes of cardiac morphogenesis are affected by the absence of TGF-β2. Analysis of serial sections revealed malformations of the outflow tract (typically a double-outlet right ventricle) in 87.5%. There was 1 case of common arterial trunk. Abnormal thickening of the semilunar valves was seen in 4.2%. Associated malformations of the atrioventricular (AV) canal were found in 62.5% and were composed of perimembranous inlet ventricular septal defects (37.5%), AV valve thickening (33.3%), overriding tricuspid valve (25.0%), and complete AV septal defects (4.2%). Anomalies of the aorta and its...

Targeted Mutation Reveals Essential Functions of the Homeodomain Transcription Factor Shox2 in Sinoatrial and Pacemaking Development

Background— Identifying molecular pathways regulating the development of pacemaking and coordinated heartbeat is crucial for a comprehensive mechanistic understanding of arrhythmia-related diseases. Elucidation of these pathways has been complicated mainly by an insufficient definition of the developmental structures involved in these processes and the unavailability of animal models specifically targeting the relevant tissues. Here, we report on a highly restricted expression pattern of the homeodomain transcription factor Shox2 in the sinus venosus myocardium, including the sinoatrial nodal region and the venous valves. Methods and Results— To investigate its function in vivo, we have generated mouse lines carrying a targeted mutation of the Shox2 gene. Although heterozygous animals did not exhibit obvious defects, homozygosity of the targeted allele led to embryonic lethality at 11.5 to 13.5 dpc. Shox2−/− embryos exhibited severe hypoplasia of the sinus venosus myocardium in the posterior heart field, including the sinoatrial nodal region and venous valves. We furthermore demonstrate aberrant expression of connexin 40 and connexin 43 and the transcription factor Nkx2.5 in vivo specifically within the sinoatrial nodal region and show that Shox2 deficiency interferes with pacemaking function in zebrafish embryos. Conclusions— From these results, we postulate a critical function of Shox2 in the recruitment of sinus venosus myocardium comprising the sinoatrial nodal region.

Development of the papillary muscles of the mitral valve: morphogenetic background of parachute-like asymmetric mitral valves and other mitral valve anomalies

OBJECTIVES To understand papillary muscle malformations, such as in parachute mitral valves or parachute-like asymmetric mitral valves, we studied the development of papillary muscles. METHODS Normal human hearts at between 5 and 19 weeks of development were studied with immunohistochemistry, three-dimensional reconstructions, and gross inspection. Scanning electron microscopy was used to study human and rat hearts. RESULTS In embryonic hearts a prominent horseshoe-shaped myocardial ridge runs from the anterior wall through the apex to the posterior wall of the left ventricle. In the atrioventricular region this ridge is continuous with atrial myocardium and covered with cushion tissue. The anterior and posterior parts of the trabecular ridge enlarge and loosen their connections with the atrial myocardium. Their lateral sides gradually delaminate from the left ventricular wall, and the continuity between the two parts is incorporated in the apical trabecular network. In this way the anterior and posterior parts of the ridge transform into the anterolateral and the posteromedial papillary muscles, respectively. Simultaneously, the cushions remodel into valve leaflets and chordae. Only the chordal part of the cushions remains attached to the developing papillary muscles. CONCLUSIONS Disturbed delamination of the anterior or posterior part of the trabecular ridge from the ventricular wall, combined with underdevelopment of chordae, seems to be the cause of asymmetric mitral valves. Parachute valves, however, develop when the connection between the posterior and anterior part of the ridge condenses to form one single papillary muscle. Thus parachute valves and parachute-like asymmetric mitral valves originate in different ways.

Cardiac malformations and myocardial abnormalities in podoplanin knockout mouse embryos: Correlation with abnormal epicardial development.

Epicardium and epicardium‐derived cells have been shown to be necessary for myocardial differentiation. To elucidate the function of podoplanin in epicardial development and myocardial differentiation, we analyzed podoplanin knockout mouse embryos between embryonic day (E) 9.5 and E15.5 using immunohistochemical differentiation markers, morphometry, and three‐dimensional reconstructions. Podoplanin null mice have an increased embryonic lethality, possibly of cardiac origin. Our study reveals impairment in the development of the proepicardial organ, epicardial adhesion, and spreading and migration of the epicardium‐derived cells. Mutant embryos show a hypoplastic and perforated compact and septal myocardium, hypoplastic atrioventricular cushions resulting in atrioventricular valve abnormalities, as well as coronary artery abnormalities. The epicardial pathology is correlated with reduced epithelial–mesenchymal transformation caused by up‐regulation of E‐cadherin, normally down‐regulated by podoplanin. Our results demonstrate a role for podoplanin in normal cardiac development based on epicardial–myocardial interaction. Abnormal epicardial differentiation and reduced epithelial–mesenchymal transformation result in deficient epicardium‐derived cells leading to myocardial pathology and cardiac anomalies. Developmental Dynamics 237:847–857, 2008.

