Coordinating tissue interactions: Notch signaling in cardiac development and disease

Abstract

The Notch pathway is a crucial cell-fate regulator in the developing heart. Attention in the past centered on Notch function in cardiomyocytes. However, recent advances demonstrate that region-specific endocardial Notch activity orchestrates the patterning and morphogenesis of cardiac chambers and valves through regulatory interaction with multiple myocardial and neural crest signals. Notch also regulates cardiomyocyte proliferation and differentiation during ventricular chamber development and is required for coronary vessel specification. Here, we review these data and highlight disease connections, including evidence that Notch-Hey-Bmp2 interplay impacts adult heart valve disease and that Notch contributes to cardiac arrhythmia and pre-excitation syndromes.

Figure 1. The Notch pathway

Membrane-bound Notch ligands (Dll1,3,4 and Jag1,2) are characterized by a Delta/Serrate/Lag2 (DSL) motif (yellow) located in the extracellular domain. The Notch receptor (Notch1-4) is processed at the S1 site by a furin protease, sugar-modified by Fringe in the Golgi, and inserted into the membrane as a heterodimer with a large extracellular domain (NECD). Ligand-receptor interaction leads to two consecutive cleavage events (at S2 and S3 sites), respectively carried out by an ADAM protease and presenilin, which release the Notch intracellular domain (NICD). NICD translocates to the nucleus and forms a transcriptional activation complex after binding to Mastermind (MAML) and CSL/RBPJk/Su(H). This ternary complex activates the transcription of a set of target genes including Hes, Hey and others.

Figure 2. Cardiac development and Notch pathway expression in the mouse

(A) E7.5, cardiac crescent. Progenitor cell populations of the first and second heart fields (FHF, yellow; SHF, light blue) have fused in the midline of the embryo. (B) Jag1, Dll4 and N1ICD are expressed in the developing endocardial tubes (blue). (C) E8.0, heart tube stage. The approximate contributions of FHF and SHF are shown. (D) The heart tube has an outer myocardial layer and an inner endocardial endothelium separated by cardiac jelly (gray). (E) Jag1 is expressed in the myocardium while Dll4, N1ICD, Notch2 and Notch4 are expressed in the endocardium. (F) E9.5, looped heart. AVC formation separates the atria from the ventricles. The approximate contributions of FHF and SHF are shown. (G) Valve primordia are formed between E9.5–10.5. The diagram shows the contributions of endocardium-derived mesenchyme to the AVC and of endocardium-derived plus neural crest (NC) mesenchyme to the OFT. Ventricular chamber development begins with trabeculae formation in the left and right ventricles. At this stage, the proepicardium arises in the inflow region of the heart and expresses Dll1,4; N2–4 and Hey1. (H) In the E9.5–10.5 heart, Jag1 is expressed in chamber myocardium and endocardium; Dll4 and N1ICD are expressed in valve and atrial endocardium and at the base of trabecular endocardium. Notch2 is expressed in chamber endocardium and valve mesenchyme. (I) E13.5-adult heart. The four chambers and four valves are shown (1, tricuspid valve; 2, pulmonary valve; 3, mitral valve; 4, aortic valve). The epicardium covering the heart and expressing various Notch elements is shown in gray and the coronary vessels in yellow. Expression of Notch elements in epicardium and coronary arteries is indicated. In all panels the ventral aspect of the heart is shown (anterior to the top). A, atria; AVC, atrio-ventricular canal; LA, left atrium, LV, left ventricle; OFT, outflow tract; RV, right ventricle.

Figure 3. Signals in cardiac patterning

(A) Top: Wild type. Notch1 activity (red) is found in the endocardium of the AVC, OFT, LA and basal region of trabeculae. Hey1, Hey2 and HeyL are differentially expressed in chamber and AVC endocardium. Myocardial Bmp2 expression (blue) is confined to the AVC and OFT myocardium by Hey1 (green) and Hey2 (brown), expressed in the chambers. Middle: Systemic Notch loss of function (Notch LOF) in RBPJk mutants leads to Hey down-regulation and ectopic Bmp2 expression in the endocardium. Endocardial AVC and chamber patterning is lost. Notch inactivation does not affect myocardial patterning or the myocardial expression domains of Hey1, Hey2 or Bmp2. Bottom: Notch gain-of-function (Notch GOF) by ectopic N1ICD expression in endocardium and myocardium (Nkx2.5-Cre; RosaN1ICD). Expression of Hey1, 2 and HeyL expands throughout the endocardium. Hey1 is expressed throughout the myocardium (green), repressing Bmp2 in the AVC. Valve patterning is lost in the myocardium and expands throughout the endocardium. (B) Pathway establishing chamber vs. valve domains in the developing heart. Cardiogenic signals activate Tbx20 expression, which drives Hey1 and Hey2 and represses Tbx2 expression in chamber myocardium. Hey1 and Hey2 confine Bmp2 expression to prospective valve tissue. Tbx2 and 3 are activated by Bmp2 and restricted to valve territory by Tbx20 and Hey. Forced Tbx2 expression represses Hey genes (dashed lines). Color codes as in (A); abbreviations as in Figure 2.

Figure 4. Tissue interactions during cardiac EMT and valve morphogenesis

(A) Diagram of an E10 looped heart. (B) Detail of the boxed area in (A) highlighting the OFT and AVC endocardial cushions where EMT takes place and valves form. Myocardium, red; endocardium, blue; extracellular matrix, gray. Signals (arrows) from endocardium and myocardium converge to trigger EMT. Migratory NC mesenchyme invades the OFT region. Its interaction with endocardial mesenchyme is crucial for valve morphogenesis. (C) Diagram of an E12.5 heart showing the color-coded expression of Notch elements during OFT and AAA remodeling. NC migrate from the dorsal neural tube around the AAA and toward the OFT cushions. They will also contribute to the SMC of the AAA.

Figure 5. Notch and ventricular trabeculation

The inner surface (endocardium) of the ventricular cavity is shown at the top, and the outer surface (epicardium) at the bottom. Note the apposition of endocardium and myocardium at the base of developing trabeculae, where active Dll4-Notch1 signaling (strong red nuclei) predominates and decays towards the apex (pink-white nuclei). Notch signals to the trabecular myocardium to promote cardiomyocyte proliferation and differentiation (yellow and blue arrows). Modified from (Grego-Bessa et al., 2007).