Differential Notch Signaling in the Epicardium Is Required for Cardiac Inflow Development and Coronary Vessel Morphogenesis

Abstract

Rationale:The proepicardium is a transient structure comprising epicardial progenitor cells located at the posterior limit of the embryonic cardiac inflow. A network of signals regulates proepicardial cell fate and defines myocardial and nonmyocardial domains at the venous pole of the heart. During cardiac development, epicardial-derived cells also contribute to coronary vessel morphogenesis.Objective: To study Notch function during proepicardium development and coronary vessel formation in the mouse. Methods and Results:Using in situ hybridization, RT-PCR, and immunohistochemistry, we find that Notch pathway elements are differentially activated throughout the proepicardial-epicardial-coronary transition. Analysis of RBPJk-targeted embryos indicates that Notch ablation causes ectopic procardiogenic signaling in the proepicardium that in turn promotes myocardial differentiation in adjacent mesodermal progenitors, resulting in a premature muscularization of the sinus venosus horns. Epicardium-specific Notch1 ablation using a Wt1-Cre driver line disrupts coronary artery differentiation, reduces myocardium wall thickness and myocyte proliferation, and reduces Raldh2 expression. Ectopic Notch1 activation disrupts epicardium development and causes thinning of ventricular walls. T he early vertebrate heart consists of 2 tissue layers, the outer myocardium and the inner endocardium. At embryonic day (E)9.5 in mice, a third tissue, the epicardium, migrates to envelop the outer surface of the myocardium, actively participating in coronary vessels (CVs) morphogenesis and compact ventricular myocardium growth. 1 Epicardial progenitors are found in the proepicardium, a grape-like structure with characteristic protrusions (proepicardial villi). The proepicardium arises by E9.0 through local proliferation of the coelomic epithelium covering the septum transversum at the posterior limit (venous pole) of the heart. 2 The mesodermal progenitors underneath the proepicardium are often referred to as "posterior pericardial mesoderm," 3 a term that refers to topology rather than an embryological relation to the pericardial membranes. The venous pole of the heart provides the developing heart with myocardium, 4 a process that in the mouse finishes with the muscularization of the sinus horns. 5 The completion of the primitive epicardial epithelium covering the myocardium is followed by the appearance of the subepicardium, 1 an abundant extracellular matrix separating epicardium and myocardium. Concomitantly, the epicardium undergoes an epithelial-tomesenchymal transition (EMT) and populates the subepicardium with mesenchymal epicardial-derived cells (EPDCs), that contribute to the development of CVs and cardiac interstitium. 1 Proepicardial cells (proEPCs) can differentiate in vivo and in vitro into vascular/fibroblastic cell types or myocardium depending on the conditions. 3 A recent study proposed that proepicardial fate depends on the balance between Bmps and Fgfs signals, Bmps promoting myocardial differentiation and Fgfs f...

Signaling During Epicardium and Coronary Vessel Development

“…23 The strategy adopted by our group to study Notch function in epicardial development is based on the detailed description of Notch elements during PE-epicardial-coronary transition ( Figure 4D-F) and specific gene deletion using the mWt1/ IRES/GFP-Cre (Wt1Cre) driver line. 80 This line recapitulates Wt1 expression in epicardium, and the driver is active in PE precusors at E8.5 and in the E9.5 PE and its derivatives from E10.5 onwards. 80 Crossing this driver line with Notch1 flox conditional mice generated Wt1Cre:Notch1 flox/flox mice, which show body and pericardial hemorrhages and a severely reduced and disorganized coronary vascular plexus at E13.5.…”

“…The EPDC produced by epicardial EMT are considered a major source of CF, 103 and several studies in avian 9,15,17,19 and mouse embryos 10,11,80 indicate that the embryonic epicardium is the first and main contributor of embryonic CF ( Figure 4B, C). Other sources of CF are the EMT that occurs in the embryo during the formation of cardiac cushions 104 or post-natally via the recruitment of circulating bone marrow cells.…”

“…These findings, based on the use of Tbx18-and Wt1-Cre drivers, 10,11 have been challenged by further research providing evidence for the expression of the epicardial marker Tbx18 in a population of chamber myocardial cells before EPDC invade the cardiac walls. 13 While research continues, it now seems that the number of epicardial cells that might spontaneously transdifferentiate into myocytes in the developing heart is small; because the so-called epicardial markers Tbx18, Gata5, and Wt1 are also expressed by nondifferentiated mesodermal cells at the venous pole of the heart before the PE forms, 80 CM genetically tagged as epicardial-derived might not, in fact, directly derive from the embryonic epicardium.…”

“…89 It is certain that epicardial-myocardial interactions are critical for coronary development 73 and that the development of arterial and venous prospective coronary domains requires fine molecular coordination. 23,80 However, it is unlikely that arterial endothelium first differentiates from a portion of the venous vasculature actively perfused by blood (the sinus venosus) and then becomes isolated from the systemic blood flow 90 before finally reconnecting to the coronary venous component to close the coronary vessel circuitry.…”

“…23,80 Various conditional mutants combined with epicardial-specific drivers have been used to ascertain the function of Notch in the epicardium and its derivatives. In the study by Grieskamp et al, a Tbx18-Cre line was bred with a conditional allele of the RBPJk gene.…”