Altered apoptosis pattern during pharyngeal arch artery remodelling is associated with aortic arch malformations in Tgfβ2 knock-out mice

OBJECTIVE The morphogenetic process underlying the remodelling of the embryonic mammalian pharyngeal arch artery system is unknown. Within this process, the right sixth, carotid ducts and the distal part of the dorsal aorta (right alpha-segment) regress. In order to unravel the underlying mechanism we studied the role of apoptosis in the normal regression of pharyngeal arch artery segments and in a mouse model that develops aortic arch malformations. METHODS Normal remodelling was studied in wild-type Swiss (CPBS) and altered remodelling in the Tgfbeta2-/- compared to the Tgfbeta2+/+ (Swiss/Bl6) strain using immunohistochemistry and morphometric analysis. RESULTS During normal remodelling, apoptosis occurs in the mesenchyme surrounding pharyngeal arch arteries before regression starts. With the onset of regression, apoptosis spreads from the mesenchyme to the media. Morphometric evaluation confirms the increase in apoptosis in the actin-positive media of the disappearing segments. In Tgfbeta2-/-, aberrant apoptosis was found in both fourth arch arteries, whereas the right dorsal aorta lacks apoptosis associated with normal regression. Fourth arch hypoplasia is the main arch abnormality. In the most severe case, the fourth arch is interrupted and the right dorsal aorta alpha-segment persists, giving rise to aortic arch interruption type-B and an aberrant right subclavian artery. CONCLUSIONS We have shown for the first time that specific vascular apoptosis patterns accompany normal regression and that the incidence of apoptosis is selectively altered in the case of arch artery abnormalities in Tgfbeta2 knock-out mice.

Loss of function of the Prx1 and Prx2 homeobox genes alters architecture of the great elastic arteries and ductus arteriosus

Abstract Prx1 (MHox) and Prx2 (S8) are non-clustered homeobox genes that are expressed in a complex, mostly mesenchyme-specific pattern throughout embryogenesis. The expression pattern and gene-targeted mice previously revealed a major role for Prx1 in skeletogenesis. In addition, specific and high expression of both Prx genes was reported in the developing cardiovascular system, predominantly in prospective connective tissues of the heart and in the great arteries and veins. We examined embryos of previously generated gene-targeted mice. Prx2-/- mutants were viable and did not show cardiovascular malformations. Intracardiac morphology of Prxl-/- and Prx1/Prx2-combined null mutants also appeared normal throughout development. However, the Prx1-/- and Prx1/Prx2 double-null mutants showed a vascular abnormality with an abnormal positioning and awkward curvature of the aortic arch in addition to a misdirected and elongated ductus arteriosus, and in two of seven combined mutants, an anomalous retro-oesophageal right subclavian artery. Generally, all great arteries appeared to run somewhat tortuously through the surrounding mesenchyme. The vascular histology and vessel wall thickness were normal in all mutants. Prx1-/- and Prx double-gene-targeted mice revealed similar spectra of vascular anomalies, but double mutants appeared to be more seriously affected. The current findings suggest that other genes may compensate for the lossof Prx in the heart, but, in contrast, our data support a role for Prx in the development of vascular and perivascular matrix.

Nkx2.5‐negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system

Recent advances in the study of cardiac development have shown the relevance of addition of myocardium to the primary myocardial heart tube. In wild‐type mouse embryos (E9.5–15.5), we have studied the myocardium at the venous pole of the heart using immunohistochemistry and 3D reconstructions of expression patterns of MLC‐2a, Nkx2.5, and podoplanin, a novel coelomic and myocardial marker. Podoplanin‐positive coelomic epithelium was continuous with adjacent podoplanin‐ and MLC‐2a‐positive myocardium that formed a conspicuous band along the left cardinal vein extending through the base of the atrial septum to the posterior myocardium of the atrioventricular canal, the atrioventricular nodal region, and the His‐Purkinje system. Later on, podoplanin expression was also found in the myocardium surrounding the pulmonary vein. On the right side, podoplanin‐positive cells were seen along the right cardinal vein, which during development persisted in the sinoatrial node and part of the venous valves. In the MLC‐2a‐ and podoplanin‐positive myocardium, Nkx2.5 expression was absent in the sinoatrial node and the wall of the cardinal veins. There was a mosaic positivity in the wall of the common pulmonary vein and the atrioventricular conduction system as opposed to the overall Nkx2.5 expression seen in the chamber myocardium. We conclude that we have found podoplanin as a marker that links a novel Nkx2.5‐negative sinus venosus myocardial area, which we refer to as the posterior heart field, with the cardiac conduction system. Anat Rec, 290:115–122, 2007.

