Contribution of mesothelium-derived cells to liver sinusoids in avian embryos.

The developing liver is vascularized through a complex process of vasculogenesis that leads to the differentiation of the sinusoids. The main structural elements of the sinusoidal wall are endothelial and stellate (Ito) cells. We have studied the differentiation of the hepatic sinusoids in avian embryos through confocal colocalization of differentiation markers, in ovo direct labeling of the liver mesothelium, induced invasion of the developing chick liver by quail proepicardial cells, and in vitro culture of chimeric aggregates. Our results show that liver mesothelial cells give rise to mesenchymal cells which intermingle between the growing hepatoblast cords and become incorporated to the sinusoidal wall, contributing to both endothelial and stellate cell populations. We have also shown that the proepicardium, a mesothelial tissue anatomically continuous with liver mesothelium, is able to form sinusoid-like vessels into the hepatic primordium as well as in cultured aggregates of hepatoblasts. Thus, both intrinsic or extrinsic mesothelium-derived cells have the developmental potential to contribute to the establishment of liver sinusoids.

Development of the coronary arteries in a murine model of transposition of great arteries.

Transposition of great arteries in humans is associated with a wide spectrum of coronary artery patterns. However, no information is available about how this pattern diversity develops. We have studied the development of the coronary arteries in mouse embryos with a targeted mutation of perlecan, a mutation that leads to ventriculo-arterial discordance and complete transposition in about 70% of the embryos. The perlecan-deficient embryos bearing complete transposition showed a coronary artery pattern consisting of right and left coronary arteries arising from the morphologically dorsal and ventral sinuses of Valsalva, respectively. The left coronary artery gives rise to a large septal artery and runs along the ventral margin of the pulmonary root. In the earliest embryos where transposition could be confirmed (12.5 d post coitum), a dense subepicardial vascular plexus is located in this ventral margin. In wild-type mice, however, capillaries are very scarce on the ventral surface of the pulmonary root and the left coronary artery runs dorsally to this root. We suggest that the establishment of the diverse coronary artery patterns is determined by the anatomical arrangement and the capillary density of the peritruncal vascular plexus, a plexus that spreads from the atrio-ventricular groove and grows around the aortic or pulmonary roots depending on the degree of the short-axis aortopulmonary rotation. This simple model, based on very few assumptions, might explain all the observed variation of the coronary artery patterns in humans with transposition, as well as our observations on the perlecan-deficient and the normal mice.

Experimental studies on the spatiotemporal expression of WT1 and RALDH2 in the embryonic avian heart: a model for the regulation of myocardial and valvuloseptal development by epicardially derived cells (EPDCs).

Epicardially derived cells (EPDCs) delaminate from the primitive epicardium through an epithelial-to-mesenchymal transformation (EMT). After this transformation, a subpopulation of cells progressively invades myocardial and valvuloseptal tissues. The first aim of the study was to determine the tissue-specific distribution of two molecules that are thought to play a crucial function in the interaction between EPDCs and other cardiac tissues, namely the Wilms' Tumor transcription factor (WT1) and retinaldehyde-dehydrogenase2 (RALDH2). This study was performed in normal avian and in quail-to-chick chimeric embryos. It was found that EPDCs that maintain the expression of WT1 and RALDH2 initially populate the subepicardial space and subsequently invade the ventricular myocardium. As EPDCs differentiate into the smooth muscle and endothelial cell lineage of the coronary vessels, the expression of WT1 and RALDH2 becomes downregulated. This process is accompanied by the upregulation of lineage-specific markers. We also observed EPDCs that continued to express WT1 (but very little RALDH2) which did not contribute to the formation of the coronary system. A subset of these cells eventually migrates into the atrioventricular (AV) cushions, at which point they no longer express WT1. The WT1/RALDH2-negative EPDCs in the AV cushions do, however, express the smooth muscle cell marker caldesmon. The second aim of this study was to determine the impact of abnormal epicardial growth on cardiac development. Experimental delay of epicardial growth distorted normal epicardial development, reduced the number of invasive WT1/RALDH2-positive EPDCs, and provoked anomalies in the coronary vessels, the ventricular myocardium, and the AV cushions. We suggest that the proper development of ventricular myocardium is dependent on the invasion of undifferentiated, WT1-positive, retinoic acid-synthesizing EPDCs. Furthermore, we propose that an interaction between EPDCs and endocardial (derived) cells is imperative for correct development of the AV cushions.

The epicardium as a source of mesenchyme for the developing heart.

