Keeping the Rhythm: Cardiac Pacemaker Cell Development

Abstract

The heart is the first organ to form and function in the developing vertebrate embryo. Its proper morphogenesis and function is crucial for survival. Here we focus on the development and characterization of a highly specialized subset of cardiac cells, the pacemaker cells. In the mammalian heart, the primary pacemaker cells are located in the sinoatrial node embedded in the upper wall of the right atrium. The pacemaker cells initiate the electric signal stimulating cardiomyocyte contraction. Thus, they constitute the topmost part of an intrinsic pacemaker and conduction system controlling and regulating heart function. Cardiac contractions commence at very early embryonic stages and coordination remains crucial for survival. Although cardiac development and function are strikingly conserved amongst animal classes, in lower vertebrates neither structural nor molecular distinguishable components of a conduction system had been identified, questioning its evolutionary origin. Here we studied pacemaker cell development in the zebrafish. We have identified the LIM-homeodomain transcription factor Islet-1 (Isl1) as central factor in pacemaker cell development. Using a transgenic reporter, we showed that Isl1+ cells harbour the characteristic electrophysiological properties of pacemaker cells. They are located at the sinoatrial junction and form a ring-shaped, interconnected structure, a remarkable contrast to the compact sinoatrial node in the mammalian heart. Isl1 had previously been described as an important factor in the development of the second heart field, a pool of cardiac progenitor cells contributing to large parts of the heart. In the mouse, loss of Isl1 leads to extensive heart defects, mainly the absence of outflow tract structures and parts of the atria. Here we show that zebrafish embryos lacking Isl1 display a milder, very specific defect in the heart. These embryos present a very slow, irregular heart rate, defects reminiscent to cardiac pacemaker dysfunction. However, this phenotype was not due to a loss of pacemaker cells in Isl1 mutants. We could show that the Isl1-expressing pacemaker cells were still present in the sinoatrial region of Isl1-/- hearts. Hence, loss of Isl1 lead to a loss of the pacemaker cell function in the Isl1+ cells. Isl1 is a transcription factor, but very little was known about its downstream target genes in pacemaker cells. We could show that expression of two genes, bmp4 and tbx2b, known to be expressed in pacemaker cells in various organisms was lost upon Isl1 knockout. To better understand the pathways regulating cardiac development and function in general and the pacemaker domain in particular, we used tomo-seq, combining high-throughput RNA sequencing with tissue sectioning, to establish a genome-wide expression dataset with high spatial resolution for the developing zebrafish heart.Analysis of the dataset revealed over 1100 genes differentially expressed in sub-compartments within the heart.Furthermore, we identified a novel role for the wnt-signalling pathway in controlling heart rate via the parasympathetic nervous system.Thus, this high-resolution transcriptome map incorporating all cell types in the embryonic heart can expose spatially-restricted molecular pathways critical for specific cardiac functions.