Epicardium development in Xenopus.

The epicardium is the outermost layer of the mature vertebrate heart. In birds and mice the epicardium originates from an extracardiac primordium - the proepicardium - and envelops the developing heart of the embryo. The epicardium of birds and mice has two main functions in normal heart development: 1) a subset of epicardial cells undergo an epithelial to mesenchymal transition, generating the pluripotent progenitors of the coronary vascular system 2) the epicardium secretes signals required for the proliferation and survival of cardiac myocytes. Whilst the first role has been the subject of considerable study, much less is understood about the role of the epicardium in maintenance and development of the heart myocardium. The heart of the amphibian, Xenopus laevis, differs from that of mammals in a number of ways. The frog myocardium has a spongy, less compact structure and completely lacks coronary vasculature. As a result, studying the epicardium in Xenopus could provide a useful model for investigating the role of this tissue in the structure and development of heart muscle. The structure of the epicardium in adult Xenopus laevis was analysed using histological methods and is shown to comprise a single cell-layered simple squamous epithelium. The embryonic origin and developmental progression of the epicardium was analysed histologically, by scanning electron microscopy, and by episcopic fluorescence image capturing. The epicardium is shown to be derived from the proepicardium, a clump of cells approximately 161 urn wide by 133pm high, located caudally to the heart on the septum transversum. The proepicardium is shown to be located asymmetrically to the right of the sagittal plane. Cell lineage analyses confirm the unilateral, right-sided origin of the epicardium. At around stage 42 in development the proepicardium can be seen to be in direct contact with the ventricular myocardium, and proepicardial cells can be observed migrating over this 'proepicardial bridge.' Once proepicardial cells contact the ventricular myocardium, they proceed to envelop the heart beginning with the atrioventricular and conoventricular canals. Epicardial cells in Xenopus laevis were identified using the expression patterns of three genes - Tbxl8, epicardin and Wilms tumour 1 - that have been shown to demarcate the avian or mammalian epicardium. Morpholino knock downs of these candidate genes were performed in order to investigate the roles of these genes in epicardium development, and therefore the wider roles of the epicardium in heart development. However, epicardium formation was found to be occurring too late in development to be accessible to morpholino knockdown analysis