PDGF-B signaling is important for murine cardiac development: Its role in developing atrioventricular valves, coronaries, and cardiac innervation

We hypothesized that PDGF‐B/PDGFR‐β‐signaling is important in the cardiac contribution of epicardium‐derived cells and cardiac neural crest, cell lineages crucial for heart development. We analyzed hearts of different embryonic stages of both Pdgf‐b−/− and Pdgfr‐β−/− mouse embryos for structural aberrations with an established causal relation to defective contribution of these cell lineages. Immunohistochemical staining for αSMA, periostin, ephrinB2, EphB4, VEGFR‐2, Dll1, and NCAM was performed on wild‐type and knockout embryos. We observed that knockout embryos showed perimembranous and muscular ventricular septal defects, maldevelopment of the atrioventricular cushions and valves, impaired coronary arteriogenesis, and hypoplasia of the myocardium and cardiac nerves. The abnormalities correspond with models in which epicardial development is impaired and with neuronal neural crest–related innervation deficits. This implies a role for PDGF‐B/PDGFR‐β‐signaling specifically in the contribution of these cell lineages to cardiac development. Developmental Dynamics 237:494–503, 2008.

Development of the atrioventricular valve tension apparatus in the human heart.

Abstract Using various microscopical techniques we studied the development of the atrioventricular valves in human hearts between 5 and 19 weeks of development. Within the atrioventricular cushions two different layers could be recognized that remained present in all ages studied. The atrial layer, being present at the side of the atrioventricular orifice, was positive for laminin while the ventricular layer, that was connected to the myocardium, was positive for fibronectin and collagen III. Fate-mapping of these two layers, morphometrics, and scanning electron microscopy, supplemented with in vivo labeling of cushion tissue in chicken hearts have lead to new insights in the process of valve development. The cushions became freely movable prevalvular leaflets by delamination of ventricular myocardium underneath the cushion tissue. This myocardium gradually retracted towards annulus and papillary muscles and finally disappeared, resulting in fibrous, non-myocardial valves. The atrial layer of the cushions remained present as a jelly-like surface on the valve leaflets while the ventricular layer of the cushions became the compact fibrous tissue of the leaflets and the chords. Chordal development was first visible at 10 weeks of development when gaps were formed in the ventricular layer of the cushions on top of the papillary muscles. These gaps enlarged into the interchordal spaces while the cushion tissue in between the gaps lengthened to form the chords. We conclude that the leaflets as well as the chords of the atrioventricular valves are derived from atrioventricular cushion tissue. Myocardium is only important for loosening of the leaflets while keeping connection with the developing papillary muscles. Errors in delamination or retraction of myocardium or remodeling of cushion tissue into chords form the basis for various congenital valve anomalies.

Dysregulation of the PDGFRA gene causes inflow tract anomalies including TAPVR: integrating evidence from human genetics and model organisms

Total anomalous pulmonary venous return (TAPVR) is a congenital heart defect inherited via complex genetic and/or environmental factors. We report detailed mapping in extended TAPVR kindreds and mutation analysis in TAPVR patients that implicate the PDGFRA gene in the development of TAPVR. Gene expression studies in mouse and chick embryos for both the Pdgfra receptor and its ligand Pdgf-a show temporal and spatial patterns consistent with a role in pulmonary vein (PV) development. We used an in ovo function blocking assay in chick and a conditional knockout approach in mouse to knock down Pdgfra expression in the developing venous pole during the period of PV formation. We observed that loss of PDGFRA function in both organisms causes TAPVR with low penetrance (approximately 7%) reminiscent of that observed in our human TAPVR kindreds. Intermediate inflow tract anomalies occurred in a higher percentage of embryos (approximately 30%), suggesting that TAPVR occurs at one end of a spectrum of defects. We show that the anomalous pulmonary venous connection seen in chick and mouse is highly similar to TAPVR discovered in an abnormal early stage embryo from the Kyoto human embryo collection. Whereas the embryology of the normal venous pole and PV is becoming understood, little is known about the embryogenesis or molecular pathogenesis of TAPVR. These models of TAPVR provide important insight into the pathogenesis of PV defects. Taken together, these data from human genetics and animal models support a role for PDGF-signaling in normal PV development, and in the pathogenesis of TAPVR.