The primitive epicardium of the vertebrate embryo has traditionally been regarded as a rather passive mesothelium, lining the embryonic myocardium and forming the adult visceral pericardium. However, in recent years, there is an increasing evidence that the primitive epicardium is a highly dynamic element which supplies cells to the developing heart through a process of epithelial-mesenchymal transition. This process seems to be more active at the atrioventricular canal and outflow tract, i.e. the cardiac segments where the endothelium transforms into mesenchyme. In this paper we review the current evidence which supports such epicardial-mesenchymal transition, namely: 1) morphological features, 2) colocalization of cytokeratin and vimentin in the epicardial and subepicardial mesenchymal cells, 3) presence of common antigens in the transforming epicardium and endocardial cushions (fibrillin-2/JB3, ES/130, Ets-1). Recendy, we have immunolocated the transcription factor Slug in the developing avian heart. Slug is a zinc-finger protein involved in the formation of the neural crest, a developmental event which implies an epithelial-mesenchymal transition. All cells of the primitive epicardium are Slug+ from their differentiation until the stage HH24. However, only a fraction of the endothelial cells from the endocardial cushions are Slug+. We speculate that the expression of Slug marks competence of the epicardial cells to transform into mesenchyme, although this transformation is only achieved where an inducing signal is produced. Regarding the developmental fate of the epicardial-derived cell population, there is strong evidence of its differentiation in fibroblasts and vascular smooth muscle cells, although a contribution to the coronary endothelium cannot be discarded.

Localization of the Wilm's tumour protein WT1 in avian embryos.

The Wilms' tumour suppressor gene WT1 encodes a zinc-finger transcription factor which is essential for the development of kidney, gonads, spleen and adrenals. WT1-null embryos lack all of these viscerae and they also show a thin ventricular myocardium and unexpectedly die from cardiac failure between 13 and 15 days post coitum. We studied the localization of the WT1 protein in chick and quail embryos between stages HH18 and HH35. In early embryos, WT1 protein was located in specific areas of the coelomic mesothelium adjacent to the nephric ducts, the myocardium or the primordia of the endodermal organs (gut, liver and lungs). These mesothelial areas also showed localized expression of Slug, a zinc-finger transcription factor involved in epithelial-mesenchymal transitions. WT1+ mesenchymal cells were always found below the immunoreactive mesothelial areas, either forming a narrow band on the surface of the endodermal organs (gut, liver and lungs) or migrating throughout the mesodermal organs (mesonephros, metanephros, gonads, spleen and heart). In the developing heart, the invasion of WTI+ cells started at stage HH26, and all the ventricular myocardium was pervaded by these cells, presumably derived from the epicardium, at HH30. We suggest that WT1 is not required for the epithelial-mesenchymal transition of the coelomic mesothelium, but it might be a marker of the mesothelial-derived cells, where this protein would be acting as a repressor of the differentiation.

Immunolocalization of the transcription factor Slug in the developing avian heart.

Slug is a transcription factor involved in processes such as the formation of mesoderm and neural crest, two developmental events that imply a transition from an epithelial to a mesenchymal phenotype. During late cardiac morphogenesis, mesenchymal cells originate from two epithelia--epicardial mesothelium and cushion endocardium. We aimed to check if Slug is expressed in these systems of epithelial-mesenchymal transition. We have immuno-located the Slug protein in the heart of quail embryos between Hamburger and Hamilton stages HH16 and HH30. In the proepicardium (the epicardial primordium), Slug was detected in most cells, mesothelial as well as mesenchymal. Slug immunoreactivity was strong in the mesenchyme of the endocardial cushions and subepicardium from its inception until HH24, but the immunoreactivity disappeared in later embryos. Only a small portion of the endocardial cells located in the areas of epithelial-mesenchymal transition (atrioventricular groove and outflow tract) were immuno-labelled, mainly between HH16 and HH20. Endocardial cells from other cardiac segments were always negative, except for a transient, weak immunoreactivity that coincided with the development of the intertrabecular sinusoids of the ventricle. In contrast, virtually all cells of the epicardial mesothelium were immunoreactive until stage HH24. The mesenchymal cells that migrate to the heart through the spina vestibuli were also conspicuously immunoreactive. The myocardium was not labelled in the stages studied. Our results stress the involvement of Slug in the epithelial to mesenchymal transition. We suggest that Slug can constitute a reliable marker of the cardiac epithelial cells that are competent to transform into mesenchyme as well as a transient marker of the epithelial-derived mesenchymal cells in the developing heart.